WO2022161282A1 - 抗cldn18.2抗体及其应用 - Google Patents

抗cldn18.2抗体及其应用 Download PDF

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WO2022161282A1
WO2022161282A1 PCT/CN2022/073207 CN2022073207W WO2022161282A1 WO 2022161282 A1 WO2022161282 A1 WO 2022161282A1 CN 2022073207 W CN2022073207 W CN 2022073207W WO 2022161282 A1 WO2022161282 A1 WO 2022161282A1
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antibody
cancer
seq
antigen
optionally
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PCT/CN2022/073207
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French (fr)
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张晓艳
徐勤枝
钱尼良
杨翠马
李宏杰
徐桂利
张坤霖
高新
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北京免疫方舟医药科技有限公司
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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C07K2317/56—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
    • C07K2317/70—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
    • C07K2317/90—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 application relates to the field of antibodies, and more particularly, the present application relates to anti-CLDN18.2 antibodies and applications thereof.
  • Cancer has biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis, and its occurrence is a complex multi-factor and multi-step process.
  • the current treatment methods include surgery, radiotherapy, chemotherapy, and immunotherapy.
  • immunotherapy has greatly improved their survival and quality of life, and antibody therapy has become a recognized immunotherapy that is effective against cancer.
  • claudin claudin
  • CLDN claudin
  • the tissue-specific expression of CLDN members indicates that this protein family is not only an important player in maintaining cell permeability and controlling tight junctions, but also in protein-protein interactions and organizing cell-cell contact signals.
  • CLDN genes There are currently 27 mammalian CLDN genes known, including 3 recently discovered distantly related members, but disagreement remains as to whether all three distantly related members should be classified as CLDNs. Not all of these genes are present in all mammals. For example, CLDN13 is present in rodents but not in humans. Therefore, a total of 26 CLDN genes have been found in humans so far.
  • CLDN18 is one of the few members of the human Claudin family that is strictly restricted to one cell line.
  • the CLDN18 molecule is a four-transmembrane protein with four transmembrane hydrophobic regions, two extracellular loops, and one intracellular loop, the ends of which are located intracellularly.
  • the molecule has two splice isoforms: CLDN18.1 and CLDN18.2, which are produced by alternative splicing of the first exon, and the sequences between the two are highly homologous with only eight amino acid differences.
  • CLDN18.1 is mainly expressed in lung tissue cells; the expression of CLDN18.2 in normal tissues is strictly limited to differentiated epithelial cells of gastric mucosa, but not expressed in gastric stem cells.
  • CLDN18.2 is preserved in malignant transformation and is expressed in a significant proportion of primary gastric cancers and their metastases. Furthermore, CLDN18.2 is frequently ectopically activated in pancreatic, esophageal, ovarian and lung tumors. Clinical data show that CLDN18.2-specific human-mouse chimeric monoclonal antibody IMAB362 has no obvious toxicity to normal tissues. The overall objective response rate of IMAB362 in gastric cancer patients was approximately 10%, and these data suggest that CLDN18.2 may be a promising therapeutic target for the treatment of gastric cancer and other CLDN18.2-positive tumors.
  • the present application provides an anti-CLDN18.2 antibody and its application. Specifically, the present application provides the following technical solutions.
  • the application provides an antibody or antigen-binding portion thereof that specifically binds CLDN18.2, comprising a heavy chain variable region comprising any one of the HCDR1, HCDR2 and HCDR3 sequences or more, wherein the HCDR1 sequence is GFSFSSFG (SEQ ID NO: 1), the HCDR2 sequence is ISSGSRTI (SEQ ID NO: 2), and the HCDR3 sequence is TRYYYGNSFDY (SEQ ID NO: 3).
  • the antibody or antigen-binding portion thereof further comprises a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence It is QSLFNTGNQKNY (SEQ ID NO: 4), the LCDR2 sequence is WAS (SEQ ID NO: 5), and the LCDR3 sequence is QNNYNFPLT (SEQ ID NO: 6).
  • the antibody is a murine antibody.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and/or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.
  • the antibody is a chimeric antibody.
  • the chimeric antibody comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO:9, and/or a light chain whose amino acid sequence is shown in SEQ ID NO:10.
  • the antibody is a humanized antibody.
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 11, and/or the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 12.
  • the application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof of the first aspect and a pharmaceutically acceptable carrier.
  • the application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of the first aspect.
  • the application provides an expression vector comprising the nucleic acid molecule of the third aspect.
  • the present application provides a host cell comprising the nucleic acid molecule of the third aspect or the expression vector of the fourth aspect.
  • the present application provides the antibody or antigen-binding portion thereof of the first aspect, the pharmaceutical composition of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, Or use of the host cell described in the fifth aspect in the preparation of a medicament for preventing and/or treating CLDN18.2-related diseases.
  • the present application provides a method for preventing and/or treating a CLDN18.2-related disease, comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-
  • the present application provides the antibody or antigen-binding portion thereof of the first aspect, the pharmaceutical composition of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, Or use of the host cell described in the fifth aspect for preventing and/or treating CLDN18.2-related diseases.
  • the CLDN18.2-related disease is a tumor.
  • the tumor is selected from one or more of the following: gastric cancer, pancreatic cancer, intestinal cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, bladder cancer, and the like, as well as the aforementioned metastases.
  • the application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof of the first aspect.
  • the antibody or antigen-binding portion thereof that specifically binds to CLDN18.2 of the present application can induce an anti-tumor immune response mediated by CLDN18.2, and/or inhibit tumor growth and the like.
  • Figure 1 shows the binding affinity of the murine anti-CLDN18.2 antibody of the present application (antibody secreted by hybridoma 7D10-1) to HEK293 cells 293T-18.2 overexpressing human CLDN18.2.
  • Figure 2 shows the binding affinity of the chimeric anti-CLDN18.2 antibody of the present application to HEK293 cells 293T-18.2 overexpressing human CLDN18.2.
  • Figure 3 shows the binding affinity of the applicant's anti-CLDN18.2 antibody to HEK293 cells 293T-18.2 overexpressing human CLDN18.2.
  • Figure 4 shows the binding affinity of the applicant's anti-CLDN18.2 antibody to human gastric cancer cells NUGC4.
  • Figure 5 shows the ADCC activity of the applicant's anthropogenic anti-CLDN18.2 antibodies.
  • the term "antibody” refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (such as IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region comprises three domains, namely CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • the light chain constant region contains one domain (CL1).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • both the light chain and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal.
  • antigen-binding portion of an antibody refers to the portion or segment of an intact antibody molecule responsible for binding an antigen.
  • the antigen binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH heavy chain variable region
  • VL light chain variable region
  • Antigen-binding portions of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others.
  • Non-limiting examples of antigen-binding moieties include: Fab fragments, F(ab') 2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and amino acids that mimic the hypervariable regions of antibodies The smallest recognition unit composed of residues (eg, isolated CDRs).
  • the term "antigen binding portion” also includes other engineered molecules such as diabodies, tribodies, tetrabodies, minibodies, and the like.
  • Fd fragments described herein refer to antibody fragments consisting of VH and CH1 domains; Fv fragments consist of VL and VH domains in a single arm of an antibody; dAb fragments (Ward et al., Nature 1989; 341:544-546 ) consists of VH domains.
  • CDRs complementarity determining regions
  • the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition.
  • the CDR sequences are defined using Kabat.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively;
  • CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.
  • variable region sequence of a given antibody the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (http://www.abysis.org/).
  • the term "specific binding” refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope, eg, an antibody with an affinity that is at least two greater than its affinity for a non-specific antigen The ability to bind to specific antigens with fold greater affinity. It will be appreciated, however, that antibodies are capable of specifically binding two or more antigens with which their sequences are related. For example, the antibodies of the present application can specifically bind to human CLDN18.2.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals.
  • Monoclonal antibodies described herein specifically include “chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they exhibit the desired (See, US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • murine antibody refers to any antibody in which all domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.
  • chimeric antibody refers to an antibody comprising segments from two or more different antibodies.
  • the one or more CDRs are derived from a mouse anti-CLDN18.2 antibody.
  • all CDRs are derived from mouse anti-CLDN18.2 antibodies.
  • CDRs from more than one mouse anti-CLDN18.2 antibody are combined in a chimeric antibody.
  • a chimeric antibody can comprise CDR1 from the light chain in a first mouse anti-CLDN18.2 antibody, CDR2 from the light chain in a second mouse anti-CLDN18.2 antibody, and CDR1 from a third mouse anti-CLDN18 antibody .2
  • the CDR3 of the light chain in the antibody, as well as the CDRs from the heavy chain, can be derived from one or more other anti-CLDN18.2 antibodies.
  • the framework regions can be from the same anti-CLDN18.2 antibody or from one or more different individuals.
  • the chimeric antibodies described herein comprise the variable regions of a murine antibody (including the heavy chain variable region VH and/or the light chain variable region VL) and the constant regions of a human antibody.
  • humanized antibody refers to a CDR-grafted antibody, specifically an antibody produced by grafting mouse CDR region sequences into the framework of human antibody variable regions. The goal is to overcome the strong immune side effects induced by chimeric antibodies in humans by carrying a large amount of protein components from other species such as mice.
  • nucleic acid molecule can refer to DNA molecules as well as RNA molecules, which can be single-stranded or double-stranded.
  • the nucleic acid molecule can also be a cDNA.
  • CLDN18.2-related disease includes diseases and/or disorders associated with the CLDN18.2 signaling pathway.
  • exemplary CLDN18.2-related diseases or disorders include tumors, cancers such as gastric cancer, pancreatic cancer, bowel cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, and bladder cancer, as well as the aforementioned metastatic cancers.
  • EC50 refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that elicits 50% of the maximal effect.
  • the term "immune response” refers to a biological response within a vertebrate to a foreign agent that protects the organism against such agent and the disease caused by it.
  • the immune response is composed of cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and by Mediated by the action of any of these cells or by liver-produced soluble macromolecules (including antibodies, cytokines, and complement) that result in selective targeting, binding, damage, destruction, and/or elimination from the vertebrate body Invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation.
  • the immune response includes activation or suppression of T cells (eg, effector T cells) or Th cells (eg, CD4 + or CD8 + T cells) or suppression of Treg cells.
  • cancer refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Dysregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.
  • treatment refers to any type of intervention or method performed on a subject or the administration of an active agent thereto, wherein the purpose is to reverse, alleviate, ameliorate, inhibit or alleviate or prevent a symptom, complication, condition or Disease-related progression, progression, severity, or recurrence.
  • prophylaxis refers to administration to a subject not suffering from a disease to prevent the occurrence of the disease or to minimize its effects, if any.
