WO2021136550A1 - 抗Claudin18.2的抗体及其应用 - Google Patents

抗Claudin18.2的抗体及其应用 Download PDF

Info

Publication number
WO2021136550A1
WO2021136550A1 PCT/CN2021/070139 CN2021070139W WO2021136550A1 WO 2021136550 A1 WO2021136550 A1 WO 2021136550A1 CN 2021070139 W CN2021070139 W CN 2021070139W WO 2021136550 A1 WO2021136550 A1 WO 2021136550A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
cells
claudin
Prior art date
Application number
PCT/CN2021/070139
Other languages
English (en)
French (fr)
Inventor
李宗海
王鹏
刘振
王华茂
Original Assignee
佧珐药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 佧珐药业有限公司 filed Critical 佧珐药业有限公司
Priority to KR1020227026732A priority Critical patent/KR20220121873A/ko
Priority to EP21736163.3A priority patent/EP4086287A4/en
Priority to AU2021204861A priority patent/AU2021204861B2/en
Priority to CA3166498A priority patent/CA3166498A1/en
Priority to CN202211674726.4A priority patent/CN117343186A/zh
Priority to US17/790,657 priority patent/US20230061005A1/en
Priority to JP2022540843A priority patent/JP7543411B2/ja
Priority to CN202180001577.2A priority patent/CN113272332B/zh
Publication of WO2021136550A1 publication Critical patent/WO2021136550A1/zh

Links

Images

Classifications

    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • 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
    • 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
    • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
    • 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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of immunology. More specifically, the present invention relates to an anti-Claudin 18.2 antibody and its application.
  • Claudin 18 (Claudin 18, CLD18) is an internal membrane protein located in the tight junction of epithelium and endothelium, with a molecular weight of about 27.9KD.
  • GenBank registration number is splice variant 1 (called CLD18A1, CLD18.1 or Claudin 18.1): NP_057453, NM016369, and splice variant 2 (called CLD18A2, CLD18.2 or Claudin 18.2): NM_001002026, NP_001002026.
  • CLD18A1 is selectively expressed in normal lungs, while CLD18A2 is expressed in normal stomachs, and its expression is limited to differentiated short-lived gastric epithelial cells.
  • CLD18A2 is strongly expressed in a variety of tumor cells. It has been found that CLAUDIN 18.2 is used in gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, breast cancer, head and neck cancer, and biliary system tumors (such as gallbladder cancer, Cholangiocarcinoma) and its metastatic cancers, gastric cancer metastasis such as Kukenberg tumor, peritoneal metastasis and lymph node metastasis.
  • NSCLC non-small cell lung cancer
  • biliary system tumors such as gallbladder cancer, Cholangiocarcinoma
  • gastric cancer metastasis such as Kukenberg tumor, peritoneal metastasis and lymph node metastasis.
  • CLDN18 is a four-pass transmembrane protein with two extracellular segments called loop 1 and loop 2.
  • Human CLDN18A2 (SEQ ID NO: 2) and human CLDN18A1 (SEQ ID NO: 1) are highly homologous. The homology comparison result between the two is shown in Figure 1. Only in the first transmembrane (TM) region of the N-terminal There are some differences from loop 1, while the rest of the amino acid sequence is the same.
  • the C-terminal polypeptide of CLDN18A2 is used to immunize mice.
  • sequence of the peptide is identical in the two splice variants, we have obtained multiple antibodies with obvious tissue specificity, which can be used for CLDN18A2 positive tumors. clinical diagnosis.
  • the present invention provides an antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment can recognize an antigen peptide comprising the sequence shown in SEQ ID NO: 7 (KNKKIYDGG); or the antibody Or the antigen-binding fragment can recognize the epitope in claudin 18.2 containing the sequence shown in SEQ ID NO: 7.
  • the antibody or antigen-binding fragment can recognize the peptide containing the sequence shown in SEQ ID NO: 6 (KNKKIYDGGART); or the antibody or antigen-binding fragment can recognize the peptide containing SEQ ID NO in claudin 18.2 : Epitope of the sequence shown in 6.
  • the antibody or antigen-binding fragment can recognize the antigen peptide comprising the sequence shown in SEQ ID NO: 8 (GFGSNTKNKKIYDGG), 9 (SNTKNKKIYDGGART) or 10 (KNKKIYDGGARTEDE); or the antibody or antigen binding The fragment can recognize the epitope in claudin 18.2 that contains the sequence shown in SEQ ID NO: 8, 9 or 10;
  • the antibody or antigen-binding fragment can recognize the antigen peptide containing the sequence shown in SEQ ID NO: 9 or 10; or the antibody or antigen-binding fragment can recognize the claudin 18.2 containing SEQ ID NO: 9 or 10 The epitope of the sequence shown.
  • the antibody is an antibody produced by a deposited hybridoma cell line with a registration number of CCTCCNO.C202007 or CCTCCNO.C202008, wherein the antibody produced by the CCTCCNO.C202007 hybridoma cell line is antibody 9, CCTCCNO.
  • the antibody produced by the C202008 hybridoma cell line is antibody 7.
  • the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antibody or antigen-binding fragment is a humanized antibody or an antibody produced by a deposited hybridoma cell line with a registration number of CCTCCNO.C202007 (antibody 9) or CCTCCNO.C202008 (antibody 7). Chimeric antibody.
  • the antibody or antigen-binding fragment is a mutant of an antibody produced by a deposited hybridoma cell line with a registration number of CCTCCNO.C202007 or CCTCCNO.C202008; wherein the CCTCCNO.C202007 hybridoma cell line is produced
  • the antibody is antibody 9, and the antibody produced by the CCTCCNO.C202008 hybridoma cell line is antibody 7.
  • the mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, and the antibody produced by the deposited hybridoma cell line with the registration number CCTCCNO.C202007 or CCTCCNO.C202008. %, 95%, 96%, 97%, 98%, or 99% homology.
  • the antibody fragment is an antibody fragment of an antibody produced by a deposited hybridoma cell line with a registration number of CCTCCNO.C202007 or CCTCCNO.C202008;
  • the antibody fragments are scFv, Fab fragments, Fd fragments, Fv fragments, F(ab')2 fragments, or single domain antibodies,
  • the antibody produced by the CCTCCNO.C202007 hybridoma cell line is antibody 9
  • the antibody produced by the CCTCCNO.C202008 hybridoma cell line is antibody 7.
  • the antibody or antigen-binding fragment binds to cells expressing claudin 18.2; preferably, the amino acid sequence of claudin 18.2 is shown in SEQ ID NO: 2, and SEQ ID NO: 2 is :
  • the antibody or antigen-binding fragment specifically binds to gastric tissue
  • the antibody or antigen-binding fragment specifically binds to stomach tissue, but not to lung tissue.
  • conjugate characterized in that the conjugate comprises the antibody or antigen-binding fragment according to any one of claims 1-9, and at least one detectable label;
  • the detectable label is a fluorescent label, a chromogenic label, a luminescent label, a chromophore label, a radioisotope label, an isotope label, an isobaric label (isobaric label) , Enzyme markers, particle markers, nucleic acids or proteins detected by the use of binding agents.
  • the deposit registration number of the hybridoma is CCTCCNO.C202007 or CCTCCNO.C202008.
  • a method for detecting the expression of claudin 18.2 in a test sample characterized in that the method comprises: binding the test sample to any one of the above-mentioned antibodies or antigens of the present invention Fragment, or contact with the above-mentioned conjugate of the present invention; detecting the condition of the antibody or antigen-binding fragment, or the complex formed by the conjugate and claudin 18.2.
  • a method for diagnosing, detecting or monitoring cancer characterized in that the method comprises:
  • the sample to be tested is contacted with the antibody or antigen-binding fragment of the present invention, or the conjugate of the present invention; detecting the antibody or antigen-binding fragment, or the conjugate and claudin 18.2 The amount of complex formed.
  • a method for analyzing whether a tumor can be treated by tumor therapy targeting claudin 18.2 characterized in that the method comprises:
  • the tumor cells are gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancer cells, or biliary system tumor cells (such as gallbladder cancer, cholangiocarcinoma cells) );
  • the tumor cells are gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, or biliary system tumor cells.
  • a detection kit which comprises the antibody or antigen-binding fragment of the present invention, or the conjugate of the present invention.
  • claudin 18.2 comprises the amino acid sequence of SEQ ID NO: 2 in the sequence listing or a variant thereof.
  • a method for producing a monoclonal antibody targeting claudin 18.2 characterized in that the method comprises using a compound containing SEQ ID NO: 6, 7, 8, 9 or 10.
  • the polypeptide of the sequence is an immunogen, or used with SEQ ID NO: 6, 7, 8, 9 or 10 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, Polypeptides with 95%, 96%, 97%, 98%, or 99% homology are used as immunogens.
  • the animal’s B cells are fused with myeloma cells to obtain hybridoma cells to produce single Cloned antibody
  • the animal is a mouse
  • the B cells are derived from spleen cells.
  • the method further includes isolating the monoclonal antibody from the hybridoma cells.
  • polypeptide shown in SEQ ID NO: 3 is used as the immunogen.
  • the monoclonal antibody specifically binds to the antigen.
  • it also includes producing a modified form of the monoclonal antibody that retains specific binding to the antigen.
  • the modified form of the monoclonal antibody is a humanized antibody.
  • Figure 1 The sequence homology comparison result of Claudin 18.2 and Claudin 18.1;
  • Figure 2A Western Blot identification result of antibody binding to 293T-Claudin 18.1 cell lysate
  • Figure 2B Western Blot identification result of antibody binding to 293T-Claudin 18.2 cell lysate
  • Figure 3 Staining results of antibody 7 and antibody 9 on gastric cancer sample F163060A1;
  • Figure 4 The staining results of antibody 7 and antibody 9 on gastric cancer sample 2018-50829E;
  • Figure 5 Staining results of antibody 7 and antibody 9 on lung cancer sample 2018-55164F;
  • Figure 6 Shows the results of epitope ELISA analysis.
  • the term "about” means to include ⁇ 20% of the specified value, or in some cases ⁇ 10%, or in some cases ⁇ 5%, or ⁇ 1% in some cases, or ⁇ 0.1% in some cases.
  • antibody herein includes intact antibodies and antigen-binding fragments.
  • a naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected between chains by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • the VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDR), separated by more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • Antibodies can be from different species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.
  • Antibodies also include chimeric antibodies, having an antibody constant region from one species (preferably human) and an antigen binding site from another species. Antibodies also include humanized antibodies that have the antigen binding sites of antibodies from non-human species and constant regions and framework regions of human origin.
  • the antibody described herein may be a monoclonal antibody, that is, a preparation of antibody molecules having a single molecular composition.
  • the system for preparing hybridomas that secrete monoclonal antibodies is preferably a mouse system. Immune procedures and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion protocols for mouse myeloma cells and mouse spleen cells are also known.
  • hybridomas that secrete monoclonal antibodies are preferably rat and rabbit systems (for example, as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995).
  • the antibodies described herein can be Recombinant antibodies, that is, all antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) whose immunoglobulin genes are transgenic or transchromosomal, or hybridomas prepared therefrom , (B) antibodies isolated from host cells transformed to express the antibodies (e.g., transfectionomas), (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) by involving immunoglobulin gene sequences Antibodies prepared, expressed, produced or isolated by any other means of splicing into other DNA sequences. Antibodies can be produced by a variety of techniques, such as the hybridoma technology of Kohler et al., Nature 256:495 (1975), and other monoclonal production methods. Antibody technology, such as phage display technology.
  • the nucleotide sequences from the heavy and light chain transcripts of hybridomas are used to design overlapping sets of synthetic oligonucleotides to produce synthetic V sequences that have the same amino acid encoding capabilities as the natural sequences.
  • the synthetic heavy chain and kappa chain sequences can be different from the natural sequence in three ways: the repeated nucleotide base string is interrupted to facilitate oligonucleotide synthesis and PCR amplification; and the optimal translation is incorporated according to Kozak rules The start site (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and the engineered HindIII site upstream of the translation start site.
  • the sequences of the optimized coding and corresponding non-coding strands are broken into 30-50 nucleotides at about the midpoint of the corresponding non-coding oligonucleotide. Therefore, for each strand, the oligonucleotides can be assembled into overlapping double-stranded groups spanning a segment of 150-400 nucleotides. This library is then used as a template to generate 150-400 nucleotide PCR amplification products. Usually a single variable region oligonucleotide component is divided into two pools, and they are each amplified to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to form a complete variable region.
  • heavy chain and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells, and then fused to form a host cell that expresses the two chains.
  • the construction of the human IgG ⁇ expression vector plasmid allows the PCR amplified V heavy chain and V ⁇ light chain cDNA sequences to be used to reconstruct complete heavy chain and light chain minigenes. These plasmids can be used to express fully human antibodies or chimeric IgG1 ⁇ or IgG4 ⁇ antibodies. Similar plasmids can be constructed to express other heavy chain isotypes, or to express antibodies containing lambda light chains.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain specific antigen-binding ability.
