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WO2020200196A1 - 抗Claudin18.2抗体及其应用 - Google Patents

抗Claudin18.2抗体及其应用 Download PDF

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Publication number
WO2020200196A1
WO2020200196A1 PCT/CN2020/082369 CN2020082369W WO2020200196A1 WO 2020200196 A1 WO2020200196 A1 WO 2020200196A1 CN 2020082369 W CN2020082369 W CN 2020082369W WO 2020200196 A1 WO2020200196 A1 WO 2020200196A1
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Prior art keywords
seq
variable region
antibody
chain variable
light chain
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PCT/CN2020/082369
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English (en)
French (fr)
Inventor
杨阳
葛虎
陶维康
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to AU2020250601A priority Critical patent/AU2020250601A1/en
Priority to EP20782022.6A priority patent/EP3950716A4/en
Priority to CN202080019073.9A priority patent/CN113544156B/zh
Priority to CN202211305192.8A priority patent/CN116063516A/zh
Priority to BR112021019135A priority patent/BR112021019135A2/pt
Priority to KR1020217034462A priority patent/KR20210148216A/ko
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to MX2021011489A priority patent/MX2021011489A/es
Priority to US17/442,939 priority patent/US20220185881A1/en
Priority to CA3134411A priority patent/CA3134411A1/en
Priority to JP2021557129A priority patent/JP7592022B2/ja
Publication of WO2020200196A1 publication Critical patent/WO2020200196A1/zh
Priority to ZA2021/06880A priority patent/ZA202106880B/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/467—Igs with modifications in the FR-residues only
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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 disclosure relates to the field of antibody drugs. Specifically, the present disclosure relates to the Claudin 18.2 antibody and its applications.
  • Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans, and belongs to the family of cellular tight junction proteins. Claudin-18 can control the molecular flow between layers of cells.
  • Claudin-18 protein structure includes four transmembrane regions, two extracellular loops, and its N-terminal and C-terminal are in the cytoplasm.
  • Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, and there are only eight amino acid differences between the two sequences in the first extracellular loop.
  • the expression distribution of Claudin 18.1 and Claudin 18.2 is different.
  • Claudin 18.1 is selectively expressed in normal lung cells, and Claudin 18.2 is highly restricted in normal cells, but in a variety of tumors (gastric cancer, lung cancer, pancreatic cancer, etc.) Frequent ectopic activation and overexpression.
  • Claudin 18.2 is considered as a potential therapeutic target for gastric cancer and other cancer types. The discovery of this target also provides a new option for the treatment of gastric cancer.
  • the present disclosure provides an anti-Claudin 18.2 antibody.
  • the anti-Claudin 18.2 antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region includes the same sequence as that shown in SEQ ID NO: 4. LCDR1, LCDR2 and LCDR3 with the same sequence of the light chain variable region shown; or
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region includes the same sequence as that shown in SEQ ID NO: 6.
  • the LCDR1, LCDR2 and LCDR3 of the same sequence of the light chain variable region are shown.
  • the anti-Claudin 18.2 antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region includes SEQ ID respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the light chain variable region includes SEQ ID LCDR1, LCDR2, and LCDR3 shown in NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
  • the anti-Claudin 18.2 antibody as described above, wherein the anti-Claudin 18.2 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the anti-Claudin 18.2 antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% of the heavy chain variable region shown in SEQ ID NO: 3 or 24 %, 100% identity, and the light chain variable region is at least 90%, 92%, 94%, 95%, 96%, 97%, and the light chain variable region shown in SEQ ID NO: 4 or 21 98%, 99%, 100% identity; or
  • the anti-Claudin 18.2 antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3 or is at least 90% identical to it, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 or is identical to it At least 90% identity;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24 or is at least 90% identical to it, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21 or has the same At least 90% identity;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5 or is at least 90% identical to it, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6 or has the same At least 90% identical; or
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31 or is at least 90% identical to it, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28 or has the same At least 90% identity.
  • the anti-Claudin 18.2 antibody as described above, wherein the anti-Claudin 18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof,
  • the framework region variants are back mutations each having at most 10 amino acids in the light chain framework region and/or the heavy chain framework region of a human antibody.
  • the heavy chain framework region of the human antibody is the same as the framework region of the heavy chain variable region shown in the amino acid sequence SEQ ID NO: 24, or the light chain variable region of the human antibody is the same as the amino acid sequence SEQ
  • the framework region of the light chain variable region shown in ID NO: 21 is the same; or the heavy chain framework region of the human antibody is the same as the framework region of the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 31, or the human antibody
  • the light chain variable region of the antibody is the same as the framework region of the light chain variable region shown in the amino acid sequence SEQ ID NO: 28.
  • the framework region variant comprises a mutation selected from the following (a) or (b):
  • the light chain variable region contains one or more amino acid back mutations in 22S, 85I or 87H, and/or the heavy chain variable region contains one selected from 48I, 82T and 69M or Multiple amino acid back mutations; or
  • the light chain variable region contains one or more amino acid back mutations selected from 4L and 22S, and/or the heavy chain variable region contains 38K, 40R, 48I, 66K, 67A, 69L, 71L And one or more amino acids in 73K are backmutated.
  • the framework region variant comprises a mutation selected from the following (a-1) or (b-1):
  • the light chain variable region contains an amino acid back mutation selected from 4L.
  • the anti-Claudin 18.2 antibody as previously described, wherein:
  • the heavy chain variable region sequence is shown in SEQ ID NO: 31, 32, 33 or 34 and the light chain variable region sequence is shown in SEQ ID NO: 28, 29 or 30.
  • the anti-Claudin 18.2 antibody as described above, wherein: the anti-Claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below:
  • the anti-Claudin 18.2 antibody as described above, wherein: the light chain variable region and the heavy chain variable region can be a combination of light and heavy chain variable regions as shown in the following table:
  • the anti-Claudin 18.2 antibody as previously described, wherein the antibody further comprises an antibody constant region.
  • the heavy chain constant region of the antibody is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and variants thereof, and the light chain constant region of the antibody is selected from human antibody ⁇ and ⁇ chain constants. Zone and its variants.
  • the antibody comprises a heavy chain constant region shown in SEQ ID NO: 7 and a light chain constant region shown in SEQ ID NO: 8.
  • the antibody comprises: at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 35 or 42 , 100% identical heavy chain, and at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with the amino acid sequence shown in SEQ ID NO: 36 or 39 Identical light chain; or
  • the amino acid sequence shown in SEQ ID NO: 38 or 46 has a light chain with at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity.
  • the anti-Claudin 18.2 antibody as previously described which comprises:
  • the antibody competes with the aforementioned anti-Claudin 18.2 antibody or antigen-binding fragment thereof to bind to human Claudin 18.2.
  • the anti-Claudin 18.2 antibody as previously described which comprises:
  • amino acid sequence is the heavy chain shown in SEQ ID NO: 44, and the light chain is shown in SEQ ID NO: 41; or
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, and the sequence of the light chain is shown in SEQ ID NO: 47.
  • Another aspect of the present disclosure also provides a nucleic acid molecule encoding the anti-Claudin 18.2 antibody as described above.
  • Another aspect of the present disclosure also provides an expression vector comprising the nucleic acid molecule as described above.
  • Another aspect of the present disclosure also provides a host cell, which comprises the nucleic acid molecule as described above or the expression vector as described above, preferably the cell is a bacterial cell, a fungal cell, an insect animal cell or a mammalian cell .
  • Another aspect of the present disclosure also provides an antibody-drug conjugate, which is an antibody-drug conjugate formed by coupling the aforementioned anti-Claudin 18.2 antibody with a cytotoxic drug.
  • Another aspect of the present disclosure also provides an antibody drug conjugate, which comprises or consists of the aforementioned anti-Claudin 18.2 antibody covalently bound to a cytotoxic drug.
  • the present disclosure provides a method for preparing the Claudin 18.2 antibody as described above.
  • the present disclosure provides a method for preparing the Claudin 18.2 antibody drug conjugate as described above.
  • the present disclosure provides a pharmaceutical composition containing a therapeutically effective amount of an anti-Claudin 18.2 antibody as described above, or a nucleic acid molecule as described above, or an antibody as described above Drug conjugates, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the present disclosure provides a method for immunodetection or determination of Claudin 18.2, the method comprising the step of contacting a sample to be tested with an anti-Claudin 18.2 antibody as described above.
  • the present disclosure provides the use of the aforementioned anti-Claudin 18.2 antibody in preparing reagents for immunodetection of human Claudin 18.2.
  • the present disclosure provides an anti-Claudin 18.2 antibody as described above for immunodetection or determination of Claudin 18.2.
  • the present disclosure provides a kit comprising an anti-Claudin 18.2 antibody according to the aforementioned.
  • the present disclosure provides the anti-Claudin 18.2 antibody as described above, or the nucleic acid molecule as described above, or the antibody drug conjugate as described above or the pharmaceutical composition as described above.
  • the cancer or tumor is preferably Claudin 18.2 positive cancer or malignant tumor, more preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glial Tumors, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer , Hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer,
  • the present disclosure provides a method of treating diseases associated with Claudin 18.2, the method comprising administering to a subject a therapeutically effective amount of an anti-Claudin 18.2 antibody as described above, or as described above
  • the aforementioned nucleic acid molecule, or the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition wherein the disease is preferably cancer or tumor; more preferably Claudin 18.2 positive cancer or malignant tumor, more preferably Selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, Nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, Mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from: non-small cell lung cancer and small cell lung cancer,
  • the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1 mg to 3000 mg or 1 mg to 1000 mg of the aforementioned anti-Claudin 18.2 antibody or the aforementioned antibody-drug conjugate.