  • Human CLDN18.2 protein is highly homologous to CLDN18.1, and it is a four-transmembrane protein. The extracellular peptide sequence is very short, and its immunogenicity is extremely low. The possibility of producing specific antibodies is very small, so screening The probability of obtaining hybridomas secreting antibodies recognizing CLDN18.2 is extremely low. A large part of the antibodies in the screened hybridoma supernatants were antibodies that could bind to both human CLDN18.2 and CLDN18.1. The inventors screened a hybridoma cell, and the antibody in the supernatant could bind to cells expressing CLDN18.2, but hardly bind to cells expressing CLDN18.1. Accordingly, the present application provides a novel anti-CLDN18.2 antibody or antigen-binding portion thereof that can specifically bind to CLDN18.2.
  • the inventors also prepared chimeric antibodies and humanized antibody forms from murine anti-CLDN18.2 antibodies by genetic engineering means, these antibodies can specifically bind to CLDN18.2 expressed on the cell surface, thereby effectively inducing CLDN18.2 mediated immune response, and play a role in the prevention or treatment of CLDN18.2-related diseases.
  • the application provides an antibody or antigen-binding portion thereof that specifically binds CLDN18.2, comprising a heavy chain variable region comprising any one of the HCDR1, HCDR2 and HCDR3 sequences or more, wherein the HCDR1 sequence is GFSFSSFG (SEQ ID NO: 1), the HCDR2 sequence is ISSGSRTI (SEQ ID NO: 2), and the HCDR3 sequence is TRYYYGNSFDY (SEQ ID NO: 3).
  • the heavy chain variable region of an antibody or antigen-binding portion thereof described herein comprises HCDR1 set forth in SEQ ID NO:1, HCDR2 set forth in SEQ ID NO:2, and SEQ ID NO:3 The HCDR3 sequence shown.
  • the antibodies or antigen-binding portions thereof described herein further comprise a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the The LCDR1 sequence is QSLFNTGNQKNY (SEQ ID NO: 4), the LCDR2 sequence is WAS (SEQ ID NO: 5), and the LCDR3 sequence is QNNYNFPLT (SEQ ID NO: 6).
  • the light chain variable region of an antibody or antigen-binding portion thereof described herein comprises LCDR1 as set forth in SEQ ID NO:4, LCDR2 as set forth in SEQ ID NO:5, and LCDR2 as set forth in SEQ ID NO:6 LCDR3 sequence shown.
  • the antibody or antigen-binding portion thereof described herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, SEQ ID NO: 1 HCDR2 shown in ID NO:2 and HCDR3 sequence shown in SEQ ID NO:3, and the light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and SEQ ID NO:5 LCDR3 sequence shown in ID NO:6.
  • the antibodies disclosed herein can be anti-human CLDN18.2 monoclonal antibodies.
  • Anti-CLDN18.2 antibody types and subtypes can be determined by any means known in the art. In general, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype.
  • Anti-CLDN18.2 antibody isotypes can be determined using an ELISA assay, eg, human Ig can be identified using murine Ig-adsorbed anti-human Ig.
  • the antibody described herein is a murine antibody, preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and/or the amino acid sequence of the light chain variable region The sequence is shown in SEQ ID NO:8.
  • the murine antibodies described herein comprise a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8.
  • the antibodies described herein are chimeric antibodies.
  • the chimeric antibodies described herein comprise the variable regions of a murine antibody (including the heavy chain variable region VH and/or the light chain variable region VL) and the constant regions of a human antibody.
  • the chimeric antibodies described herein comprise the variable regions of a murine antibody (including heavy and light chain variable regions) and the constant regions of a human antibody.
  • the chimeric antibody comprises the heavy chain shown in SEQ ID NO:9, and/or the light chain shown in SEQ ID NO:10.
  • the chimeric antibodies described herein comprise a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10.
  • the antibodies described herein are humanized antibodies.
  • the humanized antibodies described herein comprise CDR regions of murine antibodies (including any one or more of HCDR1, HCDR2 and HCDR3 and/or any one or more of LCDR1, LCDR2 and LCDR3), human antibodies Framework regions of variable regions (including any one or more of FR1, FR2, FR3, and FR4), and optionally, constant regions of human antibodies.
  • the humanized antibodies described herein comprise CDR regions (including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of murine antibodies, framework regions (including FR1, FR2) of human antibody variable regions , FR3 and FR4), and optionally the constant regions of human antibodies.
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 11
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 12.
  • the humanized antibodies described herein comprise a heavy chain variable region set forth in SEQ ID NO:11 and a light chain variable region set forth in SEQ ID NO:12.
  • the antibodies described herein may also comprise murine or human antibody constant regions.
  • the murine antibody constant region includes the heavy chain constant region of murine IgG1, IgG2a, IgG2b or IgG3, and the ⁇ or ⁇ type light chain constant region, and the like.
  • the human antibody constant region includes the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the ⁇ or ⁇ type light chain constant region, and the like.
  • the CLDN18.2 described herein is a primate CLDN18.2.
  • the primate CLDN18.2 described herein is selected from human CLDN18.2 or monkey CLDN18.2.
  • the antigen binding moieties described herein are selected from the group consisting of: Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, scFv fragments, Fd fragments, or single domain antibodies.
  • Fab fragment as used herein includes the CH1 and variable regions of the light chain as well as the heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • Fab'fragment contains a light chain as well as a portion or fragment of a heavy chain that contains a VH domain and a CH1 domain and a region between the CH1 and CH2 domains such that between two Fabs An interchain disulfide bond can be formed between the two heavy chains of the 'fragment to form an F(ab') 2 molecule.
  • F(ab') 2 fragment contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that between the two heavy chains form interchain disulfide bonds.
  • the F(ab') 2 fragment is thus composed of two Fab' fragments linked together by disulfide bonds between the two heavy chains.
  • Fv fragment as used herein comprises the variable regions from the heavy and light chains, but lacks the constant regions.
  • single-chain Fv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains exist as a single polypeptide chain.
  • Fv polypeptides also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • single domain antibody refers to an antigen-binding portion comprising one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.
  • VHH heavy chain variable region
  • scFv Single-chain antibody fragments
  • the application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof of the first aspect and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as cornstarch and potato starch; (3) cellulose and its Derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; ( 7) Lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil , olive oil, corn oil and soybean oil; (10) glycols such as prop
  • compositions described herein further comprise one or more other active ingredients, eg, agents for the treatment of CLDN18.2-related diseases such as tumors.
  • the application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of the first aspect.
  • the nucleic acids described herein may be codon-optimized nucleic acids suitable for expression in host cells. For example, depending on the degeneracy of the codons, it still encodes the same protein. Methods for codon optimization depending on the host cell used are well known to those skilled in the art.
  • the application provides an expression vector comprising the nucleic acid molecule of the third aspect.
  • prokaryotic cloning vectors include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of bacteriophage DNA such as M13 and other filamentous single-stranded DNA bacteriophages.
  • An example of a vector that can be used in yeast is the 2 ⁇ plasmid.
  • Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences and those derived from functional mammalian vectors (such as those described above) and functional plasmids and Combinatorial shuttle vectors for phage DNA.
  • Expression vectors useful in the present application contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of cloned DNA sequences. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operon and promoter regions of bacteriophage lambda, the control regions of the fd coat protein, the glycolytic promoters of yeast, such as 3-phosphate Glycerate kinase promoters, yeast acid phosphatase promoters, such as Pho5, yeast alpha-mating factor promoters, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, such as SV40 Early and late promoters and other sequences known to control gene expression in prokaryotic or eukaryotic cells and their viruses or combinations thereof.
  • useful expression control sequences are the lac system, the trp system, the tac
  • the present application provides a host cell comprising the nucleic acid molecule of the third aspect or the expression vector of the fourth aspect.
  • the host cells described herein are mammalian cells.
  • Mammalian cells can include, but are not limited to, CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select suitable host cells as required.
  • the preparation method of the anti-CLDN18.2 monoclonal antibody disclosed herein may include: culturing a host cell under expression conditions, thereby expressing the anti-CLDN18.2 monoclonal antibody; and isolating and purifying the expressed anti-CLDN18.2 monoclonal antibody. Using the above method, crude anti-CLDN18.2 monoclonal antibody can be obtained. Anti-CLDN18.2 was then purified by purification methods including CLDN18.2-based affinity purification, native gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques Monoclonal antibodies are purified to substantially homogeneous material, eg, as a single band on SDS-PAGE.
  • the present application provides the antibody or antigen-binding portion thereof of the first aspect, the pharmaceutical composition of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, Or use of the host cell described in the fifth aspect in the preparation of a medicament for preventing and/or treating CLDN18.2-related diseases.
  • the present application provides a method for preventing and/or treating a CLDN18.2-related disease, comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof of the first aspect, the second aspect
  • the pharmaceutical composition, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the host cell of the fifth aspect comprising administering to an individual in need thereof a therapeutically effective amount of the antibody or antigen-
  • the method further comprises administering a second agent that prevents and/or treats a CLDN18.2-related disease such as a tumor.
  • the term "individual” as used herein refers to mammals including, but not limited to, primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice.
  • the mammal is a non-human primate or human.
  • a particularly preferred mammal is a human.
  • "individual” and “subject” are used interchangeably.
  • Treatment refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (lessen) the target pathological state or disorder.
  • Individuals in need of treatment include those who already have the disorder, as well as those who will develop the disorder or whose disorder is to be prevented. Thus, the individual to be treated herein has been diagnosed with the disorder or is predisposed to or susceptible to the disorder.
  • the “therapeutically effective amount” used herein can be determined according to the specific situation, and can be easily grasped by those of ordinary skill in the art according to the actual required dosage, such as the patient's weight, age and disease condition.
  • the present application provides the antibody or antigen-binding portion thereof of the first aspect, the pharmaceutical composition of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, Or use of the host cell described in the fifth aspect for preventing and/or treating CLDN18.2-related diseases.
  • the CLDN18.2-related disease is a tumor.
  • the tumors described herein are primary cancers or metastatic cancers.
  • the tumor is selected from the group consisting of lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic cancer such as leukemia, breast cancer, gastric cancer, esophageal cancer, B-lymphocytic non-Hodgkin's lymphoma, Hodgkin's lymphoma Chikin lymphoma, anaplastic large cell lymphoma, head and neck cancer such as head and neck squamous cell carcinoma, glioblastoma, kidney cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreatic cancer, ovarian cancer , sarcoma, liver cancer, squamous cell carcinoma of the skin, thyroid cancer, cervical cancer, nasopharyngeal cancer, endometrial cancer, or metastatic cancer of the above tumors.
  • lung cancer such as non-small cell lung cancer, colorectal cancer, bladder
  • the methods, uses and pharmaceutical compositions described herein may further comprise administering to the individual a second agent and/or treatment, eg, as part of a combination therapy.