  • the binding fragments covered include (i) Fab fragments, monovalents composed of VL, VH, CL and CH domains. Fragment; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region; (iii) Fd fragment composed of VH and CH domains; (iv) Fv fragments composed of the VL and VH domains of one arm of the antibody; (v) single domain antibody fragments composed of the VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) optionally A combination of two or more isolated CDRs connected by a synthetic linker. It also includes the use of recombinant methods to connect the two domains VL and VH of the Fv fragment through a synthetic linker to form a single-chain antibody (or called a single-chain Fv (sc
  • binding of the antibody or antigen-binding fragment of the present invention to the target antigen Claudin 18.2 can be characterized or expressed by any method known in the art, for example, the binding can be characterized by binding affinity. Binding affinity can be determined by any method known in the art, such as ELISA, Western blot, immunofluorescence, and flow cytometry analysis. Flow cytometry can prove the presence of antibodies in the serum of immunized mice or prove that monoclonal antibodies bind to living cells.
  • the cells transiently transfected with the plasmid encoding the antigen and the fluorescent marker were stained as described above.
  • the transfected cells can be detected in a different fluorescence channel from the antibody-stained cells. Since most transfected cells express two transgenes at the same time, antigen-specific monoclonal antibodies preferentially bind to cells expressing fluorescent markers, and non-specific antibodies bind to untransfected cells in a comparable ratio.
  • the cells can be stained exactly as described above, or they can be examined by fluorescence microscopy.
  • the binding affinity of the antigen-binding portion disclosed herein can be determined.
  • the binding affinity of the test antigen binding part can be expressed by Kd, which is the equilibrium dissociation constant between the antibody and its respective antigen.
  • human monoclonal antibodies directed against CLAUDIN 18.2 can be produced using transgenic or transchromosomal mice that carry parts of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include small mice called HuMAb, respectively. Mice and KM mice. In some embodiments, the mouse is immunized with a vector-conjugated peptide from the CLAUDIN18.2 sequence, recombinantly expressed CLAUDIN18.2 antigen or enriched preparations thereof, and/or cells expressing CLAUDIN18.2 or fragments thereof . Mice can also be immunized with DNA encoding full-length human CLAUDIN 18.2 or fragments thereof.
  • mice When a purified or enriched CLAUDIN18.2 antigen preparation is used for immunization without producing antibodies, mice can be immunized with CLAUDIN18.2-expressing cells (e.g., cell lines) to promote immune response. Plasma and serum samples obtained from tail vein or retroorbital blood collection can be used to monitor the immune response during the entire immunization process. Anti-CLAUDIN18.2 immunoglobulin mice with sufficient titer can be used for fusion. The immunization can be boosted by intraperitoneal or intravenous immunization with CLAUDIN18.2-expressing cells 3 to 5 days before the mouse is sacrificed and the spleen removed to increase the rate of hybridomas that secrete specific antibodies.
  • CLAUDIN18.2-expressing cells e.g., cell lines
  • Plasma and serum samples obtained from tail vein or retroorbital blood collection can be used to monitor the immune response during the entire immunization process.
  • Anti-CLAUDIN18.2 immunoglobulin mice with sufficient titer can be
  • cells in order to prepare CLAUDIN18.2 monoclonal antibody hybridomas, cells can be isolated from lymph nodes, spleen or bone marrow of immunized mice, and combined with an appropriate immortal cell line (such as a mouse myeloma cell line) Fusion, and then screening for specific antibodies against the antigen to obtain hybridomas. Then, antibody-secreting hybridomas were screened by ELISA. By immunofluorescence or FACS analysis, antibodies that specifically bind CLAUDIN 18.2 were identified. The hybridoma that secretes the antibody can be re-plated and screened again. If the anti-CLAUDIN18.2 monoclonal antibody is still positive, it can be subcloned by limiting dilution.
  • an appropriate immortal cell line such as a mouse myeloma cell line
  • the stable subclones were cultured in vitro to generate antibodies in tissue culture medium for characterization.
  • the antibody is produced in a host cell transfected tumor, for example, using a combination of recombinant DNA technology and gene transfection methods known in the art (Morrison, S. (1985) Science 229: 1202).
  • the gene of interest (such as an antibody gene) can be ligated into an expression vector (such as a eukaryotic expression plasmid).
  • the purified plasmid carrying the cloned antibody gene can be introduced into eukaryotic host cells, such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells, or other eukaryotic cells, such as cells from plants, fungi or yeast cells.
  • the method for introducing these genes may be methods that have been described in the art, such as electroporation, lipofectine, lipofectamine or others. After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected.
  • Antibody genes can also be expressed in other expression systems, including prokaryotic cells such as microorganisms, such as E. coli.
  • antibodies can also be produced in non-human transgenic animals, such as in the milk of sheep and rabbits or in eggs, or in transgenic plants, see, for example, Verma, R., et al. (1998) J. Immunol. Meth .216:165-181; Pollock, et al. (1999) J. Immunol. Meth. 231:147-157 and Fischer, R., et al. (1999) Biol. Chem. 380:825-839.
  • Antigen refers to a substance that contains an epitope and can elicit and/or produce an immune response against the epitope.
  • Antigens may include peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • epitope refers to an antigenic determinant in a molecule, that is, a part of a molecule that is recognized by the immune system (eg, recognized by an antibody).
  • Claudins are a protein family of the most important components in tight junctions. In tight junctions, claudins constitute a paracellular barrier and control the flow of molecules in the intercellular spaces between epithelial cells. Claudin is a transmembrane protein that spans four times, and its N-terminus and C-terminus are both located in the cytoplasm.
  • the term "claudin 18" or “CLDN18” or “Claudin 18” or “CLD18” refers to human CLDN18 and includes any splice variants, such as Claudin 18.1 and Claudin 18.2 of CLDN18.
  • Claudin 18.1 is a peptide with GenBank accession number NP_057453 (mRNA: NM_016369). In some embodiments, the Claudin 18.1 comprises SEQ ID NO:
  • the Claudin 18.2 is a peptide with GenBank accession number NP_001002026 (mRNA: NM_001002026).
  • the claudin18.2 comprising SEQ ID NO: Peptide (MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSM EDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV) the amino acid sequence of the amino acid or protein / peptide sequence variants.
  • variant refers to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, especially naturally occurring variants .
  • Allelic variants involve changes in the normal sequence of genes, and their significance is usually not obvious.
  • Whole gene sequencing usually identifies a large number of allelic variants of a given gene.
  • Interspecies homologs are nucleic acid or amino acid sequences that have a different species origin from a given nucleic acid or amino acid sequence.
  • CLDN "CLDN18”, “Claudin 18.1” and “Claudin 18.2” shall cover any post-translationally modified variants and conformational variants.
  • Claudin 18.1 and Claudin 18.2 differ in the first transmembrane (TM) region and the N-terminal part of loop 1, while the C-terminal protein has the same primary sequence.
  • CLAUDIN18.2 is selectively expressed in normal gastric mucosal epithelial cells. CLAUDIN18.2 expression is limited to differentiated gastric epithelial short-lived cells, and it is expressed in a variety of tumor cells.
  • the primary tumor refers to gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, such as non-small cell lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and biliary system cancer.
  • the cells expressing CLAUDIN 18.2 refer to tumor cells, and are particularly selected from gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancer cells , And cancer cells of the biliary system.
  • a cell expressing CLAUDIN 18.2 refers to a cell in which CLAUDIN 18.2 is located or bound to the cell membrane.
  • Immunohistochemistry is a method for detecting antigens (eg, proteins) in cells of tissue sections, and is widely used to detect abnormal cells, such as tumor cells.
  • the detection antibody used can bind to an enzyme, such as peroxidase that can catalyze a reaction that produces color.
  • the detection antibody can also be labeled with a fluorescent group, such as fluorescein or rhodamine.
  • the expression of CLAUDIN 18.2 in cells is determined by immunohistochemistry.
  • a thin (approximately 4 to 40 ⁇ m) slice can be cut from the fixed tissue, or if the tissue is not very thick and penetrable, it can be used as a whole. It is usually sliced by using a microtome, and the slice is mounted on a slide. Samples may require optimization, including deparaffinization and antigen retrieval. Usually, detergents (such as Triton X-100) are used in immunohistochemistry to reduce surface tension, and related reagents can cover the sample better and more uniformly. Immunohistochemical staining can use a labeled antibody that binds directly to the stained antigen.
  • the more commonly used immunohistochemical staining is an indirect method, using antibodies directed against the antigen to be detected and a second labeled antibody directed against the first antibody.
  • a blocking buffer for example, normal serum, non-fat milk powder, BSA or gelatin, and a commercially available blocking buffer
  • the first antibody is directed against the target antigen and is usually unlabeled
  • the second antibody is directed against the immunoglobulin of the first antibody.
  • the second antibody usually binds to a linker molecule (e.g., biotin), which then recruits the reporter molecule, or the second antibody directly binds to the reporter molecule itself.
  • the most commonly used detection methods are enzyme-mediated color detection and fluorophore-mediated fluorescence detection.
  • the reaction of the enzyme label with the substrate produces a strongly colored product that can be analyzed by ordinary optical microscopy.
  • Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two most widely used as protein detection markers.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • a variety of color substrates, fluorescent substrates, and chemiluminescent substrates can be used with enzymes, including DAB or BCIP/NBT.
  • Fluorescent reporter molecules are small organic molecules used for IHC detection.
  • the optical density analysis of the signal can provide semi-quantitative and full-quantitative data, respectively, and correlate the signal level of the reporter molecule with the protein expression level or localization level.
  • a second stain for example, hematoxylin, Hoechst stain, and DAPI
  • hematoxylin, Hoechst stain, and DAPI is usually performed to provide a contrast to help highlight the first stain.
  • the present invention provides a method for detecting the presence of CLAUDIN18.2 antigen in a sample or measuring the amount of CLAUDIN18.2 antigen in a sample.
  • the sample or control sample
  • the sample is combined with CLAUDIN18 .2 Contact with the antigen of the present invention.
  • the formation of the complex is detected, and the difference between the complex formed in the sample and the complex formed in the control substance is compared to determine whether the CLAUDIN 18.2 antigen is present in the sample.
  • the detection method of the present invention can be particularly used to diagnose CLAUDIN 18.2 related diseases, such as tumors.
  • the amount of CLAUDIN18.2 antigen in the sample is higher than the amount of CLAUDIN18.2 antigen in the reference or control sample, indicating that the individual (especially human) from which the sample is derived has CLAUDIN18.2 related diseases, such as tumors.
  • the measurement sample is a cell sample, such as a sample containing cells (such as tumor cells). The complex is formed between the antibody, antigen-binding fragment or conjugate of the present invention and CLAUDIN 18.2 expressed by the cells in the above sample.
  • the "reference” (for example, a reference sample or a reference organism) can be used to correlate and compare the results from the test sample or the test organism obtained by the method of the present invention.
  • the reference organism is generally a healthy organism, especially an organism that does not suffer from a disease (e.g., a tumor).
  • the "reference value” or “reference level” can be empirically determined based on the reference object by measuring a sufficiently large number of reference objects. Preferably, by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50 or preferably at least 100 reference objects To determine the reference value.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including genomic DNA, cDNA, mRNA, recombinantly prepared or chemically synthesized molecules. Nucleic acids can be in the form of single-stranded or double-stranded molecules, and can also be linear or covalently closed into a ring.
  • RNA includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and by adding, deleting, replacing and/or changing one or more A modified RNA that differs from natural RNA by multiple nucleotides.
  • RNA can be modified by stabilizing sequences, capping, and polyadenylation.
  • transcription refers to the process in which the genetic code in a DNA sequence is transcribed into RNA.
  • Nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Therefore, the nucleic acid sequence encodes an amino acid sequence.
  • homologous means that the nucleic acids are functionally naturally linked, while the term “heterologous” means that the nucleic acids are not functionally linked naturally. In some embodiments, the nucleic acid can be functionally linked to a homologous or heterologous expression control sequence.
  • RNA includes the production of RNA or RNA and protein/peptide.
  • expression specifically refers to the production of peptides or proteins.
  • Expression can be transient or stable.
  • Expression also includes abnormal expression or abnormal expression, that is, compared with the reference (for example, individuals who do not suffer from diseases related to abnormal expression or abnormal expression of certain proteins (for example, tumor-associated antigen)), expression changes (for example, Elevated).
  • An increase in expression refers to an increase of at least 10%, especially at least 20%, at least 50% or at least 100% or more.
  • expression is only found in diseased tissues, while expression in healthy tissues is suppressed.
  • Specific expression means that the protein is basically only expressed in specific tissues or organs.
  • a tumor-associated antigen specifically expressed in the gastric mucosa means that the protein is mainly expressed in the gastric mucosa but not expressed in other tissues or not significantly expressed in other tissues or organ types.
  • the tumor-associated antigen is specifically expressed in more than one normal tissue type or organ (for example, in no more than 3 different tissues or organ types), then it can be considered that the tumor-associated antigen is in these organs.
  • Medium specific expression for example, in no more than 3 different tissues or organ types
  • isolated refers to separation from cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are usually associated in their natural state. Non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be “isolated” to distinguish them from their naturally occurring counterparts.
  • Isolated nucleic acid means that the nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced by cloning and recombination, (iii) purified, for example by cutting and gelling Fractional purified by electrophoresis, or (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid that can be manipulated by recombinant DNA technology.
  • An isolated protein or an isolated peptide refers to a protein or peptide that is separated from its natural environment and does not basically contain other substances that are combined with it in nature or in the body.
  • concentrate in addition, "concentrated”, “isolated” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof can be distinguished from their naturally occurring counterparts because the concentration or number of molecules per volume is greater than (“concentrated” ") or less (“diluted") the concentration of its naturally occurring counterpart.