  • the present disclosure provides an anti-Claudin 18.2 antibody as described above for the treatment of diseases related to Claudin 18.2, or a nucleic acid molecule as described above, or the antibody drug couple as described above
  • the disease is preferably cancer or tumor; more preferably Claudin 18.2 positive cancer or malignant tumor, more preferably selected from: head and neck squamous cell carcinoma, head and neck Cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelium Tumor, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma,
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, Mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from: non-small cell lung cancer and small cell lung cancer,
  • the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.
  • the cancer is stomach cancer, esophageal cancer, lung cancer, pancreatic cancer.
  • the aforementioned antibodies and antibody-drug conjugates can play a therapeutic effect in the aforementioned cancers with high, medium, and low expression of Claudin 18.2.
  • the Claudin 18.2 antibody and antibody-drug conjugate provided in the present disclosure have good affinity with cell surface antigens, good endocytosis efficiency and strong tumor suppression efficiency, and have a wider window of drug application, which is suitable for clinical use Drug application.
  • Figure 1 At the cellular level, the FACS test results of the binding of humanized antibody to human Claudin 18.2.
  • FIG. 1 NUGC4 endocytosis experiment of humanized antibody.
  • Figure 3A to Figure 3C Detection of ADCC effect of antibodies in NUGC4 cells with different expression levels of Claudin 18.2.
  • Figure 3A shows the ADCC effect detection of antibody in wild-type NUGC4 cells (Claudin18.2 low expression);
  • Figure 3B shows the ADCC effect detection of antibody in Claudin18.2 medium expression NUGC4 cells;
  • Figure 3C shows the high expression of antibody in Claudin 18.2 Detection of ADCC effect in NUGC4 cells.
  • cytotoxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Including toxins, chemotherapy drugs and other compounds that can be used to kill cells.
  • toxin refers to any substance that can have a harmful effect on the growth or proliferation of cells, which can be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as ixati Kang, maytansinoids and their derivatives (CN101573384) such as DM1, DM3, DM4, orlistatin F (AF) and their derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790A1, compound 7) , Diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, ⁇ -sarcin, Aleutitesfordii toxic protein, carnation (dianthin) toxic protein, Phytolaca americana toxic protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, jatropha curcin (curcin), croton protein (crotin), soap Sapaonaria officinalis inhibitor, gelon
  • chemotherapeutic agent is a compound that can be used to treat tumors. This definition also includes antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They can be hormones themselves.
  • chemotherapeutic drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN TM ); alkyl sulfonates such as busulfan, improsulfan, and pippo Piposulfan; aziridine such as benaodopa, carboquone, meturedopa and uredopa; aziridine and methyl Melamine (methylamelamine) includes altretamine, triethylenemelamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; nitrogen mustards Such as chlorambucil, chlorambucil, cholophosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride; L-phenylalanine nitrogen Melphalan, novembichin, cholesterol mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such as
  • anti-estrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone, and toremifene (Fareston); and anti-androgen preparations such as Flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts of any of the above substances, acids Or derivatives.
  • anti-estrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone, and toremifene (Fareston); and anti-androgen preparations such
  • the "antibody” in the present disclosure refers to an immunoglobulin.
  • a complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of immunoglobulin heavy chains are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies.
  • murine antibody in the present disclosure is a monoclonal antibody against human Claudin 18.2 prepared according to the knowledge and skills in the art. During the preparation, Claudin 18.2 or its epitope is used as the antigen injection test object, and then the hybridoma expressing the antibody with the desired sequence or functional characteristics is isolated.
  • the murine anti-Claudin 18.2 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chain or a variant thereof, or further comprise murine origin The heavy chain constant region of IgG1, IgG2, IgG3, or variants thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the Claudin 18.2 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or use IgG1, IgG2, or IgG4 variants with amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations).
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of CDR sequences from non-human species to the framework of human antibody variable regions, that is, different types of human species The antibody produced in the antibody framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody carrying a large amount of heterologous protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast to undergo affinity maturation mutations to CDRs.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chain or a variant thereof, or further comprise human or murine IgG1 , IgG2, IgG3, IgG4 or variants thereof; preferably comprising the heavy chain constant region of human IgG1, IgG2, or IgG4, or using amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations) IgG1, IgG2 or IgG4 variants.
  • variable region of the heavy chain constant region of the human antibody and the light chain constant region of the human antibody described in the present disclosure refer to the heavy chain constant that has been disclosed in the prior art and does not change the structure and function of the antibody variable region. Region or light chain constant region.
  • Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions; specific substitutions such as YTE mutations, L234A and/or L235A mutations known in the art, S228P mutations, and/or mutations to obtain a knob-into-hole structure (make the antibody heavy chain have a combination of knob-Fc and hole-Fc), these mutations have been confirmed to give the antibody new properties, but do not change the antibody variable region Function.
  • HumanMAb (HuMAb), “human antibody”, “fully human antibody” and “fully human antibody” can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a genetically modified organism.
  • the transgenic organism is "engineered” to produce specific human antibodies in response to antigen stimulation, and can be produced by any method known in the art.
  • human heavy and light chain locus elements are introduced into cell lines derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted for destruction.
  • Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce hybridomas that secrete human antibodies.
  • a human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to an antibody in a substantially intact form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to antibodies that contain constant regions in the light and heavy chains.
  • antibody of the present disclosure includes “full-length antibodies” and antigen-binding fragments thereof.
  • the full-length antibody of the present disclosure includes the combination of the light chain variable region and the light chain constant region and the heavy chain variable region and the heavy chain constant region in the combination of light and heavy chain variable regions in the following table. Full-length antibody formed. Those skilled in the art can select different antibody-derived light chain constant regions and heavy chain constant regions according to actual needs, such as human antibody-derived light chain constant regions and heavy chain constant regions.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen (eg, Claudin 18.2). It has been shown that fragments of full-length antibodies can be used for the antigen-binding function of antibodies.
  • binding fragment included in the term "antigen-binding fragment" of the antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including The bivalent fragment of two Fab fragments connected by a disulfide bridge on the hinge region, (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody (V) dsFv, an antigen-binding fragment formed by interchain disulfide bonds between VH and VL; (vi) diabodies, bispecific antibodies and multispecific antibodies containing fragments such as scFv, dsFv, and Fab.
  • the two domains VL and VH of the Fv fragment are connected by a synthetic linker, so that it can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); See, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single chain antibodies are also included in the term "antigen-binding fragment" of antibodies.
  • Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies.
  • the antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulin.
  • the antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment with antigen-binding activity among fragments obtained by treating IgG antibody molecules with an enzyme with the same activity as papain.
  • F(ab') 2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme with the same activity as the enzyme pepsin.
  • Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab') 2 .
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment into an expression vector and introducing the vector into a host.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Other linkers that can be used in the present disclosure are described by the following documents, such as but not limited to: Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Alfthan et al. (1995), Protein Eng.
  • Diabodies are antibody fragments in which scFv or Fab are dimerized, and are antibody fragments with bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • Bispecific antibodies and multispecific antibodies refer to antibodies that can bind to two or more antigens or antigenic determinants.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (for example, Protein Engineering, 7, 697 (1994)).
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one, two, three or one mutation on the basis of the original protein or polypeptide. Insertion, deletion or substitution of multiple amino acids.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR in the variable domain of the heavy chain (VH) The amino acid residue numbers are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acids in VL
  • the residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDR is defined by the amino acid residues 26-35 (HCDR1) in human VH. ), 50-65 (HCDR2) and 95-102 (HCDR3) and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
  • epitopes refers to a site on an antigen that is bound by an immunoglobulin or antibody (for example, a specific site on the Claudin 18.2 molecule). Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -8 M, for example, about less than 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibody of the present disclosure binds to Claudin 18.2 or its epitope with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M or 10 -9 M, for example, the antibody in the present disclosure
  • KD dissociation equilibrium constant
  • the term “competition” when used in the context of competing for antigen binding proteins of the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: in the assay, the antigen binding to be detected
  • the protein (such as an antibody or a functional fragment thereof) prevents or inhibits (such as reduces) the specific binding of a reference antigen binding protein (such as a ligand or a reference antibody) to a common antigen (such as the Claudin 18.2 antigen or a fragment thereof).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J.Immunol.137:3614-3619
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); solid-phase direct labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec.
  • the assay involves the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess.
  • the antigen binding proteins identified by the competition assay include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the Examples herein.
  • the competing antigen binding protein is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule can be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • Amino acid sequence identity refers to the alignment of amino acid sequences (introducing gaps when necessary) to achieve the maximum percentage of sequence identity, and does not consider any conservative substitutions as part of sequence identity.
  • the first sequence and the second The percentage of amino acid residues that are identical in the sequence.
  • the alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign ( DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of the host cell after being introduced into the host cell, thereby The host genome replicates together (e.g., non-episomal mammalian vectors).
  • mice can be immunized with human Claudin 18.2 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • Human FR germline sequence can be obtained from ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the family Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae).
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells, and NSO cells.
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect host cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • a stable clone was obtained by expressing an antibody that specifically binds to human Claudin 18.2. Positive clones are expanded in a bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments of the present disclosure, to a patient, the patient has one or more disease symptoms, and the therapeutic agent is known to These symptoms have a therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in the table "Exemplary Amino Acid Conservative Substitutions" below.
  • Effective amount refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the dosage of other agents required to treat the disorder , Enhance the efficacy of another agent, and/or delay the progression of the patient’s target antigen-related disorders of the present disclosure.
  • Exogenous refers to substances produced outside organisms, cells, or humans according to circumstances.
  • Endogenous refers to substances produced in cells, organisms, or human bodies according to circumstances.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, in the best sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous.
  • BLAST algorithm when two sequences are aligned, a comparison is made to give the maximum percent identity.
  • the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence.