  • second agents and/or treatments can include radiation therapy, surgery, gemcitabine, cilastatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine vindesine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate ); daunomycin; aminopterin; xelo
  • the application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof of the first aspect.
  • An antibody or antigen-binding portion thereof described herein can bind to a detectable moiety.
  • detectable moieties include, but are not limited to, radioisotopes such as iodine 125, iodine-131, cesium-137, iridium 192 and cobalt 60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphate Enzymes, chemiluminescent agents such as luminol, etc.
  • Those skilled in the art can select a suitable detectable moiety to bind to the antibody or its antigen-binding portion of the present application as required, so as to achieve different detection purposes.
  • the present invention discloses an antibody that specifically binds mammalian (human, primate, etc.) CLDN18.2, and the present invention provides the application of such protein in treatment, screening and detection, such as in cancer treatment.
  • the Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes into vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, see for example Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.
  • Example 1 Construction of high-expressing cell lines of claudin (CLDN) 18.1 and 18.2 (CLDN18.1, CLDN18.2)
  • the high expression cell lines of human CLDN18.1 and human CLDN18.2 were constructed through the stable cell line construction platform. The specific steps are as follows:
  • CLDN18.1 cDNA encoded amino acid sequence shown in SEQ ID NO: 13
  • CLDN18.2 cDNA encoded amino acid sequence shown in SEQ ID NO: 14
  • two small fragments of about 800 bp were obtained by amplification with primers, and the gel was recovered
  • the pENTER plasmid was digested with KpnI/HindIII, and the The large fragment of 7450bp was recovered; the two small fragments obtained above were respectively ligated into the large fragment of the pENTER plasmid digested with KpnI/HindIII that was recovered from the gel, and positive clones were picked and sent for sequencing, and the correctly sequenced plasmids were named pEnter-CLDN18.1- puromycin and
  • 293T cells (Concord Cell Bank) were seeded in two T25 culture flasks, and the number of cells seeded in each culture flask was 2 ⁇ 10 6 . The next day, the 293T cell culture medium was replaced with 4 mL of Opti-MEM (Thermofisher Scientific Cat31985070). Add 5 ⁇ g each of plasmids pEnter-CLDN18.1-puromycin and pEnter-CLDN18.2-puromycin respectively cloned to human CLDN18.1 and human CLDN18.2 into Opti-MEM, the final volume is 500 ⁇ L, and another 500 ⁇ L Opti-MEM is prepared.
  • Opti-MEM Thermofisher Scientific Cat31985070
  • transfection reagent PEI 3 ⁇ g/mL was added to it, the two were mixed well and allowed to stand at room temperature for 20 min, and then added to 4 ml of the cultured 293T cells above.
  • the cell culture medium was replaced with 5 mL of DMEM high glucose medium.
  • 2 ⁇ g/mL puromycin was added for screening. After 2-3 days, a large number of cells died, and after changing the fresh medium until the cells grew stably, single clones were screened, expanded and cultured, and the seeds were cryopreserved.
  • the cell lines stably expressing the target gene constructed in the present application are named as 293T-18.1 cells and 293T-18.2 cells, respectively.
  • the protein sequences used were derived from published databases, and the sequences of each protein were as follows:
  • SEQ ID NO: 13 Human CLDN18.1, NP_057453.1, Claudin-18 isoform 1 precursor [Homo sapiens]
  • SEQ ID NO: 14 Human CLDN18.2, NP_001002026.1, Claudin-18 isoform 2 [Homo sapiens]
  • the preparation method is as follows:
  • mice Five BALB/C healthy female mice aged 7-8 weeks were selected for immunization.
  • the 293T-18.2 cells were cultured, washed three times with PBS, and the blood was washed completely.
  • the cell dosage was 1 ⁇ 10 7 cells/cell, intraperitoneally injected, and 100-200 ⁇ l was injected into each mouse. Immunization every other week, each time the same amount.
  • the tail vein blood of mice was collected and centrifuged to obtain serum titer (detected by flow cytometry). When the titer reaches the fusion requirement, the optimal mouse is selected.
  • mice with high titers were selected for shock immunization by intraperitoneal injection of cells three days before fusion.
  • One day before fusion the intraperitoneal macrophages of ordinary Balb/c mice were taken as trophoblasts and seeded in 96-well plates.
  • the myeloma cells were resuscitated in SP2/0 and cultured in a 37°C, 5% CO 2 incubator. Passage or replace with fresh medium the day before fusion to maintain optimal cell status.
  • the spleen from the immunized mouse was fused with myeloma SP2/0 cells, the cells were added to a 96-well plate plated with trophoblasts in advance, and the fused cells were subjected to HAT selection (Galfre and Milstein, Methods Enzymol 1981; 73:3-46).
  • the primary screening was to determine the titer of anti-CLDN18.2 antibodies secreted by the hybridomas using an enzyme-linked immunoassay (ELISA).
  • ELISA enzyme-linked immunoassay
  • the re-screening method is as follows:
  • 293T-18.2 and 293T-18.1 cells were collected, washed once with FACS buffer, plated into 96-well deep-well plates at 2-5 ⁇ 10 5 cells/well, and 50 ⁇ L of hybridoma supernatant was added to each well, and incubated for 2 hours.
  • MAb362 Karlin Bochuang Biotechnology (Beijing) Co., Ltd. was selected as the positive control antibody.
  • Human CLDN18.2 protein is highly homologous to human CLDN18.1 protein, and it is a four-transmembrane protein with a very short extracellular peptide sequence and extremely low immunogenicity, which is unlikely to produce specific antibodies. Therefore, the probability of the obtained hybridomas secreting antibodies recognizing human CLDN18.2 is extremely low, and a large part of the antibodies in the supernatants of these hybridomas are antibodies that can bind to both human CLDN18.2 and human CLDN18.1.
  • a hybridoma cell 7D10 was obtained, and the antibody in the supernatant bound to 293T-18.2 cells, but hardly bound to 293T-18.1 cells.
  • Hybridoma cells 7D10 were plated in 96-well plates and cultured in HT medium for 7-10 days, and positive clones were further screened by FACS method. Select positive cells for expansion culture, collect the supernatant, purify the antibody (the amino acid sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively), and detect the antibody concentration. .
  • the 7D10-1 hybridoma cell cDNA was obtained, amplified and sequenced by PCR with the antibody primers kept in our laboratory, and finally the nucleotide sequences encoding VH1 and VL1 of the mouse anti-Claudin 18.2 antibody were obtained.
  • the VH1 and VL1 coding nucleic acid sequences of the anti-Claudin 18.2 antibody were constructed into plasmids pUC57 Claudin 18.2 VH1 and pUC57 Claudin 18.2 VL1 by total synthesis (General Biosystems (Anhui) Co., Ltd.), respectively pUC57 Claudin 18.2 VH1, pUC57 Claudin 18.2VL1 was used as the template, and the Gold Mix PCR kit (TSINGKE company) was used to amplify the anti-CLDN18.2 VH1 and CLDN18.2 VL1 fragments according to the instructions of the kit, and the size of the amplified product was about 0.4kb;
  • the vector plasmids pQKX1 and pQKX2 were digested with endonuclease SapI (NEB, R0569S), and the PCR amplification product and the digested vector were digested with BM seamless cloning kit (Bomed Corporation
  • 293Fv cells (Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) were diluted to 1.5 ⁇ 10 6 cells/mL, the final volume was 200 ml, and the cells were cultured at 37° C. for 24 h on a shaker.
  • the plasmids pQK CLDN18.2H1 and pQK CLDN18.2L1 were diluted with fresh medium at 10% of the transfection volume to a concentration of 1 ⁇ g/mL, calculated by the volume of transfected cells.
  • Add 200 ⁇ l of 3 mg/mL PEI to the diluted plasmid at 1/1000 of the cell volume, immediately vortex for 10 seconds, and leave it at room temperature for 15 minutes.
  • PH3.0 eluate was eluted, and collected with 1M Tris-HCl, PH9.0 neutralized and eluted antibody (the amino acid sequences of its heavy chain and light chain were as shown in SEQ ID NO:9 and SEQ ID NO:10 respectively. Show).
  • Collect 293T-18.2 cells in a T75 cell culture flask wash once with FACS buffer, resuspend and count with FACS buffer, adjust the cell concentration to 2 ⁇ 10 6 ; dilute the antibodies (including MAb362 and anti-CLDN18.2 chimeric antibodies Antibody, with MAb362 as control), specifically 20 ⁇ g/ml starting, 3.16-fold dilution, 7 gradients. 10 ⁇ L of the diluted antibodies were added to 100 ⁇ L of the above-mentioned cell samples after concentration adjustment, mixed well, and incubated at room temperature for 1 hour.
  • the anti-Claudin 18.2 humanized antibody encoding VH2 and VL2 nucleotide sequences were designed by the inventors of the present application. Among them, the anti-Claudin 18.2 humanized antibody encoding VH2 and VL2 nucleotide sequences were constructed into plasmids pUC57 Claudin 18.2 VH2 and pUC57 Claudin 18.2 VL2 by total synthesis (Universal Biosystems (Anhui) Co., Ltd.), respectively pUC57 Claudin 18.2 VH2 and pUC57 Claudin 18.2 VL2 were used as templates, and the Gold Mix PCR kit (TSINGKE company) was used to amplify the anti-CLDN18.2 VH2 and CLDN18.2 VL2 fragments according to the instructions of the kit, and the size of the amplified product was about 0.4kb; At the same time, the vector plasmids pQKX1 and pQKX2 were digested with the restriction
  • the 293Fv cells were diluted to 1.5 ⁇ 10 6 cells/mL, the final volume was 200 ml, and the cells were incubated at 37° C. for 24 h on a shaker.
  • the plasmids pQK CLDN18.2 H2 and pQK CLDN18.2L2 were diluted with fresh medium at 10% of the transfection volume to a concentration of 1 ⁇ g/mL, calculated by the volume of transfected cells.
  • Add the plasmid/PEI mixture dropwise to the cell culture medium, adding lightly while adding dropwise.
  • Collect 293T-18.2 cells in a T75 cell culture flask wash once with FACS buffer, resuspend and count with FACS buffer, adjust the cell concentration to 2 ⁇ 10 6 ; dilute antibodies (including MAb362 and anti-CLDN18.2 humanized antibody two antibody, in which MAb362 was used as a control), specifically 20 ⁇ g/ml starting, 3.16-fold dilution, 7 gradients. 10 ⁇ L of the diluted antibodies were added to 100 ⁇ L of the above-mentioned cell samples after concentration adjustment, mixed well, and incubated at room temperature for 1 hour.
  • Figure 4 shows that the anti-CLDN18.2 humanized antibody specifically binds to NUGC4 cells.