  • the technical solution of the present invention preferably has a higher degree of enrichment. Therefore, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, and 1000-fold enrichment is more preferred. It can also be artificially assembled, such as by chemical synthesis or recombinant expression, to provide "isolated" substances.
  • allelic variants refer to changes in the normal sequence of genes, whose significance is usually not obvious.
  • Whole-gene sequencing usually identifies a large number of allelic variants of a given gene.
  • Interspecies homologs refer to designated nucleic acid or amino acid sequences derived from different species.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions of specific amino acid sequences, resulting in a sequence functionally equivalent to the specific sequence.
  • the antibody after amino acid modification still binds to its target antigen. Therefore, one or more amino acid residues in the CDR region or framework region of the antibody of the present invention can be replaced with other amino acid residues of similar side chains.
  • the sequence of the CDR sequence, hypervariable region and variable region of the antibody is modified without losing the ability to bind the target antigen.
  • N-terminus and/or C-terminus truncated variants are called N-terminus and/or C-terminus truncated variants.
  • Amino acid addition variants comprise one or more amino acids fused to the amino and/or carboxy terminus, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.
  • Amino acid deletion variants remove one or more amino acids from the sequence (e.g., remove 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids).
  • An amino acid substitution variant is that at least one amino acid residue in the sequence is removed and another amino acid residue is inserted in its position.
  • non-conserved amino acid residues between homologous proteins or peptides are modified and/or replaced with amino acid residues of similar characteristics.
  • the similarity identity between the amino acid sequence and the amino acid sequence of its amino acid sequence variants is preferably at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the amino acid region targeted for similarity or identity is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about the full length of the reference amino acid sequence. 70%, at least about 80%, at least about 90%, or about 100%.
  • the reference amino acid sequence consists of 200 amino acids, it is preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180.
  • about 200 amino acids give similarity or identity, preferably consecutive amino acids.
  • similarity or identity is given for the full-length reference amino acid sequence.
  • the tools known in the art can be used to perform the alignment for determining sequence similarity (preferably sequence identity), and the best sequence alignment is preferably used, such as Align, using standard settings, preferably EMBOSS: needle; Matrix: Blosum62; Gap Open 10.0; Gap Extend 0.5.
  • Sequence similarity refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions.
  • the identity of two amino acid sequences refers to the percentage of identical amino acids between the two sequences. This percentage is completely statistically significant, and the differences between the two sequences are randomly distributed over their entire length.
  • Sequence alignment can be done in the following ways: Smith and Waterman, 1981, Ads App. Math. 2, 482 local homology algorithm, Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443 local homology algorithm , Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 similarity search method or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA, Genetics Computer Group, 575ScienceDrive, Madison, Wis.).
  • peptide includes oligopeptides and polypeptides, and refers to two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, covalently linked by peptide bonds, Preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50 (especially 100) amino acids.
  • protein refers to a large peptide, preferably a peptide having more than 100 amino acid residues. In general, the terms “peptide” and “protein” are synonymous and can be used interchangeably herein.
  • “Derivatives” of proteins and peptides are modified forms of proteins and peptides. Such modifications include any chemical modification and include single or multiple substitutions, deletions, and/or additions of any molecules related to proteins or peptides, such as carbohydrates, lipids, and/or proteins or peptides.
  • “Derivatives” of proteins or peptides include glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, prenylation, lipidation, alkylation, derivatization, and introduction of protection/blocking Modified analogs and all functional chemical equivalents of groups, proteolytic cleavage or binding to antibodies or other cellular ligands. In some embodiments, the stability and/or immunogenicity of the modified peptide is improved.
  • cell refers to a cell with a complete membrane that has not yet released its normal intracellular components (for example, enzymes, organelles or genetic material), preferably a viable cell, that is capable of performing its normal metabolic function
  • the living cell living cell.
  • prokaryotic cells for example, E. coli
  • eukaryotic cells for example, dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells.
  • they are mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, and primates.
  • the cells can be derived from a large number of tissue types and include primary cells and cell lines.
  • the cells include non-cancerous cells and cancer cells.
  • Target cells refer to cells targeted for immune response, such as tumor cells described in this patent.
  • the target cell is a cell expressing CLAUDIN 18.2.
  • cells expressing CLAUDIN 18.2 generally include cancer cells.
  • the reagents, compositions and methods in this patent can be used to diagnose whether an individual is ill.
  • the diseases that can be diagnosed include all diseases that express CLAUDIN 18.2, and preferably, refer to tumors, such as gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and biliary system cancer.
  • disease refers to any pathological condition, including tumors, particularly those types of tumors described herein.
  • normal tissue refers to a healthy tissue or a condition in a healthy individual, that is, a non-pathological condition, where "healthy” preferably means non-tumorous.
  • CLAUDIN 18.2-related diseases refers to the increased expression of CLAUDIN 18.2 in cells in tissues or organs after a disease compared with healthy tissues or organs.
  • the increase refers to an increase of at least 10%, especially at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even higher.
  • CLAUDIN 18.2 related diseases include tumors, especially those types of tumors described herein.
  • decrease refers to a decrease in the expression of CLAUDIN 18.2 of the cell at the time of the previous test, by at least 5% or higher, 10% or higher, 20% or higher, more preferably 50% or higher. And 75% or higher capacity is most preferred.
  • inhibitor refers to complete or substantially complete inhibition, that is, reduction to zero or substantially reduction to zero.
  • cancer and “tumor” are interchangeable and refer to diseases in which cells exhibit uncontrolled growth, and/or invasion, and/or metastasis.
  • diseases such as leukemia, melanoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, bowel cancer , Liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and Lung cancer, and its metastasis.
  • ENT nose and throat
  • metastasis refers to the spread of cancer cells from their original site to other parts of the body.
  • the formation of metastasis is a very complex process and depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter the body cavity and blood vessels, and subsequent blood transport to infiltrate the target organ.
  • new tumors ie, secondary tumors or metastatic tumors
  • angiogenesis Tumor metastasis often occurs even after removal of the primary tumor, because tumor cells or components may remain and develop metastatic potential.
  • metastasis refers to distant metastasis, that is, metastasis away from the primary tumor and/or local lymph node system.
  • the cells of the secondary or metastatic tumor are similar to the cells of the original tumor.
  • recurrence refers to when an individual is again affected by a past condition. For example, if an individual has suffered from a disease in the past and has been cured by treatment, and the disease recurs, the new disease can be considered as a recurrence or recurrence.
  • the recurrence or recurrence of a tumor includes cases where the tumor occurs at a site different from the original tumor and at the site of the original tumor.
  • treatment refers to the administration of a compound or composition to an individual to prevent or eliminate a disease, including reducing the size or number of the disease in the individual; blocking or slowing the progression of the disease in the individual; inhibiting or slowing the development of new diseases in the individual; Reduce the frequency or severity of symptoms and/or recurrences in individuals who currently or have previously suffered from the disease; and/or extend (ie, increase) the life span of the individual.
  • the treatment of the disease includes the cure of the disease or its symptoms, shortening the duration, alleviating, preventing, slowing or inhibiting the progression or deterioration, or preventing or delaying the onset.
  • the terms "subject”, “individual”, “organism” or “patient” are interchangeable and include vertebrates, preferably mammals.
  • mammals are humans, non-human primates, domestic animals (for example, dogs, cats, sheep, cows, goats, pigs, horses, etc.), experimental animals (for example, mice, rats, rabbits, guinea pigs). Etc.) and captive animals (e.g. zoo animals).
  • the term “animal” as used herein also includes humans.
  • the terms "subject” and “individual” include patients, ie, diseased animals, preferably humans. .
  • sample can be any sample usable according to the present invention, including biological samples, such as tissue samples (including body fluids) and/or cell samples, which can be obtained in a conventional manner, such as by surgery or tissue biopsy (including drill biopsy) and By collecting blood, bronchial extracts, sputum, urine, excrement or other body fluids.
  • sample also includes processed samples, such as biological samples or isolates, such as nucleic acid and peptide/protein isolates, formalin-treated paraffin-embedded tissues, and the like.
  • the sample contains cells or tissues of the organ to be tested (for example, cancer diagnosis is to be performed).
  • the sample may contain cells or tissues obtained from the lung.
  • the sample can be a tissue sample from a patient containing tumor cells or cancer cells or suspected of containing tumor cells or cancer cells, including blood, body fluids, primary tumors or tissue samples obtained from tumor metastasis, or any other tissue samples containing tumor cells or cancer cells sample.
  • the present invention provides antibodies or antigen-binding fragments capable of recognizing the antigen peptide comprising the sequence shown in SEQ ID NO: 7 (KNKKIYDGG); and also capable of recognizing the sequence shown in SEQ ID NO: 7 in claudin 18.2 Antibodies or antigen-binding fragments of the epitope.
  • the present invention provides antibodies or antigen-binding fragments capable of recognizing the antigen peptide comprising the sequence shown in SEQ ID NO: 6 (KNKKIYDGGART); and also capable of recognizing the sequence shown in SEQ ID NO: 6 in claudin 18.2 Antibodies or antigen-binding fragments of the epitope.
  • the present invention provides antibodies produced by a deposited hybridoma cell line (also referred to as hybridoma cell line 9G5) with the accession number CCTCCNO.C202007 or CCTCCNO.C202008.
  • a deposited hybridoma cell line also referred to as hybridoma cell line 9G5
  • the accession number CCTCCNO.C202007 or CCTCCNO.C202008 is called antibody 9
  • the antibody produced by the hybridoma cell line deposited by CCTCCNO.C202008 is called antibody 7
  • the preservation dates are all : On January 3, 2020, the preservation location is the China Type Culture Collection, the address is Wuhan, China, Wuhan University, zip code 430072.
  • the present invention provides a composition (eg, a diagnostic composition) or a detection kit, which comprises the antibody or antigen-binding fragment described herein, or a combination of antibodies and/or antigen-binding fragments.
  • a composition eg, a diagnostic composition
  • a detection kit which comprises the antibody or antigen-binding fragment described herein, or a combination of antibodies and/or antigen-binding fragments.
  • the diagnostic composition or detection kit can be used in the method of the present invention, for example, the method for diagnosis, detection or monitoring of the present invention.
  • These kits may optionally contain detectable labels, such as indicator enzymes, radioactive labels, fluorophores or paramagnetic particles.
  • the kit may include an information manual, for example, a manual explaining how to use the reagents to implement the methods disclosed herein.
  • the antibody described herein can be provided with a label that performs the following functions: (i) provides a detectable signal; (ii) interacts with a second label to modify the second label
  • the detectable signal provided by one or second label such as FRET (Fluorescence Resonance Energy Transfer); (iii) Influencing mobility (for example, electrophoretic mobility) through charge, hydrophobicity, shape, or other physical parameters ), or (iv) provide a capture moiety, such as affinity, antibody/antigen or ion complexation.
  • Suitable as labels are the following structures: for example, fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels ), enzyme markers, particle markers (especially metal particle markers, magnetic particle markers, polymer particle markers), small organic molecules (e.g., biotin, receptor ligands or binding molecules (e.g., cell Adhesin or lectin)), a marker sequence containing nucleic acid and/or amino acid residues that can be detected by using a binding agent, etc.
  • fluorescent labels for example, fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels
  • enzyme markers especially metal particle markers, magnetic particle markers, polymer particle markers
  • small organic molecules e.g., biotin, receptor ligands or binding molecules (e.g., cell Adhesin or lectin)
  • Labels include, but are not limited to, barium sulfate, iodosidic acid, iodopanic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, meglumine meglumine, sodium tyropanate, and Radiodiagnostic agents (including positron emitters (such as fluorine-18 and carbon-11), gamma emitters (such as iodine-123, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (such as , Fluorine and Gadolinium)).
  • Radiodiagnostic agents including positron emitters (such as fluorine-18 and carbon-11), gamma emitters (such as iodine-123, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (such as , Fluorine and Gadolinium)).
  • At risk means a subject (ie, a patient) who is identified as having a higher than normal probability of occurrence of a disease (such as cancer) compared with the general population.
  • a subject who has or currently suffers from a disease is a subject with an increased risk of developing the disease because the subject may continue to develop the disease.
  • Subjects who currently have or have had cancer also have an increased risk of cancer metastasis.
  • the method comprises: contacting a sample to be detected with the antibody or antigen-binding fragment or conjugate of the present invention; detecting the The amount of the antibody or antigen-binding fragment, or the complex formed by the conjugate and claudin 18.2.
  • This method can be used to detect whether a subject has a tumor or is at risk of developing a tumor, or whether a subject has or is at risk of developing a cancer.
  • the measurement sample is a tissue or cell sample, such as a sample containing cells (such as tumor cells).
  • the complex is formed between the antibody, antigen-binding fragment or conjugate of the present invention and CLAUDIN 18.2 expressed by the cells in the above sample.
  • the antibody or antigen-binding fragment or the conjugate of the present invention is used to react with a cell sample or a normal sample (or a control sample) taken from a subject, respectively,
  • a cell sample or a normal sample or a control sample taken from a subject
  • the amount of the complex obtained by the reaction with the subject's cell sample is significantly higher than the amount of the complex obtained by the reaction with the normal sample (or control sample)
  • the definitions of normal samples and significant elevations are as described herein above.
  • the first sample from the subject (the first The amount of the complex formed by cells expressing claudin 18.2 and the antibody or antigen-binding fragment, or the conjugate in the second sample (obtained at the second time) and passed twice
  • the difference between the amounts of the detected complexes is used to evaluate the changes in the subject's cancer disease, such as regression, development, etc.