  • the following references refer to the BLAST algorithm frequently used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) ) Nature Genet. 3:266-272; Madden, TL et al., (1996) Meth. Enzymol.
  • isolated means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers or salts unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredients and thus the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • Suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffer, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes an agent for treating a disease related to a target antigen (eg, Claudin 18.2) positive cell, the agent comprising the anti-Claudin 18.2 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • a target antigen eg, Claudin 18.2
  • the agent comprising the anti-Claudin 18.2 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • the disease related to Claudin 18.2 in the present disclosure is not limited, as long as it is a disease related to Claudin 18.2.
  • the therapeutic response induced by the molecule of the present disclosure includes: (1) Blocking Claudin 18 by binding to human Claudin 18.2 .2 binding to its receptor/ligand, or (2) killing tumor cells overexpressing Claudin 18.2. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of the present disclosure are very useful for people who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, Colorectal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or determination of target antigens (for example, Claudin 18.2), reagents for immunodetection or determination of target antigens (for example, Claudin 18.2), and reagents for immunodetection or determination of expression of target antigens (for example Claudin 18.2) cell method and a diagnostic agent for diagnosing a disease related to a target antigen (for example, Claudin 18.2) positive cell, which comprises the specific recognition target antigen (for example, human Claudin 18.2) of the present disclosure and interacts with the cell
  • the antibody or antibody fragment combined with the amino acid sequence of the outer region or its three-dimensional structure is used as the active ingredient.
  • the method for detecting or measuring the amount of the target antigen may be any known method.
  • it includes immunodetection or assay methods.
  • the immunoassay or measurement method is a method of detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, western blotting, physicochemical method Wait.
  • the aforementioned diseases related to Claudin 18.2 positive cells can be diagnosed by detecting or measuring Claudin 18.2 expressing cells with the antibody or antibody fragment of the present disclosure.
  • test sample used to detect or measure the target antigen for example, Claudin 18.2
  • the target antigen for example, Claudin 18.2
  • cells expressing the target antigen such as tissue Cells, blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • the reagents used to perform the antigen-antibody reaction include buffers, salts and the like.
  • the reagents used for detection include reagents commonly used in immunological detection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label.
  • Example 1 Construction of a cell line highly expressing Claudin 18.2
  • Lipofectamine 3000 transfection reagent to transfect the pCDH-hClaudin18.2 lentiviral expression vector plasmid and pVSV-G, pCMV-dR8.91 lentiviral system packaging vector into the virus packaging cell 293T; collect the virus-containing medium supernatant , Filter and perform ultra-high-speed centrifugation; use the concentrated virus to infect the human gastric signet ring cell carcinoma cell line NUGC4, screen for two to three weeks with puromycin, and then perform FACS single cell sorting.
  • the tumor IHC score to distinguish the expression degree of Claudin 18.2.
  • Cells with the same expression level of Claudin 18.2 with a tumor IHC score of 3 are high-expressing cells, and cells with the same level of Claudin 18.2 with a tumor IHC score of 2 are medium-expressing cells.
  • the FACS detection of Claudin18.2 expression on the surface of NUGC4 cells infected with lentivirus the NUGC4/hClaudin18.2 monoclonal cell line with high Claudin18.2 expression was selected.
  • the selected monoclonal cell lines will be expanded and cultured, and frozen and stored for subsequent experiments.
  • Anti-human Claudin 18.2 monoclonal antibody was produced by immunizing mice.
  • mice Female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice are purchased, they are kept in a laboratory environment for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 °C; humidity 40-60%. Mice that have adapted to the environment were immunized according to the following protocol.
  • the immune antigen was huClaudin18.2-HEK293 cells (HEK-293 stably transfected cell line transfected with human Claudin 18.2 plasmid).
  • mice with high antibody titer in the serum and the titer tending to the plateau were selected for splenocyte fusion.
  • the immunization was boosted and 1 ⁇ 10 7 cells were injected intraperitoneally (IP).
  • Hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /ml, and 100 ⁇ l/well were seeded in a 96-well plate, 37 After incubating at °C and 5% CO 2 for 3-4 days, supplement with 100 ⁇ l/well of HAT complete medium, and continue culturing for 3-4 days until clones are formed. Remove the supernatant, add 200 ⁇ l/well of HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), culture at 37°C, 5% CO 2 for 3 days, and then perform ELISA detection.
  • complete medium IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • the culture supernatant was detected by the combined ELISA method.
  • the hybridoma clones were obtained through the above experiment screening, and the antibodies were further prepared by the serum-free cell culture method.
  • the antibodies were purified according to the purification examples for use in the test examples.
  • the monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; the monoclonal antibody sequence was cloned, and then humanization, recombinant expression and activity evaluation were carried out.
  • the procedure for cloning sequences from hybridomas is as follows.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the instructions of the kit) and reverse transcription (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing.
  • the amino acid sequence SEQ ID NO: 3-6 corresponding to the obtained DNA sequence is shown in:
  • mAb1901 murine light chain variable region (SEQ ID NO: 4)
  • mAb1902 murine light chain variable region (SEQ ID NO: 6)
  • the murine heavy chain variable region and the light chain variable region are respectively connected with the heavy chain constant region and human kappa light chain constant region of the following human IgG1 antibody to form chimeric antibodies ch1901 and ch1902.
  • the constant region is selected from the following sequences:
  • the heavy chain constant region of human IgG1 antibody (SEQ ID NO: 7)
  • Human ⁇ light chain constant region (SEQ ID NO: 8)
  • the mouse monoclonal antibody is humanized as disclosed in many documents in the field.
  • human constant domains are used to replace parental (murine antibody) constant domains
  • human germline antibody sequences are selected based on the homology of murine and human antibodies
  • CDR grafting is performed.
  • candidate molecules with good activity are selected for humanization, and the results are as follows.
  • amino acid residues of the VH/VL CDR in Table 4 are determined and annotated by the Kabat numbering system.
  • the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene.
  • the chain variable region sequence is SEQ ID NO: 24 and the light chain variable region sequence is SEQ ID NO: 21; recombined with the IgG constant region to form a complete antibody.
  • the FR region in the V region of the humanized antibody is back-mutated, and exemplary back-mutation methods and combinations are as follows:
  • the corresponding heavy chain variable region in the above table can be connected with the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form a full-length antibody heavy chain, and the light chain variable region can be connected with the human ⁇ shown in SEQ ID NO: 8
  • the light chain constant regions are joined to form the light chain of a full-length antibody.
  • the variable region of the heavy chain and the variable region of the light chain can also be connected with other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.
  • the chain variable region sequence is SEQ ID NO: 31 and the light chain variable region sequence is SEQ ID NO: 28; recombined with the IgG constant region to form a complete antibody.
  • the FR region in the V region of the humanized antibody is back-mutated, and exemplary back-mutation methods and combinations are as follows:
  • the corresponding heavy chain variable region is connected to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected to the human ⁇ light chain shown in SEQ ID NO: 8
  • the chain constant regions are joined to form the light chain of the full-length antibody.
  • the full-length antibody sequence is as follows:
  • ch1901 heavy chain (SEQ ID NO: 35)
  • ch1902 heavy chain (SEQ ID NO: 37)
  • the sequence of the full-length antibody light and heavy chain is as follows:
  • the sequence of the full-length antibody light and heavy chain is as follows:
  • the positive control antibody of the present disclosure is IMAB-362 (from WO2016166122):
  • the above-mentioned antibodies were cloned, expressed and purified by conventional gene cloning and recombinant expression methods.
  • Test example 1 Cell level ELISA binding experiment
  • a cell-based ELISA experiment was used to detect the binding properties of Claudin 18.2 antibody.
  • the NUGC4 cells expressing Claudin 18.2 were cultured in a 96-well cell plate (Corning, 3599), and 4% paraformaldehyde was added to fix the cells for 1 hour when the density reached 90%.
  • the cells were fixed with PBST buffer (pH 7.4 PBS containing After washing the plate 3 times with 0.05% Tween-20), add 200 ⁇ l/well of 5% skimmed milk (bright skimmed milk powder) blocking solution diluted with PBS, and incubate in a 37°C incubator for 2.5 hours or 4°C overnight (16-18 hours) Closed.
  • sample diluent pH7.4 PBS containing 1% skim milk
  • sample diluent pH7.4 PBS containing 1% skim milk
  • HRP-labeled goat anti-human secondary antibody Jackson Immuno Research, 109-035-003
  • Test Example 2 Antibody cell-level binding experiment
  • the NUGC4 cells stably expressing Claudin 18.2 were prepared with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)) into a cell suspension of 1 ⁇ 10 6 /ml, 100 ⁇ l/well was added to a 96-well round bottom plate (Corning, 3795). After centrifugation to remove the supernatant, add 50 ⁇ l/well of the Claudin 18.2 antibody to be tested in different concentrations diluted with FACS buffer, and incubate for 1 hour in a refrigerator at 4°C in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)
  • Test Example 4 Determination of antibody affinity based on flow cytometry
  • HEK293/hClaudin18.2 cells were collected in a U-bottom 96-well plate, with 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well.
  • Add the Claudin 18.2 antibody with an initial concentration of 5 ⁇ g/ml, 2 ⁇ gradient dilution (12 concentration points), and incubate at 4°C for 1 hour.
  • the positive control is IMAB362, and a negative control without antibody is set.
  • the antibody was removed by centrifugation, and then 100 ⁇ l/well FITC anti-human IgG Fc antibody (200 ⁇ ) was added, incubated at 4°C in the dark for 30 minutes, washed twice with PBS+2% FBS, and then prepared for flow cytometry.
  • the antibody was diluted in the above-mentioned phenol red-free medium to prepare a 3 ⁇ antibody dilution, and 25 ⁇ l/well of antibody was added to the cell plate. Incubate in a 37°C, 5% CO 2 incubator for 0.5 hours.