  • NUGC4 cells One flask (T75) of NUGC4 cells was collected and adjusted to a density of 2 ⁇ 10 5 cells/ml. Add 50 ⁇ l of DMEM containing 10% low IgG FBS to each well of the real-time cell analyzer (RTCA) culture plate, use the real-time cell analyzer to check the quality of the well plate and determine the baseline, take out and discard the medium in the well. Add NUGC4 single cell suspension, 100 ⁇ l per well, after the cells settle on the bottom of the plate, place it on an RTCA instrument (placed in a cell incubator), incubate overnight and detect cell growth.
  • RTCA real-time cell analyzer
  • Human peripheral blood was collected, diluted 1:1 with PBS, and plated with equal volume on 4ml Ficoll-PagueTM plus placed in a 15ml centrifuge tube (density 1.077 ⁇ 0.001g/ml, GE Company, 17-1440-03). Centrifuge at 800 g at room temperature for 20 min, and after separation, suck the buffy coat to obtain PBMCs, wash PBMCs twice with PBS, and adjust the cell density to 4 ⁇ 10 6 cells/ml.
  • the anti-CLDN18.2 humanized antibody to be tested and the human IgG1 isotype control antibody were all diluted in PBS, with an initial concentration of 580 ⁇ g/ml (4 ⁇ M), 2.57 times of dilution, a total of 6 concentrations .
  • Pause the RTCA instrument take out the RTCA culture plate, add 50 ⁇ l PBMC and 50 ⁇ l antibodies of different types and different dilutions, parallel wells with 2 wells, and set target cell control (Blank) and no antibody control (PBMC). Static plate wells, after the cells settle to the bottom of the plate, go on the machine and continue to detect, and the EC50 of anti-CLDN18.2 humanized antibody to ADCC of human gastric cancer cells NUGC4 is calculated to be 0.18mM (see Figure 5), while the IgG1 isotype control antibody There was no ADCC effect against NUGC4, resulting in no concentration-dependent gradient trend.

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Abstract

提供了抗紧密连接蛋白18.2(claudin18.2，CLDN18.2)抗体及其用途。还提供了抗CLDN18.2抗体的嵌合抗体和人源化抗体形式。这些抗体对人CLDN18.2蛋白具有高亲和性，是用于预防和/或治疗CLDN18.2相关疾病，例如CLDN18.2相关的肿瘤的一种有前景的抗体分子。

Description

抗CLDN18.2抗体及其应用 技术领域
本申请涉及抗体领域，更具体地，本申请涉及抗CLDN18.2抗体及其应用。
背景技术
癌症具有细胞分化和增殖异常、生长失去控制、浸润性和转移性等生物学特征，其发生是一个多因子、多步骤的复杂过程，目前治疗手段主要有：手术、放疗、化疗、免疫治疗。对于许多中晚期癌症患者来说，免疫治疗极大地提高其生存期及生活质量，而其中的抗体疗法已成为公认的有效抗击癌症的免疫疗法。
全世界每年新发胃癌大约有一百万例。尽管传统癌症治疗方法已进行许多改进，但复发率仍持续增高，而且复发患者容易对传统疗法出现耐受导致治疗失败。进展期胃癌患者的5年生存率约为5％-20％，中位总生存率约为10个月。胰腺癌5年总生存率约为6～8％。这两种癌症的不良预后促使研究者们寻找新的治疗靶标。
1998年Shorichiro Tsukita等人首次发现紧密连接蛋白(claudin，CLDN)，该蛋白是人内皮和上皮紧密连接的主要跨膜蛋白成分，具有维持组织渗透平衡及细胞极性的功能。CLDN成员的组织特异性表达表明，该蛋白家族不仅是维持细胞通透性，控制紧密连接的重要角色，而且在蛋白与蛋白相互作用，组织细胞接触信号方面也至关重要。目前已知有27种哺乳动物CLDN基因，包括3种最近发现的远缘成员，但对于这三种远缘成员是否都应该被归为CLDN仍存在分歧。并非所有的这些基因都存在于所有的哺乳动物中。例如CLDN13存在于啮齿类动物，却不存在于人类当中。因此，到目前为止，在人类中共发现了26种CLDN基因。
CLDN18是人类紧密连接蛋白家族中少数严格限制于一种细胞系的成员之一。CLDN18分子是是四次跨膜蛋白，有四个跨膜疏水区， 两个胞外环，一个胞内环，其末端位于胞内。该分子有两个剪切异构体：CLDN18.1和CLDN18.2，是由第一外显子的选择性拼接产生的，两者序列之间高度同源，仅有八个氨基酸的差异。CLDN18.1主要表达在肺组织细胞；CLDN18.2在正常组织中的表达严格局限于胃粘膜的分化上皮细胞，而在胃干细胞区则不表达。CLDN18.2在恶性转化中被保留，并且在相当比例的原发性胃癌及其转移中表达。此外，CLDN18.2在胰腺、食道、卵巢和肺肿瘤中频繁异位激活。临床数据显示，CLDN18.2特异性人鼠嵌合单克隆抗体IMAB362对正常组织无明显毒性。IMAB362对胃癌患者的总体客观应答率约为10％，这些数据提示CLDN18.2可能是治疗胃癌和其他CLDN18.2阳性肿瘤的一个有前途的治疗靶点。
发明内容
为解决上述技术问题，本申请提供了一种抗CLDN18.2抗体及其应用。具体而言，本申请提供了以下技术方案。
在第一方面，本申请提供了特异性结合CLDN18.2的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3序列中的任意一项或多项，其中所述HCDR1序列为GFSFSSFG(SEQ ID NO:1)，所述HCDR2序列为ISSGSRTI(SEQ ID NO:2)，所述HCDR3序列为TRYYYGNSFDY(SEQ ID NO:3)。
在一些实施方案中，所述抗体或其抗原结合部分还包含轻链可变区，所述轻链可变区包含LCDR1、LCDR2和LCDR3序列中的任意一项或多项，其中所述LCDR1序列为QSLFNTGNQKNY(SEQ ID NO:4)，所述LCDR2序列为WAS(SEQ ID NO:5)，所述LCDR3序列为QNNYNFPLT(SEQ ID NO:6)。
在一些实施方案中，所述抗体为鼠源抗体。优选地，所述重链可变区的氨基酸序列如SEQ ID NO:7所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述抗体为嵌合抗体。优选地，所述嵌合抗体包含氨基酸序列如SEQ ID NO:9所示的重链，和/或氨基酸序列如 SEQ ID NO:10所示的轻链。
在一些实施方案中，所述抗体为人源化抗体。优选地，所述重链可变区的氨基酸序列如SEQ ID NO:11所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:12所示。
在第二方面，本申请提供了药物组合物，其包含第一方面所述的抗体或其抗原结合部分以及药学上可接受的载体。
在第三方面，本申请提供了核酸分子，其编码第一方面所述的抗体或其抗原结合部分。
在第四方面，本申请提供了表达载体，其包含第三方面所述的核酸分子。
在第五方面，本申请提供了宿主细胞，其包含三方面所述的核酸分子或第四方面所述的表达载体。
在第六方面，本申请提供了第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞在制备用于预防和/或治疗CLDN18.2相关疾病的药物中的用途。
在第七方面，本申请提供了预防和/或治疗CLDN18.2相关疾病的方法，其包括向有需要的个体施用治疗有效量的第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞。
在第八方面，本申请提供了第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞用于预防和/或治疗CLDN18.2相关疾病的用途。
在第六至第八方面的任一实施方案中，所述CLDN18.2相关疾病为肿瘤。
在一些实施方案中，所述肿瘤选自以下中的一种或多种：胃癌、胰腺癌、肠癌、食管癌、肝癌、卵巢癌、肺癌和膀胱癌等以及上述的转移瘤。
在第九方面，本申请提供了检测试剂或试剂盒，其包含第一方面 所述的抗体或其抗原结合部分。
本申请的特异性结合CLDN18.2的抗体或其抗原结合部分能够诱导CLDN18.2介导的抗肿瘤免疫应答，和/或抑制肿瘤生长等。
附图的简要说明
图1显示了本申请鼠源抗CLDN18.2抗体(杂交瘤7D10-1分泌的抗体)对过表达人CLDN18.2的HEK293细胞293T-18.2的结合亲和力。
图2显示了本申请嵌合抗CLDN18.2抗体对过表达人CLDN18.2的HEK293细胞293T-18.2的结合亲和力。
图3显示了本申请人源化抗CLDN18.2抗体对过表达人CLDN18.2的HEK293细胞293T-18.2的结合亲和力。
图4显示了本申请人源化抗CLDN18.2抗体对人胃癌细胞NUGC4的结合亲和力。
图5显示了本申请人源化抗CLDN18.2抗体的ADCC活性。
发明的详细描述