  • the detection result shows that the complex formed in the second sample is significantly reduced, which may indicate that the subject's cancer or tumor is regression or the risk of disease is reduced.
  • the detection result shows that the complex formed in the second sample is significantly increased, which may indicate that the subject's cancer or tumor metastasis, recurrence, or disease risk is increased.
  • this article also relates to a method for analyzing whether a tumor can be treated by tumor therapy targeting claudin 18.2, characterized in that the method comprises: (i) contacting a sample containing tumor cells with the antibody herein Or antigen-binding fragment, or contact with the conjugate herein; (ii) detecting the amount of the antibody or antigen-binding fragment, or the complex formed by the conjugate and claudin 18.2.
  • the tumor cells are gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancer cells, or biliary cancer cells;
  • the tumor cells are gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, or biliary system cancer cells.
  • the complex is formed between the antibody, antigen-binding fragment or conjugate and claudin 18.2 expressed by cancer cells in a sample from a subject.
  • two time points before and after targeted therapy such as the first time (before targeted therapy) and the second time (targeted therapy) After treatment
  • the first time before targeted therapy
  • the second time targeted therapy
  • the amount of the complex formed by the conjugate, and the difference between the amount of the two detected complexes to evaluate whether the subject can be treated by the tumor therapy targeting claudin 18.2.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment or a combination of antibodies and/or antigen-binding fragments as described herein.
  • the kit can be used in the method of the present invention, for example, the method for diagnosis, detection or monitoring of the present invention.
  • These kits may optionally contain detectable labels, such as indicator enzymes, radioactive labels, fluorophores or paramagnetic particles.
  • the kit may include an information manual, for example, a manual explaining how to use the reagents to implement the methods disclosed herein.
  • the C-terminal polypeptide of Claudin 18.2 (Methionine 191-Valine 261, see SEQ ID NO: 3) was synthesized in vitro as an immunogen.
  • SEQ ID NO: 3 The sequence of SEQ ID NO: 3 is as follows:
  • mice Female Balb/C mice aged 6-8 weeks were used for immunization: the immunogen was mixed with Freund’s adjuvant or Gerbu MM adjuvant at a ratio of 1:1, and the immunization dose was (50-100 ⁇ g) per mouse, subcutaneously or intraperitoneally Immunization is performed every other week or two weeks for a total of three or four immunizations.
  • the spleen cells of the immunized mice were separated and fused with mouse myeloma cells SP2/0 (ATCC, CRL 1581) according to the published standard protocol (Kohler, Milstein, 1975). After screening and culturing with HAT medium, the hybridoma supernatant was detected by ELISA, and a total of 139 positive clones were obtained, of which 11 were strong positive clones.
  • the 11 candidate antibodies were expressed by the method of serum-free culture, and purified by Protein G to prepare these 11 candidate antibodies for subsequent detection, respectively named Antibody 1, Antibody 2, Antibody 3, Antibody 4, Antibody 5, Antibody 6, Antibody 7. Antibody 8, Antibody 9, Antibody 10, Antibody 11.
  • the 11 antibodies obtained were identified by Western Blot, and their binding to 293T-Claudin 18.2 and 293T-Claudin 18.1 cell lysates were detected.
  • the Western steps are: 293T-claudin18.1, 293T-claudin18.2 cells are extracted with RIPA lysate (strong) to extract total protein (Biyuntian, P0013B), after protein quantification, load at 30 ⁇ g/well, in 12% separation gel Run the gel at a constant pressure, then transfer to the membrane with a 200mAh constant current for 1 hour, block with 5% skimmed milk powder, and then add the primary antibody (10 ⁇ g/mL) Claudin18 Polyclonal Antibody 1:200 dilution (Thermo Fisher, CAT 38-8000 or anti-flag-HRP 1 : 500 dilution or Anti-Claudin 18.2 antibody[EPR19202-244] 1:200 dilution (Abcam, ab222512); secondary antibody (goat anti-mouse IgG-HRP 1:1000, goat anti-rabbit IgG-HRP 1:1000) incubate, After fully washing the film, it was developed and photographed under a chemilumin
  • control antibody H-43-14A_2 (also called 43-14A2 in this article) is derived from the patent CN104321345A, and the sequence of the variable region of the light chain is shown in SEQ ID NO: 4 (divmtqaafsipvtlgtsasiscrssknllhsdgitylywylqrpgqspqlliyrvsnlasgvpnrfsgsesgtdfggtaevlevgvlevgrvy, variable region) The sequence is shown in SEQ ID NO: 5 (iqlvqsgpelkkfgetvkisckasgytftdysihwvkqapgkglkwmgwintetgvptyaddfkgrfafsletsastaylqinnlknedtatyfcarrtgfdywgqgttltvss).
  • Immunohistochemistry method Perform immunohistochemistry (IHC) on formalin-fixed paraffin-embedded sample slides. After the slide was deparaffinized, it was incubated with 3% hydrogen peroxide at room temperature and placed in a boiling Tris buffer of pH 9.0, heated in a microwave oven for 10 minutes for antigen retrieval, and then 0.2-2 ⁇ g/mL Claudin 18.2 antibody (antibody 4. Antibody 5, Antibody 6, Antibody 7, Antibody 8, Antibody 9, and Antibody 11) and 43-14A2 were incubated overnight at 4°C.
  • Staining intensity any intensity of cell membrane or cytoplasm staining is regarded as positive staining, and the staining intensity is divided into negative (-), weakly positive (1+), positive (2+) and strong positive (3+).
  • Antibody 4 stained the stomach tissue specifically, but did not stain the lung tissue; while the control antibody 43-14A2 has non-specific staining in the lung tissue; the control antibody EPR19202-244 is recommended in the instructions At a working concentration of 0.37 ⁇ g/mL, the specific staining intensity of gastric tissue is high, and there is no staining of lung tissue.
  • EPR19202-244 showed strong positive staining; EPR19202-244 antibody had background staining in 1 of 3 cases of myocardium and 1 of 7 cases of skeletal muscle. Antibody 9 all stained negatively.
  • the remaining 4 cases were thyroid, 3 cases of tongue, 4 cases of esophageal epithelium, 2 cases of duodenal mucosa, 6 cases of jejunum mucosa, 3 cases of ileal mucosa, 5 cases of appendix, 4 cases of colonic mucosa, 2 cases of rectal mucosa, 2 cases of liver, 2 cases of pancreas, 3 cases of trachea, 5 cases of lungs, 3 cases of skin, 1 case of seminal vesicles, 3 cases of prostate, 7 cases of testes, 4 cases of bladder, 1 case of oblongata, 2 cases of telencephalon, 2 cases of cerebellum, 1 case of brainstem 2. In the 2 cases of spleen, both antibodies stained negatively.
  • HRP horseradish peroxidase
  • the staining results of gastric cancer sample F163060A1 (purchased from Guilin Panpu) are shown in Figure 3.
  • the staining results of gastric cancer sample 2018-50829E (purchased from Guilin Panpu) are shown in Figure 4.