  • Collect effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells), centrifuge at 1000 rpm, and resuspend for counting.
  • the cells were resuspended at a density of 3 ⁇ 10 6 cells/ml in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum), and 25 ⁇ l of cells (7.5 ⁇ 10 4 Cells/well). Incubate for 6 hours in a 37°C, 5% CO 2 incubator.
  • FBS New Zealand ultra-low IgG fetal bovine serum

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Abstract

本公开涉及抗Claudin18.2抗体及其应用。具体地，本发明涉及一种抗Claudin18.2抗体；包含所述抗Claudin18.2抗体CDR的鼠源抗体、嵌合抗体、人源化抗体及其抗原结合片段，以及其作为药物的用途。特别地，本公开涉及一种抗Claudin18.2抗体在制备用于治疗Claudin18.2阳性疾病或病症的药物中的用途。

Description

抗Claudin18.2抗体及其应用
本申请要求2019年4月1日提交的中国专利申请201910257853.6的优先权，其全部内容通过引用并入此处。
技术领域
本公开涉及抗体药物领域。具体地，本公开涉及Claudin18.2抗体以及其应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
密蛋白18(Claudin-18，CLDN18)是一种在人类中由Claudin18基因编码的蛋白质，属于细胞紧密连接蛋白家族。Claudin-18可以控制层细胞之间的分子流动。
Claudin-18蛋白结构中包括四个跨膜区域、两个细胞外环，其N末端和C末端在胞浆内。Claudin-18具有两个剪接变体，分别为Claudin 18.1和Claudin 18.2，两者序列之间仅在第一个细胞外环有八个氨基酸的差异。Claudin 18.1和Claudin18.2的表达分布有所不同，Claudin 18.1在正常肺的细胞中选择性表达，Claudin 18.2在正常细胞中表达高度受限，但在多种肿瘤(胃癌、肺癌和胰腺癌等)中频繁异位激活和过表达。Claudin18.2被认为是胃癌和其他癌症类型的潜在治疗靶点，此靶点的发现也为胃癌的治疗提供了一种新的选择。
发明内容
本公开提供一种抗Claudin18.2抗体。
在一些实施方案中，如前所述抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
i)所述重链可变区包含与如SEQ ID NO：3所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：4所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3；或
ii)所述重链可变区包含与如SEQ ID NO：5所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。在一些实施方案中，如前所述抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
iii)所述重链可变区包含分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3；或
iv)所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID  NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
本领域技术人员应当理解，项目编号，例如i)、ii)、a)、b)等，其目的仅在于使所列举的技术方案或要素更加清晰、有条理，但是对跟随其后的技术方案或要素不具有任何方面的约束性。当使用相同的项目编号时，不代表跟随其后的技术方案或要素是相同的。
在一些实施方案中，如前所述抗Claudin18.2抗体，其中所述抗Claudin18.2抗体是鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，如前所述抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
(v)所述重链可变区与SEQ ID NO:3或24所示的重链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性，和所述轻链可变区与SEQ ID NO:4或21所示轻链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性；或
(vi)所述重链可变区与SEQ ID NO:5或31所示重链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性，和所述轻链可变区与SEQ ID NO:6或28所示轻链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性。
在一些实施方案中，如前所述抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区氨基酸序列如SEQ ID NO:3所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:4所示或与其有至少90％同一性；
(2)所述重链可变区氨基酸序列如SEQ ID NO:24所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:21所示或与其有至少90％同一性；
(3)所述重链可变区氨基酸序列如SEQ ID NO:5所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:6所示或与其有至少90％同一性；或
(4)所述重链可变区氨基酸序列如SEQ ID NO:31所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:28所示或与其有至少90％同一性。
在一些实施方案中，如前所述抗Claudin18.2抗体，其中所述抗Claudin18.2抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多10个氨基酸的回复突变。
在一些实施方案中，所述人抗体的重链框架区与氨基酸序列SEQ ID NO:24 所示重链可变区的框架区相同，或所述人抗体的轻链可变区与氨基酸序列SEQ ID NO:21所示轻链可变区的框架区相同；或所述人抗体的重链框架区与氨基酸序列SEQ ID NO:31所示重链可变区的框架区相同，或所述人抗体的轻链可变区与氨基酸序列SEQ ID NO:28所示轻链可变区的框架区相同。
在一些实施方案中，优选地，所述框架区变体包含选自以下(a)或(b)所述的突变：
(a)所述轻链可变区中包含22S、85I或87H中的一个或多个氨基酸回复突变，和/或所述重链可变区中包含选自48I、82T和69M中的一个或多个氨基酸回复突变；或
(b)所述轻链可变区中包含选自4L和22S一个或多个氨基酸回复突变，和/或所述重链可变区中包含38K、40R、48I、66K、67A、69L、71L和73K中的一个或多个氨基酸回复突变。
在一些实施方案中，如前所述抗Claudin18.2抗体，所述框架区变体包含选自以下(a-1)或(b-1)所述的突变：
(a-1)所述轻链可变区中包含22S、85I和87H的氨基酸回复突变，和所述重链可变区中包含48I和82T的氨基酸回复突变；或
(b-1)所述轻链可变区中包含选自4L的氨基酸回复突变。
在一些实施方案中，如前所述抗Claudin18.2抗体，其中：
(vii)所述重链可变区序列如SEQ ID NO:3所示和所述轻链可变区序列如SEQ ID NO：4所示；或
(viii)所述重链可变区序列如SEQ ID NO:24、25、26或27所示和所述轻链可变区序列如SEQ ID NO：21、22或23所示；或
(ix)所述重链可变区序列如SEQ ID NO:5所示和所述轻链可变区序列如SEQ ID NO：6所示；或
(x)所述重链可变区序列如SEQ ID NO:31、32、33或34所示和所述轻链可变区序列如SEQ ID NO：28、29或30所示。
在一些实施方案中，如前所述抗Claudin18.2抗体，其中：所述抗Claudin18.2抗体或其抗原结合片段，其包含如下所示的重链可变区和轻链可变区：
(xi)所述重链可变区序列如SEQ ID NO:31所示和所述轻链可变区序列如SEQ ID NO:29所示；或
(xii)所述重链可变区序列如SEQ ID NO:26所示和所述轻链可变区序列如SEQ ID NO:23所示。
在一些实施方案中，如前所述抗Claudin18.2抗体，其中：轻链可变区和重链可变区可以为如下表格所示的轻重链可变区的组合：
表1.mAb1901人源化抗体轻重链可变区组合
可变区 VH1 VH2 VH3 VH4
VL1 VH1VL1 VH2VL1 VH3VL1 VH4VL1
VL2 VH1VL2 VH2VL2 VH3VL2 VH4VL2
VL3 VH1VL3 VH2VL3 VH3VL3 VH4VL3
表2.mAb1902人源化抗体轻重链可变区组合
可变区 VH11 VH12 VH13 VH14
VL11 VH11VL11 VH12VL11 VH13VL11 VH14VL11
VL12 VH11VL12 VH12VL12 VH13VL12 VH14VL12
VL13 VH11VL13 VH12VL13 VH13VL13 VH14VL13
在一些实施方案中，如前所述抗Claudin18.2抗体，其中所述抗体进一步包含抗体恒定区。在一些具体的实施方案中，所述抗体的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其变体，所述抗体的轻链恒定区选自人抗体κ和λ链恒定区及其变体。在一些具体的实施方案中，所述抗体包含序列如SEQ ID NO:7所示的重链恒定区和序列如SEQ ID NO:8所示的轻链恒定区。在一些具体的实施方案中，所述抗体包含：与SEQ ID NO:35或42所示氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性的重链，和与SEQ ID NO:36或39所示氨基酸序列有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性的轻链；或
与SEQ ID NO:37或49所示氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性的重链，和/或与SEQ ID NO:38或46所示氨基酸序列有至少90％、92％、94％、95％、96％、97％、98％、99％、100％同一性的轻链。
在一些实施方案中，如前所述抗Claudin18.2抗体，其包含：
(c)序列如SEQ ID NO:35所示的重链和/或序列如SEQ ID NO：36所示的轻链；
(d)序列如SEQ ID NO:42、43、44或45所示的重链和/或序列如SEQ ID NO：39、40或41所示的轻链；
(e)序列如SEQ ID NO:37所示的重链和/或序列如SEQ ID NO：38所示的轻链；或
(f)序列如SEQ ID NO:49、50、51或52所示的重链和/或序列如SEQ ID NO：46、47或48所示的轻链。
在一些实施方案中，如前所述抗Claudin18.2抗体，所述抗体与前述的抗Claudin18.2抗体或其抗原结合片段竞争性结合人Claudin18.2。
在一些实施方案中，如前所述抗Claudin18.2抗体，其包含:
氨基酸序列如SEQ ID NO:44所示的重链，和序列如SEQ ID NO:41所示的轻链；或
氨基酸序列如SEQ ID NO:49所示的重链，和序列如SEQ ID NO:47所示的轻链。
本公开的另一方面还提供一种核酸分子，其编码如前所述的抗Claudin18.2抗体。
本公开的另一方面还提供一种表达载体，其包含如前所述的核酸分子。
本公开的另一方面还提供一种宿主细胞，其包含如如前所述的核酸分子或如前所述的表达载体，优选所述细胞为细菌细胞、真菌细胞、昆虫动物细胞或哺乳动物细胞。
本公开的另一方面还提供一种抗体药物偶联物，其为由根据如前所述的抗Claudin18.2抗体与细胞毒性药物偶联形成的抗体药物偶联物。
本公开的另一方面还提供一种抗体药物偶联物，其包含或由以下组成：共价结合有细胞毒性药物的前述抗Claudin18.2抗体。
在一些实施方案中，本公开提供一种如前所述的Claudin18.2抗体的制备方法。
在一些实施方案中，本公开提供一种如前所述的Claudin18.2抗体药物偶联物的制备方法。
在一些实施方案中，本公开提供一种药物组合物，其含有治疗有效量的根据如前所述的抗Claudin18.2抗体，或根据如前所述的核酸分子，或如前所述的抗体药物偶联物，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
在一些实施方案中，本公开提供一种用于免疫检测或测定Claudin18.2的方法，所述方法包括用如前所述的抗Claudin18.2抗体接触待测样本的步骤。
在一些实施方案中，本公开提供如前所述的抗Claudin18.2抗体在制备免疫检测人Claudin18.2的试剂中的用途。
在一些实施方案中，本公开提供一种用于免疫检测或测定Claudin18.2的如前所述的抗Claudin18.2抗体。
在一些实施方案中，本公开提供一种试剂盒，其包含根据如前所述的抗Claudin18.2抗体。
在一些实施方案中，本公开提供如前所述的抗Claudin18.2抗体，或如前所述的核酸分子，或如前所述的抗体药物偶联物或如前所述的药物组合物在制备用于治疗癌症或肿瘤的药物中的用途，其中所述癌症或肿瘤优选Claudin18.2阳性癌症或恶性肿瘤，更优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻 咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，本公开提供一种治疗与Claudin18.2相关的疾病的方法，所述方法包括向受试者施用治疗有效量的如前所述的抗Claudin18.2抗体，或如前所述的核酸分子，或如前所述的抗体药物偶联物或如前所述的药物组合物，其中所述疾病优选为癌症或肿瘤；更优选的Claudin18.2阳性癌症或恶性肿瘤，更优选选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌。更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1mg至3000mg或1mg至1000mg如前所述的抗Claudin18.2抗体或如前所述的抗体药物偶联物。