提供以下定义和方法以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，本申请的术语按照相关领域普通技术人员的常规用法理解。
定义
如本文所用的，术语“抗体”指包含四条多肽链，即通过双硫键互连的两条重链(H)及两条轻链(L)的免疫球蛋白分子，以及其多聚体(例如IgM)。各重链包含重链可变区(缩写为VH)及重链恒定区(缩写为CH)。重链恒定区包含三个域，即CH1、CH2及CH3。各轻链包含轻链可变区(缩写为VL)及轻链恒定区(缩写为CL)。轻链恒定区包含一个域(CL1)。VH及VL区可进一步细分成称为互补决定区(CDR)的高变区，其中穿插有称为构架区(FR)的保守区。在一些实施方案中，从N-末端至C-末端，轻链与重链可变结构域均包含FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
如本文所用的，术语抗体的“抗原结合部分”是指负责结合抗原的 完整抗体分子的一部分或区段。抗原结合部分可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。抗体的抗原结合部分可使用任何适合的标准技术从完整抗体分子制备，所述标准技术包括蛋白水解消化或重组遗传工程化技术等。抗原结合部分的非限制性实例包括：Fab片段、F(ab') 2片段、Fd片段、Fv片段、单链Fv(scFv)分子、单域抗体、dAb片段及由模拟抗体高变区的氨基酸残基组成的最小识别单元(例如分离的CDR)。术语“抗原结合部分”也包括其它工程化的分子，如双抗体、三抗体、四抗体及微型抗体等。例如，本文中所述Fd片段指由VH与CH1结构域组成的抗体片段；Fv片段由抗体的单臂中VL与VH结构域组成；dAb片段(Ward等人，Nature 1989；341：544-546)由VH结构域组成。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即Kabat定义和Chothia定义，例如参见Kabat等人,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，MD.(1991)；Al-Lazikani等人，J Mol Biol 273:927-948(1997)；以及Martin等人，Proc.Natl.Acad.Sci.USA 86:9268-9272(1989)。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。在本文中，重链可变区的CDR1、CDR2及CDR3分别简称为HCDR1、HCDR2及HCDR3；轻链可变区的CDR1、CDR2及CDR3分别简称为LCDR1、LCDR2及LCDR3。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
如本文所用的，术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合，例如抗体以比其对非特异性抗原的亲和性大至少两倍的亲和性结合于特异性抗原的能力。然而应了解，抗体能够特异性结合两种或更多种与其序列相关的抗原。例如， 本申请的抗体可特异性结合人CLDN18.2。
如本文所用的，术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(参见，美国专利号4,816,567；和Morrison等人,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
如本文所用的，术语“鼠源抗体”是指其中所有结构域序列均为小鼠序列的任何抗体。此类抗体可通过杂交瘤产生。
如本文所用的，术语“嵌合抗体”是指包含来自两种或更多种不同抗体的区段的抗体。在一些实施方案中，一个或多个CDR衍生自小鼠抗CLDN18.2抗体。在另一些实施方案中，所有CDR均衍生自小鼠抗CLDN18.2抗体。在一些实施方案中，在嵌合抗体中组合来自一种以上小鼠抗CLDN18.2抗体的CDR。例如，嵌合抗体可包含来自第一种小鼠抗CLDN18.2抗体中轻链的CDR1、来自第二种小鼠抗CLDN18.2抗体中轻链的CDR2、与来自第三种小鼠抗CLDN18.2抗体中轻链的CDR3，以及来自重链的CDR可衍生自一种或多种其它抗CLDN18.2抗体。此外，框架区可来自相同抗CLDN18.2抗体或来自一个或多个不同的个体。在一些实施方案中，本文所述的嵌合抗体包含鼠源抗体的可变区(包括重链可变区VH和/或轻链可变区VL)和人抗体的恒定区。
如本文所用的，术语“人源化抗体”是指CDR移植抗体，具体是指将小鼠的CDR区序列移植到人的抗体可变区框架中产生的抗体。目的是克服嵌合抗体由于携带大量其他物种例如小鼠的蛋白成分从而在人体中诱导强烈的免疫副反应。
如本文所用的，术语“核酸分子”可以指DNA分子及RNA分子，其可以是单链或双链的。核酸分子也可以为cDNA。
如本文所用的，术语“CLDN18.2相关疾病”包括与CLDN18.2信号通路相关的疾病和/或病症。示例性CLDN18.2相关疾病或病症包括肿瘤，癌例如胃癌、胰腺癌、肠癌、食管癌、肝癌、卵巢癌、肺癌和膀胱癌以及上述的转移癌。
如本文所用的，术语“EC50”是指半数最大效应浓度(concentration for 50％of maximal effect,EC50)，指能引起50％最大效应的浓度。
如本文所用的，术语“免疫反应”指脊椎动物内针对外来作用剂的生物反应，该反应保护生物体抵抗此类作用剂及由其引起的疾病。免疫反应系由免疫系统的细胞(例如，T淋巴细胞、B淋巴细胞、自然杀伤(NK)细胞、巨噬细胞、嗜酸性粒细胞、肥大细胞、树突细胞或嗜中性粒细胞)及由此类细胞中的任一者或由肝脏产生的可溶性大分子(包括抗体、细胞因子及补体)的作用介导，其导致选择性靶向、结合、损害、破坏和/或自脊椎动物身体消除侵入病原体、经病原体感染的细胞或组织、癌性或其他异常细胞或在自体免疫或病理炎症的情形下正常人类细胞或组织。免疫反应包括T细胞(例如效应T细胞)或Th细胞(例如CD4 +或CD8 +T细胞)的活化或抑制或Treg细胞的抑制。
如本文所用的，术语“癌症”指以体内异常细胞不受控生长为特点的一大类疾病。失调的细胞分裂可形成侵袭相邻组织且可经由淋巴系统或血流转移至身体的远处部分的恶性肿瘤或细胞。
如本文所用的，术语“治疗”指对受试者实施的任何类型的介入或方法或向其施用活性剂，其中目的是逆转、缓和、改善、抑制或缓解或预防症状、并发症、病况或与疾病相关的进展、发展、严重程度或复发。
如本文所用的，术语“预防”指对未患疾病的受试者施用，以防止疾病发生或使其影响(若存在)最小化。
具体实施方式
人CLDN18.2蛋白与CLDN18.1高度同源，且其为四次跨膜蛋白，胞外区肽段序列很短，免疫原性极低，能够产生特异性抗体的可能性很小，所以筛选获得的能分泌识别CLDN18.2的抗体的杂交瘤的概率 极低。而筛选的杂交瘤上清中的抗体有很大一部分是同时能结合人CLDN18.2和CLDN18.1的抗体。本发明人筛选得到了一株杂交瘤细胞，其上清中的抗体能够与表达CLDN18.2细胞结合，而与表达CLDN18.1的细胞几乎不结合。因此，本申请提供了一种能特异性结合CLDN18.2的新型抗CLDN18.2抗体或其抗原结合部分。
本发明人还通过基因工程手段从鼠源抗CLDN18.2抗体制备了嵌合抗体和人源化抗体形式，这些抗体能够与细胞表面表达的CLDN18.2特异性结合，从而有效地诱导CLDN18.2介导的免疫应答，并起到预防或治疗CLDN18.2相关疾病的作用。
在第一方面，本申请提供了特异性结合CLDN18.2的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3序列中的任意一项或多项，其中所述HCDR1序列为GFSFSSFG(SEQ ID NO:1)，所述HCDR2序列为ISSGSRTI(SEQ ID NO:2)，所述HCDR3序列为TRYYYGNSFDY(SEQ ID NO:3)。
在优选的实施方案中，本文所述的抗体或其抗原结合部分的重链可变区包含SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3序列。
在一些实施方案中，本文所述的抗体或其抗原结合部分还包含轻链可变区，所述轻链可变区包含LCDR1、LCDR2和LCDR3序列中的任意一项或多项，其中所述LCDR1序列为QSLFNTGNQKNY(SEQ ID NO:4)，所述LCDR2序列为WAS(SEQ ID NO:5)，所述LCDR3序列为QNNYNFPLT(SEQ ID NO:6)。
在优选的实施方案中，本文所述的抗体或其抗原结合部分的轻链可变区包含SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3序列。
在优选的实施方案中，本文所述的抗体或其抗原结合部分包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3序列，并且所述轻链可变区包含SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3序列。
在一些更具体的实施方案中，本文公开的抗体可以为抗人CLDN18.2单克隆抗体。抗CLDN18.2抗体类型与亚型可由本领域已知的任何方式确定。通常，抗体类型与亚型可使用特异于特定抗体类型与亚型的抗体确定。可使用ELISA测定法测定抗CLDN18.2抗体同种型，例如可使用鼠Ig吸附的抗人Ig鉴定人Ig。
在一些实施方案中，本文所述的抗体为鼠源抗体，优选地，所述重链可变区的氨基酸序列如SEQ ID NO:7所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在优选的实施方案中，本文所述的鼠源抗体包含SEQ ID NO:7所示的重链可变区和SEQ ID NO:8所示的轻链可变区。
在一些实施方案中，本文所述的抗体为嵌合抗体。本文所述的嵌合抗体包含鼠源抗体的可变区(包括重链可变区VH和/或轻链可变区VL)和人抗体的恒定区。
在优选的实施方案中，本文所述的嵌合抗体包含鼠源抗体的可变区(包括重链可变区和轻链可变区)和人抗体的恒定区。
优选地，所述嵌合抗体包含SEQ ID NO:9所示的重链，和/或SEQ ID NO:10所示的轻链。
在优选的实施方案中，本文所述的嵌合抗体包含SEQ ID NO:9所示的重链和SEQ ID NO:10所示的轻链。
在一些实施方案中，本文所述的抗体为人源化抗体。本文所述的人源化抗体包含鼠源抗体的CDR区(包括HCDR1、HCDR2和HCDR3中的任意一项或多项和/或LCDR1、LCDR2和LCDR3中的任意一项或多项)、人抗体可变区的框架区(包括FR1、FR2、FR3和FR4中的任意一项或多项)，以及任选地人抗体的恒定区。
在优选的实施方案中，本文所述的人源化抗体包含鼠源抗体的CDR区(包括HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)、人抗体可变区的框架区(包括FR1、FR2、FR3和FR4)，以及任选地人抗体的恒定区。