  • the staining results of lung cancer sample 2018-55164F (purchased from Guilin Panspectrum) are shown in Figure 5.
  • the statistical results of dyeing are shown in Table 2.
  • Antibody 9 was selected for the analysis of gastric cancer and adjacent tissue microarray staining. Antibody 9 at a working concentration of 0.2 ⁇ g/mL and Abcam EPR19202-244 at a recommended working concentration of 0.37 ⁇ g/mL in the instructions were used for 75 cases of gastric cancer and paracancerous tissue microarrays (Shanghai Xinchao, chip number: HStm-Ade150CS4-01) dyeing. See Table 3 for specific data, and Table 4 for statistics of staining results.
  • the detection rate of antibody 9 is 60.0% (45/75), and the detection rate of antibody 9 is 41.3% (31/75) for ⁇ 2+ staining degree;
  • Abcam EPR19202-244 has a detection rate of 41.3% (31/75), ⁇
  • the degree of 2+ staining is only 17.3% (13/75).
  • the detection rate of Antibody 9 was 100% (75/75), and 90.6% (68/75) were ⁇ 2+ staining; the detection rate of Abcam EPR19202-244 was 98.6% (74/75), The degree of staining was 89.3% (67/75) ⁇ 2+. This shows that antibody 9 is superior to Abcam EPR19202-244 in terms of specificity and sensitivity. Table 3. Antibody 9 staining of gastric cancer tissue chip
  • ELISA method Analyze the epitope bound by the antibody by detecting the binding of antibody 7 and antibody 9 to different peptides of the antigen.
  • Claudin 18.2 C-terminal polypeptide Based on the immunogen (Claudin 18.2 C-terminal polypeptide) sequence, it is divided into 21 peptides according to the principle of 15aa in length and 12aa in overlap. The corresponding peptides are chemically synthesized and modified with biotin at the N-terminal. Each peptide was captured with Neutravidin plate (CAT: 15128 Invitrogen), and then incubated with 10 ⁇ g/mL Claudin 18.2 antibody (antibody 7 and antibody 9).
  • CAT Neutravidin plate
  • HRP-labeled goat anti-mouse (CAT:A0168 SIGMA) as the secondary antibody, develop the substrate color solution for 5 minutes, then add 2M H2SO4 to stop the color development, and determine the OD450nm absorbance to determine the binding of the antibody and each peptide
  • the ELISA results are shown in Figure 6.
  • the sequence characteristics of the binding peptide of antibody 9 are shown in SEQ ID NO: 6: KNKKIYDGGART; the sequence characteristics of the binding peptide of antibody 7 are shown in SEQ ID NO: 7: KNKKIYDGG.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供了一种抗体或抗原结合片段，其能够识别包含SEQ ID NO：7(KNKKIYDGG)的抗原肽；或者识别claudin18.2中包含SEQ ID NO：7的表位；优选识别包含SEQ ID NO：6(KNKKIYDGGART)的肽；或者识别claudin18.2中包含SEQ ID NO：6的表位。

Description

抗Claudin18.2的抗体及其应用 技术领域
本发明属于免疫学领域，更具体地，本发明涉及抗Claudin18.2的抗体及其应用。
背景技术
密蛋白18(Claudin 18，CLD18)是位于上皮和内皮的紧密连接中的内在膜蛋白，分子量约为27.9KD。GenBank登记号为剪接变体1(称为CLD18A1、CLD18.1或Claudin 18.1)：NP_057453、NM016369，以及剪接变体2(称为CLD18A2、CLD18.2或Claudin 18.2)：NM_001002026、NP_001002026。CLD18A1在正常肺中选择性表达，而CLD18A2则在正常胃中表达，且表达局限于已分化的胃上皮短寿细胞中。但在多种肿瘤细胞中，CLD18A2呈现强烈表达。已发现，CLAUDIN18.2在胃癌、食管癌、胰腺癌、肺癌(例如非小细胞肺癌(NSCLC))、卵巢癌、结肠癌、肝癌、乳腺癌、头颈癌、和胆道系统肿瘤(如胆囊癌、胆管癌)及其转移癌中表达，胃癌转移例如库肯勃瘤、腹膜转移和淋巴结转移。
CLDN18为四次跨膜蛋白，有两个胞外段，称为环1和环2。人CLDN18A2(SEQ ID NO:2)与人CLDN18A1(SEQ ID NO:1)高度同源，两者的同源性比较结果如图1所示，仅在N端的第一个跨膜(TM)区和环1存在部分差异，而其余部分的氨基酸序列相同。
发明内容
本发明利用CLDN18A2的C端多肽免疫小鼠，虽然该肽段在两个剪切变体中的序列中完全相同，但是我们得到多个具有明显的组织特异性的抗体，可用于CLDN18A2阳性肿瘤的临床诊断。
在第一方面，本发明提供一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段能够识别包含SEQ ID NO：7(KNKKIYDGG)所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：7所示的序列的表位。
在进一步优选的方案中，所述抗体或抗原结合片段能够识别包含SEQ ID NO：6(KNKKIYDGGART)所示的序列的肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：6所示的序列的表位。
在进一步优选的方案中，所述抗体或抗原结合片段能够识别包含SEQ ID NO：8(GFGSNTKNKKIYDGG)、9(SNTKNKKIYDGGART)或10(KNKKIYDGGARTEDE)所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：8、9或10所示的序列的表位；
优选的，所述抗体或抗原结合片段能够识别包含SEQ ID NO：9或10所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：9或10所示的序列的表位。
在进一步优选的方案中，所述抗体为由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体，其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7。
在进一步优选的方案中，所述抗体或抗原结合片段是鼠源抗体、嵌合抗体、人源化的抗体、或全人抗体。
在进一步优选的方案中，所述抗体或抗原结合片段是由登记号为CCTCCNO.C202007(抗体9)或CCTCCNO.C202008(抗体7)的保藏的杂交瘤细胞株产生的抗体的人源化抗体或嵌合抗体。
在进一步优选的方案中，所述抗体或抗原结合片段是由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体的突变体；其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7
优选的，所述突变体与由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的同源性。
在进一步优选的方案中，所述抗体片段为由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体的抗体片段；
优选的，所述抗体片段是scFv、Fab片段，Fd片段，Fv片段，F(ab’)2片段、或单域抗体，
其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7。
在进一步优选的方案中，所述抗体或抗原结合片段与表达claudin18.2的细胞相结合；优选的，所述claudin18.2的氨基酸序列如SEQ ID NO：2所示，SEQ ID NO：2为：
Figure PCTCN2021070139-appb-000001
在进一步优选的方案中，所述抗体或抗原结合片段特异性结合胃组织；
优选的，所述抗体或抗原结合片段特异性结合胃组织，不结合肺组织。
在本发明的第二方面，提供一种缀合物，其特征在于，所述缀合物包含权利要求1-9任一项所述的抗体或抗原结合片段，以及至少一个可检测标记物；
优选的，所述的可检测标记物为荧光标记物、显色标记物、发光标记物、发色团标记物、放射性同位素标记物、同位素标记物、同量异位素标记物(isobaric label)、酶标记物、颗粒标记物、通过使用结合剂检测的核酸或蛋白。
在本发明的第三方面，提供一种杂交瘤，其能够产生上述本发明的抗体。
在进一步优选的方案中，所述杂交瘤的保藏登记号为CCTCCNO.C202007或CCTCCNO.C202008。
在本发明的第四方面，提供一种检测待测样品claudin18.2表达情况的方法，其特征在于，所述方法包括：使待测样品与上述本发明的任一所述的抗体或抗原结合片段，或者与上述本发明所述的缀合物相接触；检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的情况。
在本发明的第四方面，提供一种用于诊断、检测或监测癌症的方法，其特征在于，所述方法包括：
将待检测样品与上述本发明所述的抗体或抗原结合片段，或者与上述本发明所述的缀合物相接触；检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量。
在本发明的第四方面，提供一种用于分析肿瘤是否能够通过靶向claudin18.2的肿瘤疗法来治疗的方法，其特征在于，所述方法包括：
(i)使包含肿瘤细胞的样品与上述本发明所述的抗体或抗原结合片段，或者与上述本发明所述的缀合物相接触；
(ii)检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量；
优选的，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、肝癌细胞、头颈癌细胞、或胆道系统肿瘤细胞(如胆囊癌、胆管癌细胞)；
更优选的，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、或胆道系统肿瘤细胞。
在本发明的第五方面，提供一种检测试剂盒，其包含上述本发明所述的抗体或抗原结合片段，或者与上述本发明所述的缀合物。
在进一步优选的方案中，其中所述claudin18.2包含序列表的SEQ ID NO：2的氨基酸序列或其变体。
在本发明的第六方面，提供一种产生靶向claudin18.2的单克隆抗体的方法，其特征在于，所述方法包括采用含有SEQ ID NO：6、7、8、9或10所示的序列的多肽为免疫原，或者用与SEQ ID NO：6、7、8、9或10所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性的多肽为免疫原，对动物进行免疫后，取动物的B细胞与骨髓瘤细胞进行融合，得到杂交瘤细胞，以产生单克隆抗体；
优选的，所述动物为鼠；
优选的，所述B细胞来源于脾细胞。
在进一步优选的方案中，还包括从所述杂交瘤细胞中分离所述单克隆抗体。
在进一步优选的方案中，用SEQ ID NO：3(MMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV)所示的多肽为免疫原。
在进一步优选的方案中，其中所述单克隆抗体特异性结合所述抗原。
在进一步优选的方案中，还包括产生保留与所述抗原特异性结合的所述单克隆抗体的修饰形式。
在进一步优选的方案中，其中所述单克隆抗体的修饰形式是人源化抗体。
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入一般。
附图说明
图1：Claudin18.2和Claudin 18.1的序列同源性比较结果；
图2A：抗体与293T-Claudin18.1细胞裂解物结合的Western Blot鉴定结果；
图2B：抗体与293T-Claudin18.2细胞裂解物结合的Western Blot鉴定结果；
图3：抗体7和抗体9对胃癌样本F163060A1的染色结果；
图4：抗体7和抗体9对胃癌样本2018-50829E的染色结果；
图5：抗体7和抗体9对肺癌样本2018-55164F的染色结果；
图6：显示表位ELISA分析结果。
具体实施方式
以下具体说明详尽地展示了本文所公开的实施方案。应当理解，本说明书并非旨在仅限于此处所公开的具体的实施方案，而是可以发生改变。本领域技术人员将理解，本说明书中所公开的内容可以有多种改变或变化，而均涵盖于所公开的范围和原则之内。除非另有说明，每个实施方案均可与任何其他实施方案任意组合。
本文所公开的某些实施方案包含了数值范围，并且本发明的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本发明的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。例如，从1至6的范围的描述应当被认为具体公开了从1至3、1至4、1至5、2至4、2至6、3至6等的子范围，以及在这些范围内的具体的数值点，例如1、2、3、4、5、6。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语"约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