在一些实施方案中，本公开提供一种用于治疗与Claudin18.2相关的疾病如前所述的抗Claudin18.2抗体，或如前所述的核酸分子，或如前所述的抗体药物偶联物或如前所述的药物组合物，其中所述疾病优选为癌症或肿瘤；更优选的Claudin18.2阳性癌症或恶性肿瘤，更优选选自：头和颈鳞状细胞癌、头和颈癌、 脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌。更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，所述癌症为胃癌、食道癌、肺癌、胰腺癌。
在一些实施方案中，如前所述抗体、抗体药物偶联物可以在Claudin18.2高表达、中表达、低表达的如前所述癌症中发挥治疗作用。
本公开提供的Claudin18.2抗体及抗体药物偶联物具有与细胞表面抗原良好的亲和力，良好的细胞内吞效率和很强的肿瘤抑制效率，并且具有更宽的药物应用窗口，适于临床的药物应用。
附图说明
图1：在细胞水平上，人源化抗体与人Claudin18.2结合的FACS检测结果。
图2：人源化抗体的NUGC4细胞内吞实验。
图3A至图3C：在不同Claudin18.2表达程度的NUGC4细胞中，抗体的ADCC效应检测。图3A为抗体在野生型NUGC4细胞(Claudin18.2低表达)中的ADCC效应检测；图3B为抗体在Claudin18.2中等表达NUGC4细胞中的ADCC效应检测；图3C为抗体在Claudin18.2高表达NUGC4细胞中的ADCC效应检测。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“细胞毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的 物质。包括毒素、化疗药物等能用于杀伤细胞的化合物。
术语“毒素”是指能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4、奥利斯他汀F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790A1，化合物7)、白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“化疗药物”是可用于治疗肿瘤的化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXAN TM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和甲基蜜胺(methylamelamine)包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，安曲霉素(authramycin)，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin，洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin,嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)， 佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine)，依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone)，丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如亚叶酸(frolinic acid)；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；elfomithine；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
Figure PCTCN2020082369-appb-000001
雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺(spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；哌溴烷坑(pipobroman)；gacytosine；阿拉伯糖苷("Ara-C")；环磷酰胺；噻替派(thiotepa)；紫杉烷，如紫杉醇(
Figure PCTCN2020082369-appb-000002
Bristol-Myers Squibb Oncology,Princeton,NJ)和docetaxel(
Figure PCTCN2020082369-appb-000003
Rhone-Poulenc Rorer,Antony,France)；苯丁酸氮芥；吉西他滨(gemcitabine)；6-硫代鸟嘌呤；巯基嘌呤；氨甲蝶呤；铂类似物如顺铂和卡铂；长春花碱；铂；依托泊甙(etoposide)(VP-16)；异环磷航胶；丝裂霉素C；米托蒽醌；长春新碱；长春瑞宾(vinorelbine)；新霉酰胺(navelbine)；诺肖林(novantrone)；替尼泊甙(teniposide)；柔红霉素；氨基蝶呤；希罗达(xeloda)；伊拜磷酸盐(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；维甲酸esperamicins；卡培他滨(capecitabine)；以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene)，雷洛昔芬(keoxifene)，LY117018，奥那斯酮(onapristone)，和托瑞米芬(Fareston)；和抗雄激素制剂如氟他氨(flutamide)，尼鲁米特(nilutamide)，比卡鲁胺(bicalutamide)，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)；和上述任何物质的可药用盐，酸或衍生物。
本公开所述的“抗体”指免疫球蛋白，完整抗体是由两条相同的重链和两条相同 的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体和人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人Claudin18.2的单克隆抗体。制备时用Claudin18.2或其表位作为抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源抗Claudin18.2抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的Claudin18.2嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA 序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
在本公开一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区；优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变、和/或S228P突变)的IgG1、IgG2或IgG4变体。
本公开中所述人抗体的重链恒定区和人抗体的轻链恒定区的“变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体。示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体；具体替换如现有技术已知的YTE突变、L234A和/或L235A突变、S228P突变、和/或获得knob-into-hole结构的突变(使抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体，并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座元件引入到源于胚胎干细胞系的细胞株中，这些细胞株中的内源性重链和轻链基因座遭到靶向破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生分泌人抗体的杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指在轻链和重链中包含恒定区的抗体。本公开“抗体”包含“全长抗体”及其抗原结合片段。
在一些实施方案中，本公开的全长抗体包括由以下表中轻重链可变区组合中的轻链可变区与轻链恒定区连接和重链可变区与重链恒定区连接后所形成的全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区和重链恒定区。
术语抗体的“抗原结合片段”或“功能片段”是指保持特异性结合抗原(例如， Claudin18.2)的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
Fab是通过用木瓜蛋白酶相同活性的酶处理IgG抗体分子所获得的片段中具有抗原结合活性的抗体片段。
F(ab') 2是通过用酶胃蛋白酶相同活性的酶消化IgG，而获得的具有抗原结合活性的抗体片段。
Fab'是通过切割上述F(ab') 2而获得的具有抗原结合活性的抗体片段。
此外，可以通过将编码Fab'片段的DNA插入到表达载体中，并将载体导入到宿主中来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有通用结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。可用于本公开的其他接头由以下文献描述，例如但不限于：Holliger等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Alfthan等人(1995)，Protein Eng.8:725-731；Choi等人(2001)，Eur.J.Immuno l.31:94-106；Hu等人(1996)，Cancer Res.56:3055-3061；Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.。
双抗体是其中scFv或Fab被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
双特异性抗体和多特异性抗体是指能结合两个或多个抗原或抗原决定簇的抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(例如 Protein Engineering，7，697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“表位”或“抗原决定簇”是指抗原上被免疫球蛋白或抗体所结合的部位(例如，Claudin18.2分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小 于10 -9M、10 -10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合Claudin18.2或其表位，例如，在本公开中抗体与细胞表面抗原的亲和力采用FACS法测定KD值。
当术语“竞争”用于竞争相同表位的抗原结合蛋白的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如Claudin18.2抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列时(必要时引入间隙)，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序 列同一性百分比，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人Claudin18.2或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。
本公开的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染宿主细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人Claudin18.2特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或抗原结合片段的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表3.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同一性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培 养物，而不考虑传代数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“分离的”指分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本公开包括用于治疗与目标抗原(例如Claudin18.2)阳性细胞相关的疾病的药剂，所述药剂包含本公开的抗Claudin18.2抗体或其抗原结合片段作为活性成分。