优选地，所述重链可变区的氨基酸序列如SEQ ID NO:11所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:12所示。
在优选的实施方案中，本文所述的人源化抗体包含SEQ ID NO:11所示的重链可变区和SEQ ID NO:12所示的轻链可变区。
本文所述的抗体还可以包含鼠或人抗体恒定区。鼠抗体恒定区包括鼠IgG1、IgG2a、IgG2b或IgG3的重链恒定区以及κ或λ型轻链恒定区等。人抗体恒定区包括人IgG1、IgG2、IgG3或IgG4的重链恒定区以及κ或λ型轻链恒定区等。
在一些实施方案中，本文所述的CLDN18.2为灵长类CLDN18.2。优选地，本文所述的灵长类CLDN18.2选自人CLDN18.2或猴CLDN18.2。
在一些实施方案中，本文所述的抗原结合部分选自：Fab片段、Fab’片段、F(ab’) 2片段、Fv片段、scFv片段、Fd片段或单域抗体。
本文所用的术语“Fab片段”包含轻链以及重链的CH1和可变区。Fab分子的重链不能与另一个重链分子形成二硫键。
本文所用的术语“Fab’片段”含有轻链以及重链的部分或片段，所述部分或片段含有VH结构域和CH1结构域以及在CH1和CH2结构域之间的区域，使得在2个Fab’片段的两条重链之间可以形成链间二硫键，以形成F(ab’) 2分子。
本文所用的术语“F(ab’) 2片段”含有两条轻链和两条重链，所述重链含有在CH1和CH2结构域之间的恒定区的一部分，使得在两条重链之间形成链间二硫键。F(ab’) 2片段因而由两个Fab’片段组成，而两个Fab’片段通过两条重链之间的二硫键连接在一起。
本文所用的术语“Fv片段”包含来自重链和轻链的可变区，但是缺少恒定区。
本文所用的术语“单链Fv”或“scFv”，是指包含抗体的VH结构域和VL结构域的抗体片段，其中这些结构域以单一多肽链形式存在。通常，Fv多肽还包含VH结构域和VL结构域之间的多肽接头，所述接头使scFv能够形成期望的结构以进行抗原结合。
本文所用的术语“单域抗体”指包含一个重链可变区(VHH)和两个常规的CH2与CH3区的抗原结合部分。最初是在羊驼外周血液中发现的一种天然缺失轻链的抗体，该抗体虽然只包含一个重链可变区 (VHH)和两个常规的CH2与CH3区，但却不像人工改造的单链抗体片段(scFv)那样容易相互沾粘，甚至聚集成块。更重要的是单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。VHH晶体分子量只有15KDa，因此也被称作纳米抗体(Nanobody，Nb)
在第二方面，本申请提供了药物组合物，其包含第一方面所述的抗体或其抗原结合部分以及药学上可接受的载体。
药学上可接受的载体和稀释剂包括盐水、水性缓冲溶液、溶剂和/或分散介质。这种载体和稀释剂的使用是本领域熟知的。可用作药学上可接受的载体的材料的一些非限制性实例包括：(1)糖，例如乳糖、葡萄糖和蔗糖；(2)淀粉，例如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，例如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和乙酸纤维素；(4)粉末状黄蓍胶；(5)麦芽；(6)明胶；(7)润滑剂，例如硬脂酸镁、十二烷基硫酸钠和滑石；(8)赋形剂，例如可可脂和栓剂蜡；(9)油，例如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；(10)二醇，例如丙二醇；(11)多元醇，例如甘油、山梨糖醇、甘露糖醇和聚乙二醇(PEG)；(12)酯，例如油酸乙酯和月桂酸乙酯；(13)琼脂；(14)缓冲剂，例如氢氧化镁和氢氧化铝；(15)藻酸；(16)无热原水；(17)等渗盐水；(18)林格氏溶液；(19)乙基酒精；(20)pH缓冲溶液；(21)聚酯、聚碳酸酯和/或聚酸酐；(22)填充剂，例如多肽和氨基酸；(23)血清组分，例如血清白蛋白、HDL和LDL；(22)C 2-C 12醇类，例如乙醇；以及(23)药物制剂中所用的其他无毒的相容物质。湿润剂、着色剂、脱模剂、包衣剂、甜味剂、调味剂、芳香剂、防腐剂和抗氧化剂也可以存在于药物制剂中。
在一些实施方案中，本文所述的药物组合物还包含一种或多种其他活性成分，例如用于治疗CLDN18.2相关疾病如肿瘤的药剂。
在第三方面，本申请提供了核酸分子，其编码第一方面所述的抗体或其抗原结合部分。
在优选的实施方案中，本文所述的核酸可以是适合在宿主细胞中表达的密码子优化的核酸。例如根据密码子的简并性，其仍然编码同 样的蛋白质。根据所用宿主细胞进行密码子优化的方法是本领域技术人员公知的。
在第四方面，本申请提供了表达载体，其包含第三方面所述的核酸分子。
可以使用任何合适的表达载体。例如，原核克隆载体包括来自大肠杆菌的质粒，如colEl、pCRl、pBR322、pMB9、pUC、pKSM和RP4。原核载体还包括噬菌体DNA如M13和其它丝状单链DNA噬菌体的衍生物。可用于酵母的载体的实例是2μ质粒。用于在哺乳动物细胞中表达的合适载体包括以下众所周知的衍生物：SV-40、腺病毒、逆转录病毒衍生的DNA序列以及衍生自功能性哺乳动物载体(如上述那些)和功能性质粒和噬菌体DNA的组合的穿梭载体。
另外的真核表达载体为本领域已知的(例如，P J.Southern&P.Berg,J.Mol.Appl.Genet,1:327-341(1982)；Subramani等人,Mol.Cell.Biol,1:854-864(1981)；Kaufinann&Sharp,"Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene,"J.Mol.Biol,159:601-621(1982)；Kaufhiann&Sharp,Mol.Cell.Biol,159:601-664(1982)；Scahill等人,"Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"Proc.Nat'l Acad.Sci USA,80:4654-4659(1983)；Urlaub&Chasin,Proc.Nat'l Acad.Sci USA,77:4216-4220,(1980)，将其全部通过引用并入本文)。
可用于本申请的表达载体含有至少一个表达控制序列，其与待表达的DNA序列或片段可操作连接。将控制序列插入载体中以控制和调节克隆的DNA序列的表达。有用的表达控制序列的实例是lac系统，trp系统，tac系统，trc系统，噬菌体λ的主要操纵子和启动子区，fd外壳蛋白的控制区，酵母的糖酵解启动子，例如3-磷酸甘油酸激酶的启动子，酵母酸性磷酸酶的启动子，例如Pho5，酵母α-交配因子的启动子，以及来源于多瘤病毒、腺病毒、逆转录病毒和猿猴病毒的启动子，例如SV40的早期和晚期启动子和已知控制原核或真核细胞及其病毒或其组合的基因表达的其它序列。
在第五方面，本申请提供了宿主细胞，其包含三方面所述的核酸分子或第四方面所述的表达载体。
在一些实施方案中，本文所述的宿主细胞为哺乳动物细胞。哺乳动物细胞可以包括但不限于CHO细胞、NS0细胞、SP2/0细胞、HEK293细胞、COS细胞和PER.C6细胞。本领域技术人员能够根据需要选择适合的宿主细胞。
本文公开的抗CLDN18.2单克隆抗体的制备方法可包括：在表达条件下培养宿主细胞，从而表达抗CLDN18.2单克隆抗体；分离和纯化表达的抗CLDN18.2单克隆抗体。利用上述方法，可以获得粗抗CLDN18.2单克隆抗体。然后通过纯化方法，包括基于CLDN18.2的亲和纯化、非变性凝胶纯化、HPLC或RP-HPLC、分子排阻、在蛋白A柱上纯化、或这些技术的任何组合，将抗CLDN18.2单克隆抗体纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
在第六方面，本申请提供了第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞在制备用于预防和/或治疗CLDN18.2相关疾病的药物中的用途。
在第七方面，本申请提供了预防和/或治疗CLDN18.2相关疾病的方法，其包括向有需要的个体施用治疗有效量的第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞。
在一些实施方案中，所述方法还包括施用预防和/或治疗CLDN18.2相关疾病如肿瘤的第二药剂。
本文使用的术语“个体”是指哺乳动物，包括但不限于灵长类动物、牛、马、猪、绵羊、山羊、狗、猫以及诸如大鼠和小鼠的啮齿类动物。优选地，哺乳动物为非人类的灵长类或者人类。特别优选的哺乳动物是人。本文使用的“个体”和“受试者”可以互换使用。
“治疗”既指治疗性处理，也指预防性或防止性的措施，其目的就是预防或减缓(减轻)目标病理状态或病症。需要治疗的个体包括那些已经存在所述病症的个体，还包括那些将发展为该病症的或欲对其病 症进行预防的个体。因此，本文中欲被治疗的个体已经被诊断为患有该病症或倾向于或易患该病症。
本文中所用的“治疗有效量”可以根据具体情况而定，本领域普通技术人员根据实际所需药量可以很容易地掌握，如可根据患者体重、年龄和病症情况来确定。
在第八方面，本申请提供了第一方面所述的抗体或其抗原结合部分、第二方面所述的药物组合物、第三方面所述的核酸分子，第四方面所述的表达载体、或者第五方面所述的宿主细胞用于预防和/或治疗CLDN18.2相关疾病的用途。
在第六至第八方面的任一实施方案中，所述CLDN18.2相关疾病为肿瘤。
在一些实施方案中，本文所述的肿瘤为原发性癌症或转移性癌症。在具体的实施方案中，肿瘤选自肺癌例如非小细胞肺癌、结直肠癌、膀胱癌、造血系统癌症例如白血病、乳腺癌、胃癌、食管癌、B淋巴细胞型非霍奇金淋巴瘤、霍奇金淋巴瘤、间变大细胞淋巴瘤、头颈癌例如头颈部鳞状细胞癌、恶性胶质瘤、肾癌、黑色素瘤、前列腺癌、骨癌、骨巨细胞瘤、胰腺癌、卵巢癌、肉瘤、肝癌、皮肤鳞癌、甲状腺癌、宫颈癌、鼻咽癌、子宫内膜癌，或上述肿瘤的转移癌。
在任一方面的实施方案中，本文所述的方法、用途和药物组合物还可包括向个体施用第二药剂和/或治疗，例如作为联合疗法的一部分。第二药剂和/或治疗的非限制性实例可包括放射疗法、手术、吉西他滨、西司他丁、紫杉醇、卡铂、硼替佐米、AMG479、伏立诺他、利妥昔单抗、替莫唑胺、雷帕霉素、苯丁酸氮芥；吉西他滨；6-硫鸟嘌呤(6-thioguanine)；巯基嘌呤；甲氨蝶呤；铂类似物，诸如顺铂、奥沙利铂和卡铂；长春碱；长春地辛；依托泊苷(VP-16)；异环磷酰胺；米托蒽醌；长春新碱；长春瑞滨；诺肖林(novantrone)；替尼泊苷；依达曲沙(edatrexate)；正定霉素(daunomycin)；氨基蝶呤；希罗达(xeloda)；降低细胞增殖的PKC-α、Raf、H-Ras、EGFR(例如，埃罗替尼
Figure PCTCN2022073207-appb-000001
)和VEGF-A的抑制剂以及上述任何的药学上可接受的盐、酸或衍生物。
在第九方面，本申请提供了检测试剂或试剂盒，其包含第一方面 所述的抗体或其抗原结合部分。