术语“抗体”在本文中包括完整的抗体和抗原结合片段。天然存在的“抗体”是包含通过二硫键链间连接的至少2个重(H)链和2个轻(L)链的糖蛋白。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。
轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为称为互补决定区(CDR)的具有高可变性的区域，其间隔以更保守的称为框架区(FR)的区域。每一个VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体可来自不同物种，包括但不限于小鼠、大鼠、兔、豚鼠和人。抗体还包括嵌合抗体，具有来自一个物种(优选人)的抗体恒定区与来自另一物种的抗原结合位点。抗体还包括人源化的抗体，具有来自非人物种的抗体的抗原结合位点与人来源的恒定区和构架区。本文所述的抗体可以是单克隆抗体，即具有单分子组成的抗体分子制备物。用于制备分泌单克隆抗体的杂交瘤的系统优选小鼠系统。分离用于融合的免疫化的脾细胞的免疫操作和技术为本领域所已知。小鼠骨髓瘤细胞和小鼠脾细胞融合方案也是已知的。用于制备分泌单克隆抗体的杂交瘤的其他系统优选大鼠和兔系统(例如Spieker-Polet等，Proc.Natl.Acad.Sci.U.S.A.92：9348(1995)所述。本文所述的抗体可以是重组抗体，即通过重组手段制备、表达、产生或分离的所有抗体，例如(a)从免疫球蛋白基因为转基因或转染色体的动物(例如小鼠)或由其制备的杂交瘤分离的抗体，(b)从经转化以表达所述抗体的宿主细胞(例如转染瘤)分离的抗体，(c)从重组的组合抗体文库分离的抗体，和(d)通过涉及将免疫球蛋白基因序列剪接成其他DNA序列的任何其他手段制备、表达、产生或分离的抗体。抗体可通过多种技术产生，例如Kohler等，Nature 256：495(1975)的杂交瘤技术，也可以采用其他产生单克隆抗体的技术，例如噬菌体展示技术。
使用来自杂交瘤的重链和轻链转录本的核苷酸序列来设计合成寡核苷酸的重叠组，以产生与天然序列的氨基酸编码能力相同的合成V序列。合成的重链和κ链序列可以以三种方式不同于天然序列：中断了重复的核苷酸碱基串，以便于寡核苷酸合成和PCR扩增；根据Kozak规则掺入了最佳翻译起始位点(Kozak，1991，J.Biol.Chem.266：19867-19870)；以及翻译起始位点上游的经改造HindIII位点。
对于重链和轻链可变区而言，经优化的编码链及相应非编码链的序列在相应非编码寡核苷酸的约中点处打断成30-50个核苷酸。因此，对于每条链，可将所述寡核 苷酸装配成跨越150-400个核苷酸的区段的重叠双链组。接着将该库用作模板产生150-400个核苷酸的PCR扩增产物。通常将单个可变区寡核苷酸组分为两个库，将它们各自扩增以产生两个重叠的PCR产物。接着通过PCR扩增来组合这些重叠产物，以形成完整的可变区。在PCR扩增中还可能包括重链或轻链恒定区的重叠片段，以产生易克隆进表达载体的构建体片段。接着将重建的嵌合或人源化重链及轻链可变区与克隆的启动子序列、前导序列、翻译起始序列、恒定区序列、3’非翻译序列、多聚腺苷酸化序列和转录终止序列组合，以形成表达载体构建体。可将重链及轻链表达构建体组合进单个载体，共转染、连续转染或分别转染进宿主细胞中，接着将其融合形成表达这两种链的宿主细胞。构建人IgGκ表达载体的质粒使得PCR扩增的V重链及Vκ轻链cDNA序列可用于重建完整的重链及轻链小基因(minigene)。这些质粒可用于表达全人抗体或者嵌合IgG1κ或IgG4κ抗体。可以构建类似的质粒用于表达其他重链同种型，或者表达包含λ轻链的抗体。
术语“抗原结合片段”指抗体中保留特异性抗原结合能力的一个或更多个片段，所涵盖的结合片段的包括(i)Fab片段，由VL、VH、CL和CH结构域组成的一价片段；(ii)F(ab′)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(iii)由VH和CH结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(v)由VH结构域组成的单域抗体片段；(vi)分离的互补决定区(CDR)，和(vii)可任选地通过合成接头连接的两个或更多个分离的CDR的组合。还包括用重组方法将Fv片段的两个结构域VL和VH通过合成接头连接在一起，形成为单链抗体(或称单链Fv(scFv))。
本发明的抗体或抗原结合片段与靶抗原Claudin18.2的结合可通过本领域已知的任何方法表征或表达,例如，结合可以通过结合亲和力来表征。结合亲和力可以通过本领域已知的任何方法测定，例如ELISA、Western印迹、免疫荧光和流式细胞术分析。流式细胞术可证明经免疫接种小鼠的血清中存在抗体或者证明单克隆抗体结合活细胞。将天然表达或在转染后表达抗原的细胞系以及缺乏抗原表达的阴性对照(在标准生长条件下生长)与杂交瘤上清液中或含1％FBS的PBS中多种浓度的单克隆抗体混合，4℃下孵育30分钟。洗涤后，在与第一抗体染色相同的条件下使APC或Alexa647标记的抗IgG抗体与结合了抗原的单克隆抗体结合。用FACS仪器分析样品，其中使用光散射和侧散射特性对单个活细胞设置门控(gate)。使用共转染法可以在单个测量中区分抗原特异性单克隆抗体与非特异性结合物。按照上述对用编码抗原和荧光标记 物的质粒瞬时转染的细胞进行染色。可在与抗体染色细胞不同的荧光通道中检测到经转染的细胞。由于大部分转染细胞同时表达两种转基因，因此抗原特异性单克隆抗体优先与表达荧光标记的细胞结合，非特异性抗体以相当的比率与未转染细胞结合。可以完全如上所述对细胞进行染色，也可以通过荧光显微术来检查细胞。
当使用表达Claudin18.2的细胞的体外结合测定中测定时，可以确定本文公开的抗原结合部分的结合亲和力。受试抗原结合部分的结合亲和力可以用Kd表示，Kd是抗体与其各自抗原之间的平衡解离常数。
在一些实施例中，针对CLAUDIN18.2的人单克隆抗体可使用携带部分人免疫系统而不是小鼠系统的转基因或转染色体小鼠来产生，这些转基因和转染色体小鼠包括分别称为HuMAb小鼠和KM小鼠的小鼠。在一些实施例中，用来自CLAUDIN18.2序列的载体缀合肽、重组表达的CLAUDIN18.2抗原或其片段的富集制备物和/或表达CLAUDIN18.2或其片段的细胞来免疫接种小鼠。也可以用编码全长人CLAUDIN18.2或其片段的DNA免疫接种小鼠。当使用纯化或富集的CLAUDIN18.2抗原制备物进行免疫接种而未产生抗体时，可以用表达CLAUDIN18.2的细胞(例如细胞系)免疫接种小鼠，以促进免疫应答。可以用由尾静脉或眶后取血获得的血浆和血清样品在免疫全程中监测免疫应答。可将效价足够的抗CLAUDIN18.2免疫球蛋白小鼠用于融合。可在小鼠处死并摘除脾之前3至5天用表达CLAUDIN18.2的细胞进行腹腔内或静脉来加强免疫，以提高分泌特异性抗体的杂交瘤的比率。在一些实施例中，为了制备CLAUDIN18.2单克隆抗体的杂交瘤，可从免疫接种小鼠的淋巴结、脾或骨髓分离细胞，并与适当的无限增殖细胞系(如小鼠骨髓瘤细胞系)融合，然后筛选针对抗原的特异性抗体来得到杂交瘤。接着通过ELISA筛选分泌抗体的杂交瘤。通过免疫荧光或FACS分析，鉴定出特异性结合CLAUDIN18.2的抗体。可将分泌该抗体的杂交瘤重新铺板，再次筛选，如果抗CLAUDIN18.2单克隆抗体仍为阳性则可以通过有限稀释进行亚克隆。体外培养稳定的亚克隆，以在组织培养基中产生抗体用于表征。在一些实施例中，抗体在宿主细胞转染瘤中产生，例如使用本领域公知的重组DNA技术与基因转染方法的组合来进行(Morrison，S.(1985)Science 229：1202)。
在一些实施例中，可将目的基因(如抗体基因)连接进表达载体(例如真核表达质粒)中。可将带有所克隆抗体基因的纯化质粒引入真核宿主细胞中，例如CHO细胞、NS/0细胞、HEK293T细胞或HEK293细胞，或者其他真核细胞，例如来自植物的细胞、真菌或酵母细胞。用于引入这些基因的方法可以是本领域已描述过的方法，例如 电穿孔、lipofectine、lipofectamine或其他。将这些抗体基因引入宿主细胞后，可以鉴定并选择表达该抗体的细胞。重组抗体可从这些培养物上清液和/或细胞中分离和纯化。抗体基因也可以在其他表达系统中表达，包括原核细胞例如微生物，例如大肠杆菌(E.coli)。此外，抗体也可以在非人转基因动物中产生，例如在绵羊和兔的乳汁中或在鸡蛋中产生，或者在转基因植物中产生，参阅例如Verma，R.，等(1998)J.Immunol.Meth.216：165-181；Pollock，等(1999)J.Immunol.Meth.231：147-157以及Fischer，R.，等(1999)Biol.Chem.380：825-839。
本文所用的“抗原”指包含表位，并且针对所述表位可引起和/或产生免疫应答的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。
术语“表位”指分子中的抗原决定簇，即分子中被免疫系统识别(例如被抗体识别)的部分。
密蛋白是紧密连接中最重要组分的一个蛋白质家族，在紧密连接中，密蛋白构成了细胞旁屏障，控制着上皮细胞之间的细胞间隙中的分子流动。密蛋白是四次跨膜的跨膜蛋白，其N端和C端均位于细胞质中。在一些实施方案中，术语“密蛋白18”或“CLDN18”或“Claudin 18”或“CLD18”是指人CLDN18并且包括任何剪接变体，例如CLDN18的Claudin18.1和Claudin 18.2。在一些实施方案中，Claudin 18.1是具有GenBank登记号NP_057453的肽(mRNA:NM_016369)。在一些实施方案中，所述Claudin 18.1是包含SEQ ID NO:
Figure PCTCN2021070139-appb-000002
所述氨基酸序列变体的蛋白质/肽。所述Claudin 18.2是具有GenBank登记号NP_001002026的肽(mRNA:NM_001002026)。在一些实施方案中，所述claudin18.2是包含SEQ ID NO:2(MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSM EDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV)的氨基酸序列的肽或所述氨基酸序列变体的蛋白质/肽。术语“变体”是指突变体、剪接变体、构象、同种型、等位基因变体、种变体(species variant)和种同源物(species homolog)，特别是天然存在的变体。等位基因变体涉及基因正常序列中的改变，其显著性通常不明显。全基因测序通常鉴定出给定基因的大量等位基因变体。种间同源物是与给定核酸或氨基酸序列具有不同物种起源的核酸或氨基酸序列。术语“CLDN”、“CLDN18”、“Claudin18.1”和“Claudin 18.2”应涵盖任何翻译后修饰的变体和构象变体。Claudin18.1和Claudin 18.2在包含第一个跨膜(TM)区和环1的N端部分存在差异，而C端的蛋白质一级序列相同。
CLAUDIN18.2在正常胃粘膜上皮细胞中选择性表达。CLAUDIN18.2表达仅局限于已分化的胃上皮短寿细胞，在多种肿瘤细胞中表达。在一些实施例中，原发肿瘤是指胃癌、食管癌、胰腺癌、肺癌例如非小细胞肺癌、卵巢癌、结肠癌、肝癌、头颈癌和胆道系统癌症等。在一些实施例中，表达CLAUDIN18.2的细胞是指肿瘤细胞，并且特别地选自胃癌细胞、食管癌细胞、胰腺癌细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、肝癌细胞、头颈癌细胞、和胆道系统癌症细胞。在一些实施例中，表达CLAUDIN18.2的细胞是指CLAUDIN18.2位于或结合于细胞膜的细胞。
免疫组化(IHC)是检测组织切片的细胞中的抗原(例如，蛋白质)的方法，广泛用于检测异常细胞，例如肿瘤细胞。所用的检测抗体可结合酶，例如可催化产生颜色之反应的过氧化物酶。也可以用荧光基团标记检测抗体，例如荧光素或罗丹明。在本发明中，用免疫组化测定细胞中CLAUDIN18.2表达。使用取自肿瘤患者常规手术中的非癌组织或癌组织样品的多聚甲醛或丙酮固定冷冻切片或多聚甲醛固定的石蜡包埋组织切片，或者切片取自表达CLAUDIN18.2的异种移植瘤小鼠。所述切片先与CLAUDIN18.2抗体进行孵育，再与缀合了辣根过氧化物酶的山羊抗小鼠或山羊抗兔抗体孵育，来进行免疫染色。在这个检测中，样品的制备对于维持细胞形态、组织结构和靶抗原表位的抗原性是至关重要的。通常使用多聚甲醛来固定组织。根据实验目的和实验样品的厚度，从固定后的组织切取薄(约4至40μm)的切片，或者如果组织不是非常厚并且可穿透，则可整体使用。通常通过使用显微切片机切片，并且将切片封片于载片上。样品可能需要优化处理，包括脱石蜡化和抗原修复。通常，在免疫组 化中使用去污剂(例如Triton X-100)来降低表面张力，相关试剂可更好且更均匀的覆盖样品。免疫组化染色可使用一种直接与所染抗原相结合标记抗体。免疫组化染色更常用的是间接方法，使用针对待检测抗原的抗体和针对第一抗体的第二标记抗体。用封闭缓冲液(例如，正常血清、非脂奶粉、BSA或明胶以及市售封闭缓冲剂)孵育样品，封闭反应位点以减少IHC中的背景染色。第一抗体针对靶抗原并且通常未标记的，第二抗体针对第一抗体的免疫球蛋白。第二抗体通常与接头分子(例如，生物素)相结合，其然后募集报告分子，或者第二抗体直接与报告分子本身相结合。最常用的检测方法分别是用酶介导的呈色检测和荧光团介导的荧光检测。酶标记物与底物反应产生可以用普通光学显微镜进行分析的强烈着色的产物。碱性磷酸酶(AP)和辣根过氧化物酶(HRP)是最广泛地用作蛋白质检测标记物的两种酶。多种呈色底物、荧光底物和化学致发光底物可与酶一起使用，包括DAB或BCIP/NBT。荧光报告分子是用于IHC检测的小的有机分子。对于生色和荧光检测方法，信号的光密度分析可分别提供半定量和全定量数据，将报告分子信号的水平与蛋白表达水平或定位水平相关联。在对靶抗原进行免疫组化染色后，通常进行第二染色(例如，苏木精、Hoechst染色剂和DAPI)以提供帮助凸显第一染色的反差。
本发明提供用于检测样品中CLAUDIN18.2抗原的存在或者测量样品中CLAUDIN18.2抗原量的方法，在抗体与CLAUDIN18.2抗原形成复合物时，使所述样品(或对照样品)与结合CLAUDIN18.2抗原的本发明抗体相接触。随后检测复合物的形成，通过对比样品中形成的复合物与对照品中形成复合物的差异来判断所述样品中是否存在CLAUDIN18.2抗原。本发明的检测方法可特别地用于诊断CLAUDIN18.2相关疾病，例如肿瘤。在一些实施例中，样品中CLAUDIN18.2抗原的量高于参照或对照样品中CLAUDIN18.2抗原的量指示所述样品来源的个体(特别是人)患有CLAUDIN18.2相关疾病，例如肿瘤。在一些实施例中，所述测量样品是细胞样品，例如包含细胞(如肿瘤细胞)的样品。所述复合物是由本发明的抗体、抗原结合片段或缀合物与上述样品中的细胞所表达的CLAUDIN18.2之间形成的。
本发明中“参照”(例如参照样品或参照生物体)可用于与通过本发明方法获得的来自受试样品或受试生物体的结果进行关联和比较。所述参照生物体一般是健康生物体，尤其是未患有疾病(例如，肿瘤)的生物体。“参照值”或“参照水平”可通过测量足够大量的参照物而根据参照物来经验性地确定。优选地，通过测量至少2个、优选至 少3个、优选至少5个、优选至少8个、优选至少12个、优选至少20个、优选至少30个、优选至少50个或优选至少100个参照物来确定参照值。
术语“核酸”，是指脱氧核糖核酸(DNA)或核糖核酸(RNA)，包括基因组DNA、cDNA、mRNA、重组制备的或化学合成的分子。核酸可以是单链或双链分子形式，也可以是线性的或者共价闭合成环。RNA包括双链RNA、单链RNA、分离的RNA(例如，部分纯化的RNA)、基本上纯的RNA、合成的RNA、重组产生的RNA以及通过添加、缺失、替换和/或改变一个或更多个核苷酸而不同于天然RNA的经改变RNA。RNA可通过稳定化序列、加帽和多聚腺苷酸化来修饰。术语“转录”是指其中将DNA序列中的遗传密码转录成RNA的过程。“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。术语“同源”指核酸功能性地天然连接，而术语“异源”指所述核酸不是功能性地天然连接。在一些实施例中，核酸可以与同源或异源的表达控制序列功能性连接。