本公开中与Claudin18.2相关的疾病没有限制，只要它是与Claudin18.2相关的疾病即可，例如，本公开的分子诱导的治疗反应包括：(1)通过结合人类Claudin18.2，阻遏Claudin18.2与其受体/配体的结合，或(2)杀伤过表达Claudin18.2的肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本公开涉及用于免疫检测或测定目标抗原(例如Claudin18.2)的方法、用于免疫检测或测定目标抗原(例如Claudin18.2)的试剂、用于免疫检测或测定表达目标抗原(例如Claudin18.2)的细胞的方法和用于诊断与目标抗原(例如Claudin18.2)阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别目标抗原(例如人Claudin18.2)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定目标抗原(例如Claudin18.2)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与Claudin18.2阳性细胞相关的疾病可以通过用本公开的抗体或抗体片段检测或测定表达Claudin18.2的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原(例如Claudin18.2)的待测样品没有特别限制，只要它具有包含表达目标抗原(例如Claudin18.2)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本发明一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本发明，但是以下描述优选的方法和材料。通过说明书和权利要求书，本发明的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本发明所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本发明的优选实施方式。这些实施例不应以任何方式理解为限制本发明的范围，本发明的范围由权利要求书限定。
实施例
实施例1：构建高表达Claudin18.2的细胞株
用Lipofectamine 3000转染试剂，将pCDH-hClaudin18.2慢病毒表达载体质粒与pVSV-G,pCMV-dR8.91慢病毒系统包装载体转染至病毒包装细胞293T中；收集含有病毒的培养基上清，过滤并进行超高速离心；使用浓缩后的病毒感染人胃印戒细胞癌细胞株NUGC4，经puromycin筛选两至三周，再进行FACS单细胞分选。
根据肿瘤IHC评分来区分Claudin18.2表达程度。与肿瘤IHC评分为3分的肿瘤Claudin18.2表达水平相当的细胞为高表达细胞，与肿瘤IHC评分为2分的肿瘤 Claudin18.2表平水平相当的细胞为中等表达细胞。根据通过FACS检测慢病毒感染的NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量高的NUGC4/hClaudin18.2单克隆细胞株。同时通过FACS检测野生型NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量中等的NUGC4克隆细胞株，野生型NUGC4为Claudin18.2低表达量细胞。
将挑选出的单克隆细胞株扩大培养，冻存备库以便后续实验。
Claudin18.2序列Genbank:NP_001002026：(SEQ ID NO:1)
Figure PCTCN2020082369-appb-000004
Claudin18.2DNA序列：(SEQ ID NO:2)
Figure PCTCN2020082369-appb-000005
Figure PCTCN2020082369-appb-000006
实施例2：抗人claudin18.2单克隆抗体产生
1免疫
通过免疫小鼠产生抗人Claudin18.2单克隆抗体。
实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为huClaudin18.2-HEK293细胞(转染人Claudin18.2质粒的HEK-293稳转细胞株)。
免疫方案：首次免疫细胞前，用
Figure PCTCN2020082369-appb-000007
Gold Adjuvant(Sigma Cat No.T2684)0.1ml/只注射小鼠腹膜内(IP)；半小时后每只小鼠腹膜内(IP)注射0.1ml生理盐水稀释至1×10 8/ml浓度的细胞液。细胞吹散均匀后进行接种，时间为第0、14、28、42、56天。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射1×10 7细胞。
2脾细胞融合
采用PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2020082369-appb-000008
CRL-8287 TM)进行融合得到杂交瘤细胞。杂交瘤细胞以0.5-1×10 6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMDM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成克隆。去除上清，加入200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法检测培养上清。选择与huClaudin18.2-HEK293细胞结合能力强，同时与HEK293细胞没有结合的细胞，及时进行扩增冻存；经过二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞均需进行细胞结合实验。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
实施例3：鼠源抗体的人源化
挑选出体外活性高的单克隆杂交瘤细胞株mAb1901，mAb1902；克隆其中的单克隆抗体序列，再进行人源化、重组表达和活性评价。
从杂交瘤中克隆序列的过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA(按照试剂盒说明书步骤)并进行反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增，送测序公司测序。得到的DNA序列对应的氨基酸序列SEQ ID NO:3-6所示：
mAb1901鼠源重链可变区(SEQ ID NO:3)
Figure PCTCN2020082369-appb-000009
mAb1901鼠源轻链可变区(SEQ ID NO:4)
Figure PCTCN2020082369-appb-000010
mAb1902鼠源重链可变区(SEQ ID NO:5)
Figure PCTCN2020082369-appb-000011
mAb1902鼠源轻链可变区(SEQ ID NO:6)
Figure PCTCN2020082369-appb-000012
上述鼠源重链可变区和轻链可变区，分别与下述人IgG1抗体的重链恒定区和人源κ轻链恒定区连接，形成嵌合抗体ch1901和ch1902。
恒定区选自以下序列：
人IgG1抗体的重链恒定区：(SEQ ID NO:7)
Figure PCTCN2020082369-appb-000013
人源κ轻链恒定区：(SEQ ID NO:8)
Figure PCTCN2020082369-appb-000014
Figure PCTCN2020082369-appb-000015
如本领域许多文献公示的方法，对鼠源单克隆抗体进行人源化。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。本发明选择活性好的候选分子进行人源化，结果如下。
1.鼠源抗体的CDR区
表4中VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体的CDR序列如表4所述：
表4.鼠源抗体的CDR序列
抗体 mAb1901
HCDR1 DYGIH(SEQ ID NO:9)
HCDR2 YISRGSSTIYYADTVKG(SEQ ID NO:10)
HCDR3 GGYDTRNAMDY(SEQ ID NO:11)
LCDR1 KSSQSLLNSGNQKNYLA(SEQ ID NO:12)
LCDR2 GASTRAS(SEQ ID NO:13)
LCDR3 QNDLYYPLT(SEQ ID NO:14)
抗体 mAb1902
HCDR1 SYWMH(SEQ ID NO:15)
HCDR2 MIHPNSGSTNYNEKFKGR(SEQ ID NO:16)
HCDR3 LKTGNSFDY(SEQ ID NO:17)
LCDR1 KSSQSLLNSGNQKNYLT(SEQ ID NO:18)
LCDR2 WASTRES(SEQ ID NO:19)
LCDR3 QNAYTYPFT(SEQ ID NO:20)
2.选择人种系FR区序列
在所获得的鼠源抗体VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因。
2.1 mAb1901的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1901鼠源抗体进行人源化改造，将鼠源抗体mAb1901的CDR区移植到选择的人源化模板上，得到人源化可变区，其人源化重链可变区序列为SEQ ID NO:24和轻链可变区序列为SEQ ID NO:21；再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表5.mAb1901人源化抗体及回复突变 *
Figure PCTCN2020082369-appb-000016
*表格中所有氨基酸位置编号为Kabat编号规则的编号，重链可变区的N82T中，82为Kabat规则的第82A位。
表6.mAb1901人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2020082369-appb-000017
上表中对应重链可变区可与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中，重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.2 mAb1902的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1902鼠源抗体进行人源化改造，将鼠源抗体mAb1902的CDR区移植到选择的人源化模板上，得到人源化可变区，其人源化重链可变区序列为SEQ ID NO:31和轻链可变区序列为SEQ ID NO:28；再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表7.mAb1902人源化抗体及其回复突变设计 *
Figure PCTCN2020082369-appb-000018
Figure PCTCN2020082369-appb-000019
*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表8.mAb1902人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2020082369-appb-000020
上表中对应重链可变区与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。
示例性的，抗体全长序列如下：
嵌合抗体ch1901：
ch1901重链：(SEQ ID NO:35)
Figure PCTCN2020082369-appb-000021
Figure PCTCN2020082369-appb-000022
ch1901轻链(SEQ ID NO:36)
Figure PCTCN2020082369-appb-000023
嵌合抗体ch1902：
ch1902重链(SEQ ID NO:37)
Figure PCTCN2020082369-appb-000024
ch1902轻链(SEQ ID NO:38)
Figure PCTCN2020082369-appb-000025
表9.mAb1901人源化抗体
Figure PCTCN2020082369-appb-000026
全长抗体轻重链序列如下所示：
表10.mAb1901人源化抗体轻链和重链序列
Figure PCTCN2020082369-appb-000027
Figure PCTCN2020082369-appb-000028
Figure PCTCN2020082369-appb-000029
表11.mAb1902人源化抗体
Figure PCTCN2020082369-appb-000030
全长抗体轻重链序列如下所示：
表12.mAb1901人源化抗体轻链和重链序列
Figure PCTCN2020082369-appb-000031
Figure PCTCN2020082369-appb-000032
本公开阳性对照抗体为IMAB-362(来自WO2016166122)：
IMAB-362重链(SEQ ID NO:53)
Figure PCTCN2020082369-appb-000033
IMAB-362轻链(SEQ ID NO:54)
Figure PCTCN2020082369-appb-000034
用常规的基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体。
体外活性生物学评价
测试例1：Cell水平ELISA结合实验
基于细胞的ELISA实验被用来检测Claudin18.2抗体的结合特性。将稳转表达 Claudin18.2的NUGC4细胞培养于96孔细胞板(Corning，3599)中，待生长至90％密度时加入4％多聚甲醛固定细胞1小时，用PBST缓冲液(pH 7.4 PBS含0.05％Tween-20)洗板3次后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入50μl/孔用样品稀释液(pH7.4 PBS含1％脱脂乳)稀释的不同浓度待测抗体，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research,109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL,52-00-03)，于室温孵育10-15min，加入50μl/孔1M H 2SO 4终止反应，用MD Versa Max TM酶标仪在450nm处读取吸收值，计算Claudin18.2抗体对Claudin18.2的结合EC50值(结果见下表)。
表13.抗体的结合活性
抗体 IMAB362 ch1901 ch1902
Emax 1.175 1.399 1.272
EC50(nM) 0.108 0.098 0.074
表14-1.mAb1901人源化抗体的结合活性
抗体 Emax EC50(nM)
IMAB362 1.115 0.086
h1901-2 1.039 0.076