本文所述的抗体或其抗原结合部分可与可检测部分结合。示例性的可检测部分包括但不限于放射性同位素例如碘125、碘-131、铯-137、铱192和钴60、辣根过氧化物酶、异硫氰酸荧光素、生物素、碱性磷酸酶、化学发光剂如鲁米诺类等。本领域技术人员可以根据需要选择适合的可检测部分与本申请的抗体或其抗原结合部分结合，从而实现不同的检测目的。
本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。
本发明公开了特异性结合哺乳动物(人、灵长类动物等)CLDN18.2的抗体，本发明提供此类蛋白质在治疗、筛选和检测等方面的应用，如在癌症治疗中的用途。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明的范围内。本领域技术人员能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合实施例对本发明作进一步的详细说明。
实施例
以下实施例用于说明本申请，但不用来限制本申请的范围。在不背离本申请精神和实质的情况下，对本申请方法、步骤或条件所作的修改或替换，均属于本申请的范围。
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到载体和质粒的方法或将质粒引入 宿主细胞的方法。这样的方法对于本领域具有普通技术的人员来说是众所周知的，并且在许多出版物中都有所描述，例如参见Sambrook,J.,Fritsch,EF.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1.紧密连接蛋白(claudin，CLDN)18.1和18.2(CLDN18.1，CLDN18.2)高表达细胞株的构建
通过稳定细胞株构建平台构建人CLDN18.1和人CLDN18.2的高表达细胞株，具体步骤如下：
质粒pEnter-CLDN18.1-puromycin和pEnter-CLDN18.2-puromycin的构建：分别以CLDN18.1cDNA(其编码的氨基酸序列如SEQ ID NO：13所示)和CLDN18.2cDNA(其编码的氨基酸序列如SEQ ID NO：14所示)((通用生物系统(安徽)有限公司))为模板，用引物扩增得到两种约800bp的小片段，胶回收；将pENTER质粒用KpnI/HindIII酶切，胶回收大片段7450bp；将上述所得的两种小片段分别连接入胶回收的用KpnI/HindIII酶切的pENTER质粒大片段，挑阳性克隆送测序，测序正确的质粒分别命名为pEnter-CLDN18.1-puromycin和pEnter-CLDN18.2-puromycin。
将293T细胞(协和细胞库)接种于两个T25培养瓶，每个培养瓶接种的细胞数为2×10 6。第二天将293T细胞培养液更换为4mL Opti-MEM(Thermofisher Scientific Cat31985070)。将分别克隆有人CLDN18.1和人CLDN18.2的质粒pEnter-CLDN18.1-puromycin和pEnter-CLDN18.2-puromycin各5μg分别加入到Opti-MEM中，终体积为500μL，另准备500μL Opti-MEM，并在其中加入7μL转染试剂PEI(3μg/mL)，将二者混匀并室温静置20min后，加入到上述培养好的4ml 293T细胞中。第三天将细胞培养液更换为5mL DMEM高糖培养基。第四天加入2μg/mL嘌呤霉素进行筛选。2-3天后细胞大量死亡，更换新鲜培养基至细胞稳定生长后，进行单克隆筛选，扩增培养并冻存保种。
本申请构建的稳定表达目的基因的细胞株分别命名为293T-18.1 细胞和293T-18.2细胞。所用蛋白序列源自公开发表的数据库，各蛋白质的序列如下：
SEQ ID NO：13：人CLDN18.1，NP_057453.1，紧密连接蛋白-18同种型1前体[智人(Homo sapiens)]
Figure PCTCN2022073207-appb-000002
SEQ ID NO：14：人CLDN18.2，NP_001002026.1，紧密连接蛋白-18同种型2[智人]
Figure PCTCN2022073207-appb-000003
实施例2.产生抗CLDN18.2抗体的杂交瘤的制备
制备方法如下：
选择7～8周的BALB/C健康雌性小鼠5只进行免疫。培养293T-18.2细胞，用PBS将细胞清洗三次，将血清洗尽，细胞用量为1×10 7个细胞/只，腹腔注射，每只小鼠注射100-200μl。隔周免疫，每次用量相同。第3次免疫两周后采小鼠尾静脉血离心取血清测效价(用流式细胞仪方法检测)。效价达到可融合要求，选取最优的小鼠。
选择效价高的小鼠在融合前三天腹腔注射细胞进行冲击免疫。进行融合前1天，取普通Balb/c小鼠腹腔内巨噬细胞作为滋养层，接种于96孔板中。
效价检测前一周复苏骨髓瘤细胞SP2/0,37℃，5％CO 2培养箱培 养。融合前一天传代或更换新鲜培养基，使细胞状态保持最佳。
取来自经免疫接种的小鼠的脾与骨髓瘤SP2/0细胞融合，将细胞加入到提前铺有滋养层的96孔板中，对融合的细胞进行HAT选择(Galfre and Milstein,Methods Enzymol 1981；73：3-46)。
抗CLDN18.2杂交瘤初筛(ELISA)
初次筛选是利用酶联免疫分析法(ELISA)确定杂交瘤分泌的抗CLDN18.2抗体的滴度。表1显示了部分杂交瘤上清的检测结果。
表1
初始杂交瘤克隆号 293T-18.2细胞结合活性
3C1 1.314
6B4 0.087
6E7 0.432
7B1 0.091
7D1 1.116
抗CLDN18.2杂交瘤复筛(FACS)
复筛方法如下：
收集293T-18.2和293T-18.1细胞，用FACS缓冲液洗涤一次，以2-5×10 5个/孔细胞铺到96孔深孔板，每孔加入50μL杂交瘤上清，孵育2个小时。选用MAb362(康源博创生物科技(北京)有限公司)作为阳性对照抗体。
每孔加入400μl FACS缓冲液洗涤两次，加入PE抗小鼠IgG Fc二抗(Biolegend，Cat405307)，孵育1小时后上流式细胞仪检测。
结果如表2所示，获得5个候选单克隆抗体，分别标记为6E7、7D10、7E12、9A9、3C1。
表2抗CLDN18.2杂交瘤复筛结果
初始杂交瘤克隆号 293T-18.2细胞结合活性 293T-18.1细胞结合活性
3C1 3297 2451
6E7 1359 2673
7D10 2517 7.65
7E12 2620 1893
9A9 3267 3647
空白 6.97 7.22
二抗 7.09 7.7
阳性对照 1587 61.3
人CLDN18.2蛋白与人CLDN18.1蛋白高度同源，且其为四次跨膜蛋白，胞外区肽段序列很短，免疫原性极低，能够产生特异性抗体的可能性很小，所以筛选获得的能分泌识别人CLDN18.2的抗体的杂交瘤的概率极低，而这些杂交瘤上清中的抗体有很大一部分是同时能结合人CLDN18.2和人CLDN18.1的抗体。本实施例中得到了一株杂交瘤细胞7D10，其上清中的抗体与293T-18.2细胞结合，而与293T-18.1细胞几乎不结合。
杂交瘤细胞7D10亚克隆筛选及抗体纯化
将杂交瘤细胞7D10铺96孔板，并于HT培养基中培养7-10天，用FACS方法进一步筛选阳性克隆。选择阳性细胞进行扩大培养，收取上清，进行抗体(其重链可变区和轻链可变区的氨基酸序列分别如SEQ ID NO:7和SEQ ID NO:8所示)纯化，检测抗体浓度。
结果如表3所示，进一步筛选后得到了一株杂交瘤单克隆细胞株7D10-1，该克隆上清中的抗体能特异地结合293T-18.2细胞，并且与293T-18.1细胞几乎不结合。这意味着该杂交瘤细胞分泌的抗CLDN18.2的抗体是一种有前景的抗体。
表3杂交瘤细胞7D10亚克隆筛选
初始杂交瘤克隆号 293T-18.2细胞结合活性(FACS) 293T-18.1细胞结合活性(FACS)
7D10-1 1458 9.65
7D10-2 609 9.32
7D10-3 817 9.47
7D10-4 531 9.87
7D10-5 267 9.15
实施例3. 7D10-1杂交瘤分泌的抗CLDN18.2单克隆抗体和人CLDN18阳性细胞的结合活性
取含有293T-18.1和293T-18.2细胞的T75细胞培养瓶各一瓶，FACS缓冲液洗涤一次，用FACS缓冲液重悬计数，调整细胞浓度为2×10 6；稀释抗体(包括MAb362和抗CLDN18.2单克隆抗体两种抗体，其中MAb362作为对照)，具体为20μg/ml起始，3.16倍稀释，7个梯度。取10μL稀释后的各抗体分别加入上述调整浓度后的每种细胞样品(即293T-18.1和293T-18.2细胞)100μL，混匀，室温孵育1小时。
每孔加入400μl FACS缓冲液洗涤两次，加入PE抗小鼠IgG Fc二抗，孵育1小时，上流式细胞仪检测，结果见表4和图1。表4的结果表明，抗CLDN18.2单克隆抗体和对照抗体MAb362均与293T-18.2细胞特异性结合，并且抗CLDN18.2单克隆抗体的EC50比对照抗体MAb362强得多，约为对照抗体的三倍。
表4抗CLDN18.2单克隆抗体和人CLDN18阳性细胞的结合活性
Figure PCTCN2022073207-appb-000004
实施例4.抗CLDN18.2嵌合抗体和人CLDN18阳性细胞的结合活性(FACS检测)
抗CLDN18.2嵌合抗体的制备
获取7D10-1杂交瘤细胞cDNA，用本实验室保存的抗体引物通过PCR扩增测序，最终获得鼠源抗Claudin 18.2抗体编码VH1和VL1的核苷酸序列。其中抗Claudin 18.2抗体的VH1、VL1编码核酸序列通过全合成的方式构建成质粒pUC57 Claudin 18.2 VH1、pUC57 Claudin 18.2 VL1中(通用生物系统(安徽)有限公司)，分别以pUC57 Claudin 18.2 VH1、pUC57 Claudin 18.2VL1为模板，使用金牌Mix PCR试剂盒(TSINGKE公司)，按照试剂盒的说明书，扩增抗CLDN18.2 VH1、CLDN18.2 VL1片段，其扩增产物大小约为0.4kb；同时以限制性内切酶SapI(NEB,R0569S)对载体质粒pQKX1和pQKX2进行酶切，将所得的PCR扩增产物和酶切载体以BM无缝克隆试剂盒(博迈德公司)，按照试剂盒的说明书，进行重组连接以获得重链表达载体pQK CLDN18.2H1和轻链表达载体pQK CLDN18.2L1。
将293Fv细胞(中国医学科学院基础医学研究所细胞资源中心)稀释到1.5×10 6个细胞/mL，终体积为200ml，37℃摇床培养24h。用转染体积10％的新鲜培养基稀释质粒pQK CLDN18.2H1和pQK CLDN18.2L1，按转染细胞体积计算，至浓度为1μg/mL。向稀释后质粒中按细胞体积的1/1000加入3mg/mL的PEI 200μl，立即涡旋震荡10秒，室温放置15分钟，将质粒/PEI混合物滴加到细胞培养基中，边滴加边轻轻摇动培养瓶，放入摇床培养，7天后收集培养上清，0.4μm的滤膜过滤后，使用Mab sure Lx 5ml纯化柱从培养上清中捕获抗体，流速设定为3ml/min，用5个柱体积的20mM PB+150mM Nacl,PH7.4平衡液平衡纯化柱，柱平衡稳定后上样，上样结束选择20mM PB+150mM Nacl,PH7.4淋洗，淋洗结束使用50mM柠檬酸PH3.0洗脱液进行洗脱，收集用1M Tris-Hcl,PH9.0中和洗脱的抗体(其重链和轻链的氨基酸序列分别如SEQ ID NO:9和SEQ ID NO:10所示)。