术语“表达”，包括生成RNA或者RNA和蛋白质/肽。就RNA而言，术语“表达”或“翻译”特别指产生肽或蛋白质。表达可以是瞬时的或稳定的。表达还包括异常表达或不正常表达，即与参照相比(例如未患有与某些蛋白质(例如，肿瘤相关抗原)的异常表达或不正常表达相关的疾病之个体)，表达改变(比如，升高)。表达的升高指升高至少10％，特别是至少20％，至少50％或至少100％或者更多。在一个实施方案中，表达仅见于患病的组织，而健康组织中的表达被抑制。特异性表达是指蛋白质基本上只在特定组织或器官中表达。例如，特异性地在胃粘膜中表达的肿瘤相关抗原，指所述蛋白质主要在胃粘膜中表达而在其他组织中不表达或者在其他组织或器官类型中不显著表达。在一些实施方案中，肿瘤相关抗原在多于一种正常组织类型或器官中(例如，在不多于3种不同的组织或器官类型中)特异性表达，那么可以认为肿瘤相关抗原在这些器官中特异性表达。
如本文所用，术语“分离的”是指与细胞成分或其他成分相分离，在这些成分中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在自然状态通常是相关联的。非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”以将其与天然存在的对应物区分开。分离的核酸指该核酸是(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增，(ii)通过克隆重组产生的，(iii)纯化的，例如通过切割和凝胶电泳分级纯化的，或者(iv)合成的，例如通过化学合成。分离的核酸是可通过重组DNA技术操作 的核酸。分离的蛋白质或分离的肽是指从其天然环境中被分离出来的蛋白质或肽，基本上不含与其在自然界中或在体内相结合的其它物质。此外，“浓缩”、“分离”或“稀释”的多核苷酸、肽、多肽、蛋白质、抗体或其片段可与其天然存在的对应物区分开，因为每体积分子的浓度或数量大于(“浓缩”)或小于(“稀释”)其天然存在的配对物的浓度。在一些实施方案中，本发明的技术方案优选的富集程度更高。因此，例如，优选2倍富集、更优选10倍富集、更优选100倍富集、更优选1000倍富集。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。
术语“变体”，包括突变体、剪接变体、构象、同种型、等位基因变体、种变体和种同源物，特别是天然存在。等位基因变体指基因正常序列中的改变，其显著性通常不明显。全基因测序通常鉴定出指定基因的大量等位基因变体。种间同源物是指有不同物种来源的指定核酸或氨基酸序列。
术语“氨基酸修饰”(或修饰的氨基酸、氨基酸序列的变体)包括对特定氨基酸序列的氨基酸取代、插入和/或缺失，产生与所述特定序列功能相当的序列。在一些实施例中，氨基酸修饰后的抗体仍与其靶抗原结合。因而，可以用其他相似侧链的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基。优选地，修饰抗体的CDR序列、高变区和可变区的序列而不丧失结合靶抗原的能力。本领域技术人员可通过重组RNA操作容易地制备氨基酸变体。例如，Sambrook等(1989)中详细描述了用于制备具有替换、添加、插入或缺失的蛋白质和肽的DNA序列操作。此外，本文所述的肽和氨基酸变体可以借助于已知的肽合成技术(例如，固相合成和类似方法)容易地制备。例如仅替换1至5、1至4、1至3、或1或2被认为是高度同源。在蛋白质的N端和/或C端包含缺失的氨基酸缺失变体称为N端和/或C端截短变体。氨基酸添加变体包含氨基和/或羧基端融合一个或更多个氨基酸，例如1、2、3、5、10、20、30、50或更多个氨基酸。氨基酸缺失变体是从序列中除去一个或更多个氨基酸(例如，除去1、2、3、5、10、20、30、50或更多个氨基酸)。氨基酸替换变体是序列中至少一个氨基酸残基被除去并在其位置中插入另一个氨基酸残基。在一个实施例中，将同源的蛋白质或肽之间非保守的氨基酸残基进行修饰和/或置换为相似特性的氨基酸残基。优选地，氨基酸序列与其氨基酸序列变体的氨基酸序列之间的相似度同一性，优选为至少约60％、65％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。优选地相似度或同一性针对的氨基酸区域为参照氨基酸序列全长 的至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％或约100％。例如，如果参照氨基酸序列由200个氨基酸组成，优选地针对至少约20、至少约40、至少约60、至少约80、至少约100、至少约120、至少约140、至少约160、至少约180或约200个氨基酸给出相似度或同一性，优选连续的氨基酸。在一些优选的实施方案中，针对全长的参照氨基酸序列给出相似度或同一性。可用本领域已知的工具进行用于确定序列相似性(优选序列同一性)的比对，优选使用最佳序列比对，例如使用Align，采用标准设置，优选EMBOSS：needle；Matrix：Blosum62；Gap Open 10.0；Gap Extend 0.5。序列相似性指相同或表示保守性氨基酸替换的氨基酸的百分比。两条氨基酸序列的同一性指两条序列间相同的氨基酸的百分比，这一百分比完全是统计学意义上的，且两序列之间的差异随机分布在其全长上。序列比对可通过以下方式：Smith和Waterman，1981，Ads App.Math.2，482的局部同源性算法，Neddleman和Wunsch，1970，J.Mol.Biol.48，443的局部同源性算法，Pearson和Lipman，1988，Proc.Natl Acad.Sci.USA 85，2444的相似性检索法或者使用这些算法的计算机程序(在Wisconsin Genetics软件包中的GAP，BESTFIT，FASTA，BLAST P，BLAST N和TFASTA，Genetics Computer Group，575ScienceDrive，Madison，Wis.)。
术语“肽”包括寡肽和多肽，指包含通过肽键共价连接的两个或更多个、优选3个或更多个、优选4个或更多个、优选6个或更多个、优选8个或更多个、优选9个或更多个、优选10个或更多个、优选13个或更多个、优选16个或更多个、优选21个或更多个以及高达优选8、10、20、30、40或50个(尤其是100个)氨基酸的物质。术语“蛋白质”指大的肽，优选指具有超过100个氨基酸残基的肽，一般来说，术语“肽”和“蛋白质”是同义词，并可在本文中可互换使用。
蛋白质和肽的“衍生物”为蛋白质和肽的经修饰形式。这样的修饰包括任何化学修饰并且包括与蛋白质或肽相关的任何分子(例如碳水化合物、脂质和/或蛋白质或肽)的单个或多个替换、缺失和/或添加。蛋白质或肽的“衍生物”包括经糖基化、乙酰化、磷酸化、酰胺化、棕榈酰化、豆蔻酰化、异戊二烯化、脂化、烷化、衍生化、引入保护/封闭基团、蛋白质水解切割或与抗体或者其他细胞配体相结合的经修饰的类似物及全部功能性化学等同物。在一些实施例中，经修饰的肽的稳定性和/或免疫原性得到提高。
术语“细胞”指具有完整膜的尚未释放其正常的细胞内组分(例如，酶、细胞器或 遗传物质)的细胞，优选地是有活力的细胞(viable cell)，即能够进行其正常代谢功能的活细胞(living cell)。包括原核细胞(例如，大肠杆菌(E.coli))或真核细胞(例如，树突细胞、B细胞、CHO细胞、COS细胞、K562细胞、HEK293细胞、HELA细胞、酵母细胞和昆虫细胞)。优选为哺乳动物的细胞，例如来自人、小鼠、仓鼠、猪、山羊和灵长类的细胞。所述细胞可以源自大量组织类型，并且包括原代细胞和细胞系。在一些实施例中，细胞包括非癌细胞和癌细胞。靶细胞是指作为免疫应答靶向的细胞，例如本专利所述的肿瘤细胞。在一些实施例中，靶细胞是表达CLAUDIN18.2的细胞。在一些实施例中，表达CLAUDIN18.2的细胞通常包括癌细胞。
本专利中试剂、组合物和方法可用于诊断个体是否患病。可被诊断的疾病涵盖所有表达CLAUDIN18.2的疾病,优选的,是指肿瘤，例如胃癌、胰腺癌、食道癌、卵巢癌、胆道系统癌等。
术语“疾病”指任何病理状态，包括肿瘤，特别是本文所述的那些类型的肿瘤。
术语“正常组织”或“正常条件”中所用的术语“正常”指健康组织或者健康个体中的条件，即，非病理条件，其中“健康”优选地意为非肿瘤的。
本文中“CLAUDIN18.2相关的疾病”，是指与健康组织或器官相比，患病后的组织或器官中的细胞的CLAUDIN18.2的表达升高。所述升高指升高至少10％，特别是至少20％、至少50％、至少100％、至少200％、至少500％、至少1000％、至少10000％或者甚至更高。在本发明中，CLAUDIN18.2相关的疾病包括肿瘤，特别是本文所述的那些类型的肿瘤。
在本文中，“降低”是指与之前检测时的细胞的CLAUDIN18.2的表达降低，降低至少5％或更高，10％或更高，20％或更高，更优选50％或更高并且最优选75％或更高的能力。术语“抑制”是指完全或基本完全的抑制，即降低至0或基本降低至0。
在本文中，术语“癌症”和“肿瘤”可互换，指其中细胞表现出不受控生长、和/或侵袭、和/或转移的疾病。例如白血病、黑色素瘤、淋巴瘤、成神经细胞瘤、神经胶质瘤、直肠癌、子宫内膜癌、肾癌、肾上腺癌、甲状腺癌、血液癌症、皮肤癌、脑癌、宫颈癌、肠癌、肝癌、结肠癌、胃癌、肠癌、头颈癌、胃肠癌、淋巴结癌、食管癌、结肠直肠癌、胰腺癌、耳鼻喉(ENT)癌、乳腺癌、前列腺癌、子宫癌、卵巢癌和肺癌，及其转移。
术语“转移”指癌细胞从其原始部位扩散到身体的其他部位。转移的形成是非常复杂的过程，并依赖于恶性细胞从原发肿瘤中脱离、侵袭胞外基质、透过内皮基底膜 以进入体腔和血管以及其后由血液转运后浸润靶器官。最后，新肿瘤(即继发性肿瘤或转移性肿瘤)在靶部位的生长依赖于血管发生。肿瘤转移经常甚至在切除原发肿瘤后发生，因为肿瘤细胞或组分可能残留并发展出转移潜力。在一个实施例中，转移是指远端转移，即远离原发肿瘤和/或局部淋巴结系统的转移。继发性或转移性肿瘤的细胞与原始肿瘤中的细胞类似。
术语“再发”或“复发”，是指当个体再次被过去所患病症影响。例如，个体曾经患有某疾病，且已接受治疗痊愈，而再次发生所述疾病，则所述新发疾病可被认为是再发或复发。肿瘤的再发或复发，包括所述肿瘤发生在不同于原始肿瘤的部位以及发生在原始肿瘤的部位的情况。
术语“治疗”，是指向个体施用化合物或组合物以预防或消除疾病，包括降低个体中的疾病尺寸或疾病数目；阻滞或减缓个体中的疾病进程；抑制或减缓个体中新疾病的发展；降低目前患有或先前曾患有疾病的个体的症状和/或复发的频率或严重程度；和/或延长(即，提高)个体的寿命。在一些实施例中，疾病的治疗包括疾病或其症状的治愈、缩短持续时间、减轻、预防、减缓或抑制进展或恶化，或者预防或延缓发病。
在文本中，术语“对象”、“个体”、“生物体”或“患者”可互换，包括脊椎动物，优选哺乳动物。本发明中，哺乳动物是人、非人灵长类、家养动物(例如，犬、猫、绵羊、牛、山羊、猪、马等)、实验动物(例如，小鼠、大鼠、兔、豚鼠等)以及圈养的动物(例如，动物园的动物)。本文使用的术语“动物”还包括人。术语“对象”、“个体”包括患者，即，患病的动物，优选人。。
术语“样品”可以是根据本发明可用的任何样品，包括生物样品，例如组织样品(包括体液)和/或细胞样品，可以以常规方式获得，例如通过手术或组织活检(包括钻孔活检)和通过采集血液、支气管抽出物、痰、尿、排泄物或其他体液。根据本发明，术语“样品”还包括经加工的样品，例如生物样品或分离物，例如核酸和肽/蛋白质分离物以及福尔马林处理石蜡包埋组织等。优选地，样品包含待检测(例如，待进行癌症诊断)器官的细胞或组织。例如，如果待诊断癌症为肺癌，则样品可包含从肺获得的细胞或组织。样品可以是来自含有肿瘤细胞或癌细胞或者怀疑含有肿瘤细胞或癌细胞的患者组织样品，包括血液、体液、原发肿瘤或从肿瘤转移获得的组织样品、或者任何其他包含肿瘤细胞或癌细胞的样品。
在一个方面，本发明提供了能够识别包含SEQ ID NO：7(KNKKIYDGG)所示的序列的抗原肽的抗体或抗原结合片段；还能够识别claudin18.2中包含SEQ ID NO：7所示的序列的表位的抗体或抗原结合片段。
在一个方面，本发明提供了能够识别包含SEQ ID NO：6(KNKKIYDGGART)所示的序列的抗原肽的抗体或抗原结合片段；还能够识别claudin18.2中包含SEQ ID NO：6所示的序列的表位的抗体或抗原结合片段。
在一个方面，本发明提供了由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株(也称为杂交瘤细胞株9G5)产生的抗体。其中，CCTCCNO.C202007保藏的杂交瘤细胞株(也称为杂交瘤细胞株14F8)产生的抗体称为抗体9，CCTCCNO.C202008保藏的杂交瘤细胞株产生的抗体称为抗体7，保藏日期均为：2020年1月3日，保藏地点为中国典型培养物保藏中心，地址为中国武汉，武汉大学，邮编430072。
在一个方面，本发明提供了组合物(例如，诊断组合物)或检测试剂盒，其包含本文所述的抗体或抗原结合片段，或者抗体和/或抗原结合片段的组合。该诊断组合物或检测试剂盒可用于本发明的方法，例如本发明的用于诊断、检测或监测的方法。这些试剂盒可任选地包含可检测标记物，例如指示酶、放射性标记物、荧光团或顺磁性颗粒。所述试剂盒可包含信息手册，例如，用于说明如何使用试剂来实施本文所公开的方法的手册。
当用于上文所述的方法中时，可为本文所述的抗体提供发挥以下功能的标记物：(i)提供可检测信号；(ii)与第二标记物相互作用以修饰所述第一或第二标记物所提供的可检测信号，例如FRET(荧光共振能量转移，Fluorescence Resonance Energy Transfer)；(iii)通过电荷、疏水性、形状或其他物理参数影响迁移率(例如，电泳迁移率)，或者(iv)提供捕获部分，例如亲和力、抗体/抗原或离子络合。
适合作为标记物的为以下结构：例如荧光标记物、发光标记物、发色团标记物、放射性同位素标记物、同位素标记物，优选稳定的同位素标记物、同量异位素标记物(isobaric label)、酶标记物、颗粒标记物(特别是金属颗粒标记物、磁性颗粒标记物、聚合物颗粒标记物)、小有机分子(例如，生物素、受体的配体或结合分子(例如，细胞黏附蛋白或凝集素))、可通过使用结合剂检测的包含核酸和/或氨基酸残基的标记物序列等。标记物非限制性地包括硫酸钡、碘西他酸、碘番酸、胺碘苯丙酸钙(calcium ipodate)、泛影酸钠、泛影葡胺、甲泛葡胺、酪泮酸钠和放射诊断剂(包括正电子发射 体，(例如氟-18和碳-11)、γ发射体(例如，碘-123、锝-99m、碘-131和铟-111)、核磁共振核素(例如，氟和钆))。
“处于风险”意为被鉴定为与一般人群相比具有高于疾病(如癌症)发生正常几率的对象(即患者)。此外，已患有或目前患有疾病(特别是癌症)的对象是具有升高的疾病发生风险的对象，因为该对象可能继续发生疾病。目前已患有或曾患有癌症的对象还具有升高的癌转移风险。
在本文中，还涉及用于诊断、检测或监测癌症的方法，其特征在于，所述方法包括：将待检测样品与本发明的抗体或抗原结合片段，或者缀合物相接触；检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量。可以使用该方法来检测对象是否患有肿瘤或者是否处于发生肿瘤的风险中，或者是否患有癌疾病或是否处于发生癌疾病风险中。在一个实施例中，所述测量样品是组织或细胞样品，例如包含细胞(如肿瘤细胞)的样品。所述复合物是由本发明的抗体、抗原结合片段或缀合物与上述样品中的细胞所表达的CLAUDIN18.2之间形成的。
在本文所述的诊断、检测癌症的方法中，使用本发明的抗体或抗原结合片段、或者所述缀合物与取自受试者的细胞样品、正常样品(或对照样品)分别进行反应，当与受试者的细胞样品反应得到的复合物的量显著高于与正常样品(或对照样品)反应得到的复合物的量时，提示受试者可能罹患癌症。正常样品和显著升高的定义如上本文所述。
在本文所述的监测癌症的方法中，针对受试者，在两个相隔一段时间的两个时间点，例如第一时间和第二时间，分别检测来自受试者的第一样品(第一时间获取的)和第二样品(第二时间获取的)中的表达claudin18.2的细胞与所述抗体或抗原结合片段、或者所述缀合物形成的复合物的量，并通过两次检测的复合物的量之间的差异，来评估受试者癌疾病的变化，例如消退、发展等。