h1901-3 1.1055 0.22
h1901-4 0.986 0.201
h1901-6 0.937 0.091
h1901-7 0.921 0.166
h1901-8 1.047 0.091
h1901-11 1.44 0.076
h1901-12 1.22 0.116
表14-2.mAb1902人源化抗体的结合活性
抗体 Emax EC50(nM)
IMAB362 0.88 0.187
h1902-1 0.87 0.113
h1902-2 0.88 0.107
h1902-3 0.84 0.175
h1902-4 0.82 0.087
h1902-5 0.9 0.098
h1902-6 0.78 0.141
h1902-7 0.75 0.121
h1902-8 0.89 0.132
h1902-9 0.75 0.137
h1902-10 0.89 0.133
测试例2：抗体细胞水平结合实验
将稳转表达Claudin18.2的NUGC4细胞用FACS缓冲液(2％胎牛血清(Gibco，10099141)pH7.4 PBS(Sigma,P4417-100TAB))制备成1×10 6/ml的细胞悬液，100μl/孔加入96孔圆底板(Corning，3795)中。离心去除上清后加入50μl/孔用FACS缓冲液稀释的不同浓度待测Claudin18.2抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488包被的抗人IgG(H+L)(invitrogen,A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的结合EC50值，结果见图1。
测试例3：抗体内吞实验
将预标记了DyLight 488NHS Ester(thermofisher，46403)的待测Claudin18.2抗体，以5μg/ml终浓度加入1×10 6/ml稳转表达Claudin18.2的NUGC4细胞中，放于冰上避光孵育1小时，以预冷的FACS缓冲液(pH7.4 PBS，2％胎牛血清)离心洗涤3次，去上清后加入预热的完全培养基，放入37℃5％CO 2细胞培养箱。分别在0、0.5、1、2、4小时后取出细胞，放置于冰上避光保存。待样品全部收集后，300g低温离心去除上清，加入洗脱缓冲液(pH1.7 0.05M甘氨酸，0.1M氯化钠)后，室温孵育7分钟，以FACS缓冲液300g离心洗涤1次，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的内吞效率。结果显示(见图2)，人源化抗体具有良好的细胞内吞效率。
测试例4：基于流式细胞技术测定抗体亲和力
实验当天收集HEK293/hClaudin18.2细胞于U底96孔板中，每孔1×10 5至2×10 5个细胞。加入起始浓度5μg/ml，2×梯度稀释(12个浓度点)的Claudin18.2抗体，4℃孵育1小时，阳性对照为IMAB362，同时设置不加抗体的阴性对照。离心去除抗体，再加入100μl/孔FITC抗人IgG Fc抗体(200×)，4℃避光孵育30分钟，用PBS+2％FBS清洗两遍后准备进行流式细胞检测。启动BD FACS CantoII，预热完成后打开BD FACSDiva软件，建立一个新的实验，检测HEK293/hClaudin18.2阴性对照样品，调节FSC及SSC电压至适当的数值并保存。根据Quantum TM FITC-5MESF Kit说明书，分别检测空白样品B及标准曲线1，调节FITC电压至适当的数值并保存。在保存的电压下检测U底96孔板中的样品，记录数据。使用Flowjo软件分析实验数据得到Geo Mean数值，根据Quantum TM FITC-5MESF Kit说明书拟合MESF-Geo Mean标准曲线，根据FITC抗人IgG Fc抗体的浓度荧光值计算出与HEK293/hClaudin18.2细胞结合的Claudin18.2抗体的摩尔浓度及游离抗体浓度， 利用Scatchard作图法计算抗体的Bmax和解离常数KD。结果见表15。
表15.人源化抗体细胞水平亲和力
抗体 IMAB362 h1901-11 h1902-5
KD(nM) 10.2 6.8 1.64
测试例5：抗体的ADCC效应评价
消化各种NUGC4细胞(高中低表达Claudin18.2)，1000rpm离心后，重悬计数。将细胞以3×10 5细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清，Gibco,1921005PJ)的无酚红RPMI 1640中(Gibco,11835-030)。在96孔板(Corning,3903)中，每孔加入25μl细胞(7500个/孔)。将抗体稀释在上述无酚红培养基中，配制成3×的抗体稀释液，向细胞板中加入25μl/孔的抗体。在37℃、5％CO 2培养箱中孵育0.5小时。
收集效应细胞(FcrR3A-V158-NFAT-RE-Jurkat细胞)，1000rpm离心后，重悬计数。将细胞以3×10 6细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清)的无酚红RPMI 1640中，在实验板中每孔加入25μl细胞(7.5×10 4个细胞/孔)。在37℃、5％CO 2培养箱中孵育6小时。
向实验板的每个孔中加入75μl/孔的Bright-Glo(Promega,E2610)，用酶标仪(PerkinElmer,VITOR3)检测化学发光(luminescence)。
结果显示(见表16和图3A-图3C)，在低中高不同程度Claudin18.2表达的NUGC4细胞中，抗体h1901-11和h1902-5均显示出很强的ADCC活性。
表16.抗体在Claudin18.2不同表达程度的NUGC4细胞中的ADCC效应
Figure PCTCN2020082369-appb-000035

Claims (17)

  1. 一种抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
    i)所述重链可变区包含与如SEQ ID NO：3所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：4所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3；或
    ii)所述重链可变区包含与如SEQ ID NO：5所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
    iii)所述重链可变区包含分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，
    所述轻链可变区包含分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3；或
    iv)所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，
    所述轻链可变区包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2所述的抗Claudin18.2抗体，其是鼠源抗体、嵌合抗体或人源化抗体。
  4. 根据权利要求1至3任一项所述的抗Claudin18.2抗体，其包含重链可变区和轻链可变区，其中：
    v)所述重链可变区，其氨基酸序列与SEQ ID NO:3或24具有至少90％同一性，和
    所述轻链可变区，其氨基酸序列与SEQ ID NO:4或21具有至少90％同一性；或
    vi)所述重链可变区，其氨基酸序列与SEQ ID NO:5或31具有至少90％同一性，和
    所述轻链可变区，其氨基酸序列与SEQ ID NO:6或28具有至少90％同一性；
    优选地：
    1)所述重链可变区，其氨基酸序列如SEQ ID NO:3所示或与其有至少90％同一性，和所述轻链可变区，其氨基酸序列如SEQ ID NO:4所示或与其有至少90％ 同一性；
    2)所述重链可变区，其氨基酸序列如SEQ ID NO:24所示或与其有至少90％同一性，和所述轻链可变区，其氨基酸序列如SEQ ID NO:21所示或与其有至少90％同一性；
    3)所述重链可变区，其氨基酸序列如SEQ ID NO:5所示或与其有至少90％同一性，和所述轻链可变区，其氨基酸序列如SEQ ID NO:6所示或与其有至少90％同一性；或
    4)所述重链可变区，其氨基酸序列如SEQ ID NO:31所示或与其有至少90％同一性，和所述轻链可变区，其氨基酸序列如SEQ ID NO:28所示或与其有至少90％同一性。
  5. 根据权利要求4所述的抗Claudin18.2抗体，其为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体在人抗体的轻链框架区和/或重链框架区上分别具有1至10个回复突变；
    优选地，所述框架区变体包含选自以下a)或b)所述的突变：
    a)所述轻链可变区中包含选自22S、85I和87H中的一个或多个回复突变，和/或所述重链可变区中包含选自48I、82T和69M中的一个或多个回复突变；或
    b)所述轻链可变区中包含选自4L和22S中的一个或多个回复突变，和/或所述重链可变区中包含选自38K、40R、48I、66K、67A、69L、71L和73K中的一个或多个回复突变；
    更优选地，所述框架区变体包含选自以下a-1)或b-1)所述的突变：
    a-1)所述轻链可变区中包含22S、85I和87H的回复突变，和所述重链可变区中包含48I和82T的回复突变；或
    b-1)所述轻链可变区中包含4L的回复突变；
    重链可变区的82T中，82为Kabat规则的第82A位。
  6. 根据权利要求3所述的抗Claudin18.2抗体，其包含如下所示的重链可变区和轻链可变区：
    vii)所述重链可变区序列如SEQ ID NO:3所示和所述轻链可变区序列如SEQ ID NO：4所示；或
    viii)所述重链可变区序列如SEQ ID NO:24、25、26或27所示和所述轻链可变区序列如SEQ ID NO：21、22或23所示；或
    ix)所述重链可变区序列如SEQ ID NO:5所示和所述轻链可变区序列如SEQ ID NO：6所示；或
    x)所述重链可变区序列如SEQ ID NO:31、32、33或34所示和所述轻链可变区序列如SEQ ID NO：28、29或30所示；
    优选地，所述抗Claudin18.2抗体或其抗原结合片段，其包含如下所示的重链可变区和轻链可变区：
    xi)所述重链可变区序列如SEQ ID NO:31所示和所述轻链可变区序列如SEQ ID NO:29所示；或
    xii)所述重链可变区序列如SEQ ID NO:26所示和所述轻链可变区序列如SEQ ID NO:23所示。
  7. 根据权利要求1至6任一项所述的抗Claudin18.2抗体，其进一步包含重链恒定区和轻链恒定区；
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其变体，
    优选地，所述轻链恒定区选自人抗体κ和λ链恒定区及其变体；
    更优选地，所述抗Claudin18.2抗体包含SEQ ID NO:7所示的重链恒定区和SEQ ID NO:8所示的轻链恒定区；
    最优选地，所述抗Claudin18.2抗体包含：与SEQ ID NO:35或42具有至少90％同一性的重链，和与SEQ ID NO:36或39具有至少90％同一性的轻链；或
    与SEQ ID NO:37或49具有至少90％同一性的重链，和与SEQ ID NO:38或46具有至少90％同一性的轻链。
  8. 根据权利要求1至7任一项所述的抗Claudin18.2抗体，其包含：
    c)SEQ ID NO:35所示的重链和SEQ ID NO：36所示的轻链；
    d)SEQ ID NO:42、43、44或45所示的重链和SEQ ID NO：39、40或41所示的轻链；
    e)SEQ ID NO:37所示的重链和SEQ ID NO：38所示的轻链；或
    f)SEQ ID NO:49、50、51或52所示的重链和SEQ ID NO：46、47或48所示的轻链。
  9. 根据权利要求1至8中任一项所述的抗Claudin18.2抗体，其包含:
    SEQ ID NO:44所示的重链，和SEQ ID NO:41所示的轻链；或
    SEQ ID NO:49所示的重链，和SEQ ID NO:47所示的轻链。
  10. 一种分离的抗Claudin18.2抗体，其与权利要求1至9任一项所述的抗Claudin18.2抗体竞争性结合人Claudin18.2。
  11. 一种核酸分子，其编码权利要求1至10任一项所述的抗Claudin18.2抗体。
  12. 一种宿主细胞，其包含如权利要求11所述的核酸分子。
  13. 一种抗体药物偶联物，其为由根据权利要求1-10任一项所述的抗Claudin18.2抗体与细胞毒性药物偶联形成的抗体药物偶联物。
  14. 一种药物组合物，其含有治疗有效量的根据权利要求1至10任一项所述的抗Claudin18.2抗体，或根据权利要求11所述的核酸分子，或权利要求13所述的抗体药物偶联物，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  15. 一种用于免疫检测或测定Claudin18.2的方法，所述方法包括：
    使权利要求1至10任一项所述的抗Claudin18.2抗体接触待测样本的步骤。
  16. 一种试剂盒，其包含根据权利要求1至10任一项所述的抗Claudin18.2抗体。
  17. 一种治疗与Claudin18.2相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至10任一项所述的抗Claudin18.2抗体，或权利要求11所述的核酸分子，或权利要求13所述的抗体药物偶联物或权利要求14所述的药物组合物，优选地，所述疾病为肿瘤；
    更优选地，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
    更优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；
    所述肺癌选自：非小细胞肺癌和小细胞肺癌；
    所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021115426A1 (zh) 2019-12-12 2021-06-17 江苏恒瑞医药股份有限公司 抗密蛋白抗体药物偶联物及其医药用途