收集一个T75细胞培养瓶的293T-18.2细胞，FACS缓冲液洗涤一 次，用FACS缓冲液重悬计数，调整细胞浓度为2×10 6；稀释抗体(包括MAb362和抗CLDN18.2嵌合抗体两种抗体，其中MAb362作为对照)，具体为20μg/ml起始，3.16倍稀释，7个梯度。取10μL稀释后的各抗体分别加入上述调整浓度后的细胞样品100μL，混匀，室温孵育1小时。
每孔加入400μl FACS缓冲液洗涤两次，加入PE抗人IgG Fc二抗(Biolegend，Cat409304)，孵育1小时，上流式细胞仪检测，结果见表5和图2。表5结果表明抗CLDN18.2嵌合抗体和对照抗体MAb362均与293T-18.2细胞特异性结合，并且抗CLDN18.2嵌合抗体的EC50比对照抗体MAb362强得多，约为对照抗体的十倍。
表5抗CLDN18.2嵌合抗体和人CLDN18.2阳性细胞结合活性
Figure PCTCN2022073207-appb-000005
实施例5.抗CLDN18.2人源化抗体和人CLDN18.2阳性细胞的结合活性(FACS检测)
抗CLDN18.2人源化抗体的制备
抗Claudin 18.2人源化抗体编码VH2和VL2核苷酸序列由本申请的发明人设计。其中抗Claudin 18.2人源化抗体编码VH2、VL2核苷酸序列通过全合成的方式构建成质粒pUC57 Claudin 18.2 VH2和pUC57 Claudin 18.2 VL2中(通用生物系统(安徽)有限公司)，分别以pUC57 Claudin 18.2 VH2和pUC57 Claudin 18.2 VL2为模板，使用金牌Mix PCR试剂盒(TSINGKE公司)，按照试剂盒的说明书，扩增抗CLDN18.2 VH2和CLDN18.2 VL2片段，其扩增产物大小约为0.4kb；同时以限制性内切酶SapI(NEB,R0569S)对载体质粒pQKX1和pQKX2进行酶切，将所得的PCR扩增产物和酶切载体以BM无缝克隆试剂盒(博迈德公司)，按照试剂盒的说明书，进行重组连接以 获得重链表达载体pQK CLDN18.2H2和pQK CLDN18.2L2。
将293Fv细胞稀释到1.5×10 6个细胞/mL，终体积为200ml，37℃摇床培养24h。用转染体积10％的新鲜培养基稀释质粒pQK CLDN18.2 H2和pQK CLDN18.2L2，按转染细胞体积计算，至浓度为1μg/mL。向稀释后质粒中按细胞体积的1/1000加入3mg/mL的PEI 200μl，立即涡旋震荡10秒，室温放置15分钟，将质粒/PEI混合物滴加到细胞培养基中，边滴加边轻轻摇动培养瓶，放入摇床培养，7天后收集培养上清，0.4μm的滤膜过滤后，使用Mab sure Lx 5ml纯化柱从培养上清中捕获抗体，流速设定为3ml/min，用5个柱体积的20mM PB+150mM Nacl,PH7.4平衡液平衡纯化柱，柱平衡稳定后上样，上样结束选择20mM PB+150mM Nacl,PH7.4淋洗，淋洗结束使用50mM柠檬酸PH3.0洗脱液进行洗脱，收集用1M Tris-Hcl,PH9.0中和洗脱的抗体(其重链可变区和轻链可变区的氨基酸序列分别如SEQ ID NO:11和SEQ ID NO:12所示)。
收集一个T75细胞培养瓶的293T-18.2细胞，FACS缓冲液洗涤一次，用FACS缓冲液重悬计数，调整细胞浓度为2×10 6；稀释抗体(包括MAb362和抗CLDN18.2人源化抗体两种抗体，其中MAb362作为对照)，具体为20μg/ml起始，3.16倍稀释，7个梯度。取10μL稀释后的各抗体分别加入上述调整浓度后的细胞样品100μL，混匀，室温孵育1小时。
每孔加入400μl FACS缓冲液洗涤两次，加入PE抗人IgG Fc二抗，孵育1小时，上流式细胞仪检测，结果见表6和图3。表6的结果表明抗CLDN18.2人源化抗体和对照抗体MAb362均与293T-CLDN18.2细胞特异性结合，并且抗CLDN18.2人源化抗体的EC50比对照抗体MAb362强得多，约为对照抗体的四倍。
表6抗CLDN18.2人源化抗体和人CLDN18.2阳性细胞结合活性
Figure PCTCN2022073207-appb-000006
Figure PCTCN2022073207-appb-000007
实施例6.抗CLDN18.2人源化抗体的活性
抗CLDN18.2人源化抗体与人胃癌细胞NUGC4的结合活性
收集一个T75细胞培养瓶的NUGC4细胞(中美冠科生物技术北京有限公司)，FACS缓冲液洗涤一次，用FACS缓冲液重悬计数，调整细胞浓度为2×10 6；稀释抗体，具体地，用FACS缓冲液将抗CLDN18.2人源化抗体从20μg/ml起始进行系列十倍稀释，共计3个梯度，每100μL上述调整浓度后的NUGC4细胞样品加入10μL抗体混匀，室温孵育1小时。
每孔加入400μl FACS缓冲液洗涤两次，加入PE抗人IgG Fc二抗，孵育1小时，上流式细胞仪检测。结果如图4所示。图4表明抗CLDN18.2人源化抗体与NUGC4细胞特异性结合。
抗CLDN18.2人源化抗体对人胃癌细胞NUGC4的ADCC活性
收集一瓶(T75)NUGC4细胞，调节密度为2×10 5个/ml。在实时细胞分析仪(RTCA)培养板的每孔中加入50μl含10％低IgG FBS的DMEM，上实时细胞分析仪检测孔板好坏并测定基线，取出后吸弃孔内培养基。加入NUGC4单细胞悬液，每孔100μl，待细胞沉降于板底后置于RTCA仪(置于细胞培养箱中)，孵育过夜并检测细胞生长情况。
采集人外周血，采用PBS1:1稀释，等体积平铺于15ml离心管中放置的4ml Ficoll-PagueTM plus上(密度1.077±0.001g/ml，GE公司，17-1440-03)。800g室温离心20min，待分离后吸取白膜层，获得PBMC，将PBMC用PBS清洗两遍后调节细胞密度为4×10 6个/ml。
待测抗体抗CLDN18.2人源化抗体及人IgG1同型对照抗体(Biolegend，Cat403502)均采用PBS倍比稀释，起始浓度为580μg/ml(4μM)，2.57倍倍比稀释，共6个浓度。
暂停RTCA仪运行，取出RTCA培养板，加入50μl PBMC及50μl 不同种类不同稀释度的抗体，平行孔2孔，同时设置靶细胞对照(Blank)及无抗体对照(PBMC)。静止板孔，待细胞沉入板底后上机持续检测，并计算得到抗CLDN18.2人源化抗体对人胃癌细胞NUGC4的ADCC的EC50为0.18mM(参见图5)，而IgG1同型对照抗体不会产生针对NUGC4的ADCC效应，结果无浓度依赖的梯度趋势。
可以理解，尽管本申请以上述具体形式描述了所涉及的发明，但这些发明并不局限于这些具体形式描述的特定内容。对本领域的技术人员显而易见的是，在不偏离本申请所描述的发明精神的前提下，还可对其中所涉及的发明包含的技术特征进行各种等同变化，这些变化都应该属于所述发明的范围之内。

Claims (12)

  1. 特异性结合CLDN18.2的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3序列中的任意一项或多项，其中所述HCDR1序列为GFSFSSFG(SEQ ID NO:1)，所述HCDR2序列为ISSGSRTI(SEQ ID NO:2)，所述HCDR3序列为TRYYYGNSFDY(SEQ ID NO:3)，优选地，所述抗原结合部分选自：Fab片段、Fab’片段、F(ab’) 2片段、片段、scFv片段、Fd片段或单域抗体。
  2. 如权利要求1所述的抗体或其抗原结合部分，其还包含轻链可变区，所述轻链可变区包含LCDR1、LCDR2和LCDR3序列中的任意一项或多项，其中所述LCDR1序列为QSLFNTGNQKNY(SEQ ID NO:4)，所述LCDR2序列为WAS(SEQ ID NO:5)，所述LCDR3序列为QNNYNFPLT(SEQ ID NO:6)。
  3. 如权利要求1或2所述的抗体或其抗原结合部分，其中所述抗体为鼠源抗体，任选地，所述重链可变区的氨基酸序列如SEQ ID NO:7所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示，任选地，其中所述抗体为嵌合抗体，任选地，所述嵌合抗体包含氨基酸序列如SEQ ID NO:9所示的重链，和/或氨基酸序列如SEQ ID NO:10所示的轻链，任选地，其中所述抗体为人源化抗体，任选地，所述重链可变区的氨基酸序列如SEQ ID NO:11所示，和/或所述轻链可变区的氨基酸序列如SEQ ID NO:12所示。
  4. 如权利要求1或2所述的所述的抗体或其抗原结合部分，其中所述CLDN18.2为灵长类CLDN18.2；优选地，所述灵长类CLDN18.2选自人CLDN18.2或猴CLDN18.2。
  5. 药物组合物，其包含权利要求1-4中任一项所述的抗体或其抗原结合部分以及药学上可接受的载体，
    任选地，所述药物组合物还包含一种或多种其他活性成分，如治疗肿瘤的药剂。
  6. 核酸分子，其编码权利要求1-4中任一项所述的抗体或其抗原 结合部分。
  7. 表达载体，其包含权利要求6所述的核酸分子。
  8. 宿主细胞，其包含权利要求6所述的核酸分子或权利要求7所述的表达载体。
  9. 权利要求1-4中任一项所述的抗体或其抗原结合部分、权利要求5所述的药物组合物、权利要求6所述的核酸分子，权利要求7所述的表达载体、或者权利要求8所述的宿主细胞在制备用于预防和/或治疗CLDN18.2相关疾病的药物中的用途，任选地，所述CLDN18.2相关疾病为肿瘤，任选地，所述肿瘤选自以下中的一种或多种：胃癌、胰腺癌、肠癌、食管癌、肝癌、卵巢癌、肺癌和膀胱癌以及上述的转移瘤。
  10. 检测试剂或试剂盒，其包含权利要求1-4中任一项所述的抗体或其抗原结合部分。
  11. 预防和/或治疗CLDN18.2相关疾病的方法，其包括向有需要的个体施用治疗有效量的权利要求1-4中任一项所述的抗体或其抗原结合部分、权利要求5所述的药物组合物、权利要求6所述的核酸分子，权利要求7所述的表达载体、或者权利要求8所述的宿主细胞，任选地，所述CLDN18.2相关疾病为肿瘤，任选地，所述肿瘤选自以下中的一种或多种：胃癌、胰腺癌、肠癌、食管癌、肝癌、卵巢癌、肺癌和膀胱癌以及上述的转移瘤。
  12. 权利要求1-4中任一项所述的抗体或其抗原结合部分、权利要求5所述的药物组合物、权利要求6所述的核酸分子，权利要求7所述的表达载体、或者权利要求8所述的宿主细胞用于预防和/或治疗CLDN18.2相关疾病的用途，任选地，所述CLDN18.2相关疾病为肿瘤，任选地，所述肿瘤选自以下中的一种或多种：胃癌、胰腺癌、肠癌、食管癌、肝癌、卵巢癌、肺癌和膀胱癌以及上述的转移瘤。
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