CN113416260A (zh) * 2021-04-14 2021-09-21 南京凯地医疗技术有限公司 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
WO2022111633A1 (en) * 2020-11-27 2022-06-02 Sunshine Lake Pharma Co., Ltd. Cldn18.2 antibody and use thereof
WO2022122709A1 (en) 2020-12-07 2022-06-16 Sotio Biotech A.S. Antibody-drug conjugates based on humanized cldn18.2 antibodies
CN114634566A (zh) * 2020-12-16 2022-06-17 广州百暨基因科技有限公司 一种抗Claudin18_2的抗原结合片段、抗体及其应用
WO2022136642A1 (en) 2020-12-23 2022-06-30 Sotio Biotech A.S. Tumor-specific claudin 18.2 antibody-drug conjugates
CN114685682A (zh) * 2020-12-31 2022-07-01 上海莱馥医疗科技有限公司 一种靶向表达cldn 18.2的细胞的嵌合抗原受体
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
WO2022188740A1 (en) * 2021-03-08 2022-09-15 Genequantum Healthcare (Suzhou) Co., Ltd. Antibody-immune agonist conjugate and applications thereof
WO2023076998A1 (en) * 2021-10-27 2023-05-04 Atyr Pharma, Inc. COMPOSITIONS AND METHODS COMPRISING ANTI-NRP2a ANTIBODIES
US11807687B2 (en) 2019-10-03 2023-11-07 Atyr Pharma, Inc. Therapeutic compositions comprising anti-NRP2 antibodies
US12065495B2 (en) 2018-04-06 2024-08-20 Atyr Pharma, Inc. Compositions and methods comprising anti-NRP2 antibodies

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220411492A1 (en) * 2019-05-16 2022-12-29 Qilu Pharmaceutical Co., Ltd. Antibody against claudin 18a2 and use thereof
CN115947855B (zh) * 2022-05-20 2023-10-27 杭州邦顺制药有限公司 抗cd24抗体的制备及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101573384A (zh) 2006-10-27 2009-11-04 健泰科生物技术公司 抗体和免疫偶联物及其用途
CN104321345A (zh) * 2012-05-09 2015-01-28 加尼梅德药物公司 用于癌症诊断的针对密蛋白18.2的抗体
CN104379166A (zh) * 2012-05-23 2015-02-25 加尼梅德药物公司 用于治疗癌症的涉及针对密蛋白18.2之抗体的联合治疗
WO2016127790A1 (zh) 2015-02-15 2016-08-18 江苏恒瑞医药股份有限公司 配体-细胞毒性药物偶联物、其制备方法及其应用
WO2016166122A1 (en) 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Drug conjugates comprising antibodies against claudin 18.2
WO2018006882A1 (zh) * 2016-07-08 2018-01-11 科济生物医药（上海）有限公司 抗密蛋白18a2的抗体及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1997832A1 (en) * 2007-05-29 2008-12-03 Ganymed Pharmaceuticals AG Monoclonal antibodies against Claudin-18 for treatment of cancer
WO2018054484A1 (en) * 2016-09-23 2018-03-29 Biontech Ag Bispecific trivalent antibodies binding to claudin 6 or claudin18.2 and cd3 for treatment of claudin expressing cancer diseases
BR112022011032A2 (pt) * 2019-12-12 2022-08-16 Jiangsu Hengrui Medicine Co Conjugado de anticorpo-fármaco anti-claudina e uso farmacêutico do mesmo

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101573384A (zh) 2006-10-27 2009-11-04 健泰科生物技术公司 抗体和免疫偶联物及其用途
CN104321345A (zh) * 2012-05-09 2015-01-28 加尼梅德药物公司 用于癌症诊断的针对密蛋白18.2的抗体
CN104379166A (zh) * 2012-05-23 2015-02-25 加尼梅德药物公司 用于治疗癌症的涉及针对密蛋白18.2之抗体的联合治疗
WO2016127790A1 (zh) 2015-02-15 2016-08-18 江苏恒瑞医药股份有限公司 配体-细胞毒性药物偶联物、其制备方法及其应用
WO2016166122A1 (en) 2015-04-15 2016-10-20 Ganymed Pharmaceuticals Ag Drug conjugates comprising antibodies against claudin 18.2
WO2018006882A1 (zh) * 2016-07-08 2018-01-11 科济生物医药（上海）有限公司 抗密蛋白18a2的抗体及其应用

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
ALFTHAN ET AL., PROTEIN ENG., vol. 8, 1995, pages 725 - 731
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
ALTSCHUL, SF ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL, SF ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHEUNG ET AL., VIROLOGY, vol. 176, 1990, pages 546 - 552
CHOI ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 94 - 106
EPITOPE MAPPING PROTOCOLS IN METHODS IN MOLECULAR BIOLOGY, vol. 66, 1996
GISH, W. ET AL., NATURE GENET, vol. 3, 1993, pages 266 - 272
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PROC. NATL. ACAD. SCI USA, vol. 85, 1988, pages 5879 - 5883
J. BIOL. CHEM, vol. 243, 1968, pages 3558
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
KABAT, EA ET AL., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 1991
KIPRIYANOV ET AL., J. MOL. BIOL., vol. 293, 1999, pages 41 - 56
KIRKLAND ET AL., J. IMMUNOL., vol. 137, 1986, pages 3614 - 3619
LEFRANC MP, IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
LEFRANC, MP, DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
MADDEN, TL ET AL., METH. ENZYMOL., vol. 266, 1996, pages 131 - 141
MOLDENHAUER ET AL., SCAND. J. IMMUNOL., vol. 32, 1990, pages 77 - 82
MOREL ET AL., MOLEC. IMMUNOL., vol. 25, 1988, pages 7 - 15
ROOVERS ET AL., CANCER IMMUNOL, 2001
STAHLI ET AL., METHODS IN ENZYMOLOGY, vol. 9, 1983, pages 242 - 253
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224
ZHANG, J. ET AL., GENOME RES, vol. 7, 1997, pages 649 - 656

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12065495B2 (en) 2018-04-06 2024-08-20 Atyr Pharma, Inc. Compositions and methods comprising anti-NRP2 antibodies
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
US11807687B2 (en) 2019-10-03 2023-11-07 Atyr Pharma, Inc. Therapeutic compositions comprising anti-NRP2 antibodies
EP4074345A4 (en) * 2019-12-12 2023-10-25 Jiangsu Hengrui Pharmaceuticals Co., Ltd. ANTI-CLAUDINE ANTIBODY-DRUG CONJUGATE AND ITS PHARMACEUTICAL USE
WO2021115426A1 (zh) 2019-12-12 2021-06-17 江苏恒瑞医药股份有限公司 抗密蛋白抗体药物偶联物及其医药用途
EP4251652A4 (en) * 2020-11-27 2024-11-27 Sunshine Lake Pharma Co., Ltd. Cldn18.2 antibody and use thereof
WO2022111633A1 (en) * 2020-11-27 2022-06-02 Sunshine Lake Pharma Co., Ltd. Cldn18.2 antibody and use thereof
WO2022122709A1 (en) 2020-12-07 2022-06-16 Sotio Biotech A.S. Antibody-drug conjugates based on humanized cldn18.2 antibodies
WO2022126687A1 (zh) * 2020-12-16 2022-06-23 广州百暨基因科技有限公司 一种抗Claudin18.2的抗原结合片段、抗体及其应用
CN114634566A (zh) * 2020-12-16 2022-06-17 广州百暨基因科技有限公司 一种抗Claudin18_2的抗原结合片段、抗体及其应用
CN114634566B (zh) * 2020-12-16 2023-09-19 广州百暨基因科技有限公司 一种抗Claudin18_2的抗原结合片段、抗体及其应用
WO2022136642A1 (en) 2020-12-23 2022-06-30 Sotio Biotech A.S. Tumor-specific claudin 18.2 antibody-drug conjugates
CN114685682A (zh) * 2020-12-31 2022-07-01 上海莱馥医疗科技有限公司 一种靶向表达cldn 18.2的细胞的嵌合抗原受体
CN114685682B (zh) * 2020-12-31 2023-11-10 上海莱馥医疗科技有限公司 一种靶向表达cldn 18.2的细胞的嵌合抗原受体
WO2022188740A1 (en) * 2021-03-08 2022-09-15 Genequantum Healthcare (Suzhou) Co., Ltd. Antibody-immune agonist conjugate and applications thereof
CN113416260A (zh) * 2021-04-14 2021-09-21 南京凯地医疗技术有限公司 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
WO2023076998A1 (en) * 2021-10-27 2023-05-04 Atyr Pharma, Inc. COMPOSITIONS AND METHODS COMPRISING ANTI-NRP2a ANTIBODIES

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