例如与第一时间获取的第一样品相比，检测结果显示第二样品中形成的复合物明显减少，则可以提示受试者的癌症或肿瘤的消退或患病风险降低。例如与第一时间获取的第一样品相比，检测结果显示第二样品中形成的复合物明显升高，则可以提示受试者的癌症或肿瘤的转移、复发或患病风险升高。
在本文中，还涉及一种用于分析肿瘤是否能够通过靶向claudin18.2的肿瘤疗法来治疗的方法，其特征在于，所述方法包括：(i)使包含肿瘤细胞的样品与本文的抗体 或抗原结合片段，或者与本文的缀合物相接触；(ii)检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量。
其中，在一个具体的实施例中，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、肝癌细胞、头颈癌细胞、或胆道系统癌症细胞；优选的，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、或者胆道系统癌症细胞。在一个实施例中，所述复合物是所述抗体、抗原结合片段或缀合物与来自受试者的样品中的癌细胞所表达的claudin18.2之间形成的。
在本文所述的分析方法中，针对同一受试者，在进行治疗之前和进行靶向治疗之后的两个时间点，例如第一时间(进行靶向治疗之前)和第二时间(进行靶向治疗之后)，分别检测来自受试者的第一样品(第一时间获取的)和第二样品(第二时间获取的)中的表达claudin18.2的细胞与所述抗体或抗原结合片段、或者所述缀合物形成的复合物的量，并通过两次检测的复合物的量之间的差异，来评估受试者是否能够通过靶向claudin18.2的肿瘤疗法来治疗。
在一个方面，本发明提供了试剂盒，其包含本文所述的抗体或抗原结合片段或者抗体和/或抗原结合片段的组合。该试剂盒可用于本发明的方法，例如本发明的用于诊断、检测或监测的方法。这些试剂盒可任选地包含可检测标记物，例如指示酶、放射性标记物、荧光团或顺磁性颗粒。所述试剂盒可包含信息手册，例如，用于说明如何使用试剂来实施本文所公开的方法的手册。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.抗体的筛选
体外合成Claudin18.2 C端多肽(第191位甲硫氨酸-第261位缬氨酸，参见SEQ ID NO:3)作为免疫原。
SEQ ID NO:3的序列如下所示：
Figure PCTCN2021070139-appb-000003
其次选用6～8周龄的雌性Balb/C小鼠进行免疫：免疫原与弗氏佐剂或Gerbu MM佐剂按1:1混合，免疫剂量为(50-100μg)/只，经皮下或腹腔进行免疫，每隔一周或两周免疫一次，共免疫三次或四次。在脾切除术前两天，对小鼠进行加强免疫。分离免疫小鼠的脾细胞，与小鼠骨髓瘤细胞SP2/0(ATCC,CRL 1581)按照公开的标准方案进行融合(Kohler、Milstein，1975)。经HAT培养基筛选培养后，通过ELISA检测杂交瘤上清，一共得到139个阳性克隆，其中有11个强阳性克隆。
通过无血清培养的方法表达这11个候选抗体，并通过Protein G纯化制备这11个候选抗体用于后续检测分别命名为抗体1、抗体2、抗体3、抗体4、抗体5、抗体6、抗体7、抗体8、抗体9、抗体10、抗体11。将所得的11个抗体进行Western Blot鉴定，检测与293T-Claudin18.2以及293T-Claudin18.1细胞裂解物的结合。Western步骤为：293T-claudin18.1、293T-claudin18.2细胞经RIPA裂解液(强)提取总蛋白(碧云天，P0013B)，蛋白定量后以30μg/孔进行上样，在12％分离胶中以恒压跑胶，然后200mAh恒流转膜1h，5％脱脂奶粉封闭然后顺序加入一抗(10μg/mL)Claudin18 Polyclonal Antibody 1:200稀释(Thermo Fisher,CAT 38-8000或者anti-flag-HRP 1:500稀释或者Anti-Claudin18.2 antibody[EPR19202-244]1:200稀释(Abcam，ab222512)；二抗(羊抗鼠IgG-HRP 1:1000，羊抗兔IgG-HRP 1:1000)孵育，充分洗膜后在化学发光成像仪下进行显像并拍照。B-actin作为内参对照。检测结果如图2A和图2B显示。
根据检测结果，挑取7个候选抗体通过免疫组化的方式进行进一步的筛选。并且挑取抗体7和抗体9两株杂交瘤进行了菌种保藏。保藏信息为：两株杂交瘤细胞保藏于中国典型培养物保藏中心(中国武汉)命名和登记号如下：抗体7(也称为抗体9G5)，保藏号：CCTCCNO.C202008，保藏日期：2020年1月3日。抗体9(也称为抗体14F8)，保藏号：CCTCCNO.C202007，保藏日期：2020年1月3日。
实施例2.组织学分析
从这11株抗体中筛选出7株抗体(抗体5、抗体6、抗体9、抗体11、抗体4、抗体8和抗体7)通过免疫组化评价这些抗体与福尔马林固定的石蜡包埋组织(FFPE)中的其抗原的结合能力的组织特异性。
对照抗体H-43-14A_2(在本文中也称43-14A2)的的序列来源于专利CN104321345A，轻链可变区的序列如SEQ ID NO：4(divmtqaafsipvtlgtsasiscrssknllhsdgitylywylqrpgqspqlliyrvsnlasgvpnrfsgsesgtdftlrisrveae dvgvyycvqvlelpftfgggtkleikr)所示，重链可变区的序列如SEQ ID NO：5(iqlvqsgpelkkfgetvkisckasgytftdysihwvkqapgkglkwmgwintetgvptyaddfkgrfafsletsastaylqinnlknedtatyfcarrtgfdywgqgttltvss)所示，通过体外重组表达的方式制备了含有该抗体轻重链可变区的鼠单克隆抗体。对照抗体EPR19202-244自Abcam。
免疫组化方法：在福尔马林固定的石蜡包埋样品载片上进行免疫组化(IHC)。在载片脱蜡后，用3％过氧化氢室温孵育后置于煮沸的pH9.0的Tris缓冲液中用微波炉加热10min进行抗原修复，然后用0.2–2μg/mL的Claudin18.2抗体(抗体4、抗体5、抗体6、抗体7、抗体8、抗体9、和抗体11)以及43-14A2在4℃孵育过夜。使用基于聚合物的抗体(Dako REAL TM EnVision TM Detection System,Peroxidase/DAB+,Rabbit/Mouse)和底物显色液(Dako REAL TM EnVision TM Detection System,Peroxidase/DAB+)用辣根过氧化物酶结合的第二抗体对抗体结合进行可视化。然后用苏木素复染，再由评价者进行评价。
用抗体4、抗体5、抗体6、抗体7、抗体8、抗体9、抗体11、和43-14A2对恒河猴(恒河猴GenBank登记号XP_001114708.1，与人CLDN18.2的序列(NP_001002026.1)同源性是98.85％)的胃及肺组织切片进行染色，对染色结果特异性进行评价；用正常人组织芯片进行免疫组化染色，确认抗体对正常人组织染色结果的特异性；用人胃癌和肺癌切片进行免疫组化反应，确认抗体对人肿瘤组织的特异性和灵敏度；用人胃癌组织芯片进行免疫组化染色，评价抗体的灵敏度。
组织学评价：染色结果从染色强度和染色阳性肿瘤细胞％两个方面进行评价。染色强度：任何强度的细胞膜或细胞浆染色均认为阳性染色，染色强度分为阴性(-)、弱阳性(1+)、阳性(2+)和强阳性(3+)。
1.抗体对恒河猴的胃及肺组织切片的染色
抗体对恒河猴胃及肺组织染色的结果如表1所示，抗体4、抗体5、抗体6、抗体7、抗体8、抗体9、和抗体11对恒河猴胃黏膜上皮细胞有不同程度的染色，染色强度从1+到3+不等，个别抗体会产生非特异性染色/背景染色；对恒河猴肺组织有阴性及阳性反应。抗体4、抗体5、抗体7、抗体8和抗体9对胃组织特异性染色，且对肺组织没有染色；而对照抗体43-14A2在肺组织有非特异性染色；对照抗体EPR19202-244在说明书推荐的工作浓度0.37μg/mL下对胃组织特异性染色强度高，且对肺组织没有染色。
表1.Claudin18.2抗体对恒河猴胃及肺组织染色
Figure PCTCN2021070139-appb-000004
Figure PCTCN2021070139-appb-000005
2.抗体9对正常人组织的染色
抗体9在工作浓度0.2μg/mL下，与对照抗体EPR19202-244在工作浓度0.37μg/mL下，分别对来源于7例正常人的正常器官共计24种，每种2－6例，每例1点的组织芯片(上海芯超，芯片编号：HorgN090PT02)进行染色，抗体9仅在胃粘膜上皮细胞呈现阳性染色，其他组织均为阴性染色，但是EPR19202-244在骨骼肌和心肌组织呈现较明显的背景染色。6例胃粘膜中的胃粘膜上皮细胞，抗体9和EPR19202-244均呈强阳性染色；EPR19202-244抗体在3例心肌中的1例、7例骨骼肌中的1例中有背景染色，而抗体9均为阴性染色。其余4例甲状腺、3例舌、4例食管上皮、2例十二指肠粘膜、6例空肠粘膜、3例回肠粘膜、5例阑尾、4例结肠粘膜、2例直肠粘膜、2例肝脏、2例胰腺、3例气管、5例肺、3例皮肤、1例精囊、3例前列腺、7例睾丸、4例膀胱、1例延脑、2例端脑、2例小脑、1例脑干、2例脾中，2个抗体均呈阴性染色。
3.抗体对人胃癌/肺癌组织染色
抗体7、抗体9、对照抗体43-14A2在工作浓度0.2μg/mL，与对照抗体Abcam EPR19202-244在说明书推荐工作浓度0.37μg/mL下对人胃癌或肺癌的组织样本进行染色，染色主要步骤为：将组织切片经过脱石蜡化和抗原修复，然后用CLDN18A2抗体孵育，接着用封闭缓冲液孵育样品，然后用辣根过氧化物酶(HRP)标记的二抗进行孵育，最后加入显色底物进行显色。
对胃癌样本F163060A1(购自桂林泛谱)的染色结果如图3所示。对胃癌样本2018-50829E(购自桂林泛谱)的染色结果如图4所示。对肺癌样本2018-55164F(购自桂林泛谱)的染色结果如图5所示。染色的统计结果见表2。
表2.Claudin18.2抗体对人胃癌/肺癌组织染色
Figure PCTCN2021070139-appb-000006
4.抗体9对胃癌组织芯片的染色
选择抗体9对胃癌及癌旁组织芯片染色分析。抗体9在工作浓度0.2μg/mL与Abcam EPR19202-244在说明书推荐工作浓度0.37μg/mL下，分别对75例胃癌及癌旁组织芯片(上海芯超，芯片编号：HStm-Ade150CS4-01)进行染色。具体数据见表3，染色结果统计见表4。
对于癌组织，抗体9检出率为60.0％(45/75)，≥2+染色程度的有41.3％(31/75)；Abcam EPR19202-244检出率为41.3％(31/75)，≥2+染色程度的仅有17.3％(13/75)。
对于癌旁组织，抗体9检出率为100％(75/75)，≥2+染色程度的有90.6％(68/75)；Abcam EPR19202-244检出率为98.6％(74/75)，≥2+染色程度的有89.3％(67/75)。由此可见在特异性和灵敏度上，抗体9均优于Abcam EPR19202-244。表3.抗体9对胃癌组织芯片的染色
Figure PCTCN2021070139-appb-000007
Figure PCTCN2021070139-appb-000008
Figure PCTCN2021070139-appb-000009
Figure PCTCN2021070139-appb-000010
Figure PCTCN2021070139-appb-000011
表4.Claudin18.2诊断抗体对胃癌组织芯片的染色结果统计
Figure PCTCN2021070139-appb-000012
实施例4.表位分析
ELISA方法：通过检测抗体7和抗体9与抗原的不同肽段的结合情况，分析抗体结合的表位。
基于免疫原(Claudin18.2 C端多肽)序列，按照长度15aa、重叠12aa的原则分成21个肽段，化学合成相应肽段并在N端加生物素修饰。用Neutravidin plate(CAT:15128 Invitrogen)捕获各肽段，然后用10μg/mL的Claudin18.2抗体(抗体7和抗体9)孵育。二抗使用HRP标记羊抗鼠(CAT:A0168 SIGMA)，底物显色液显色5分钟，然后加2M H2SO4终止显色，通过测定OD450nm吸光度定性抗体和个各肽段的结合情况
ELISA结果见图6，抗体9的结合肽段序列特征如SEQ ID NO：6所示：KNKKIYDGGART；抗体7的结合肽段序列特征如SEQ ID NO：7所示：KNKKIYDGG。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (23)

  1. 抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段能够识别包含SEQ ID NO：7(KNKKIYDGG)所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：7所示的序列的表位；
    优选的，所述抗体或抗原结合片段能够识别包含SEQ ID NO：6(KNKKIYDGGART)所示的序列的肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：6所示的序列的表位。
  2. 如权利要求1所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段能够识别包含SEQ ID NO：8(GFGSNTKNKKIYDGG)、9(SNTKNKKIYDGGART)或10(KNKKIYDGGARTEDE)所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：8、9或10所示的序列的表位；
    优选的，所述抗体或抗原结合片段能够识别包含SEQ ID NO：9或10所示的序列的抗原肽；或者所述抗体或抗原结合片段能够识别claudin18.2中包含SEQ ID NO：9或10所示的序列的表位。
  3. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述抗体为由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体，其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7。
  4. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段是鼠源抗体、嵌合抗体、人源化的抗体、或全人抗体。
  5. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段是由登记号为CCTCCNO.C202007(抗体9)或CCTCCNO.C202008(抗体7)的保藏的杂交瘤细胞株产生的抗体的人源化抗体或嵌合抗体。
  6. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段是由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体的突变体；其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7
    优选的，所述突变体与由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的同源性。
  7. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述抗体片段为由登记号为CCTCCNO.C202007或CCTCCNO.C202008的保藏的杂交瘤细胞株产生的抗体的抗体片段；
    优选的，所述抗体片段是scFv、Fab片段，Fd片段，Fv片段，F(ab’)2片段、或单域抗体，
    其中CCTCCNO.C202007杂交瘤细胞株产生的抗体为抗体9，CCTCCNO.C202008杂交瘤细胞株产生的抗体为抗体7。
  8. 如权利要求1-7任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段与表达claudin18.2的细胞相结合；
    优选的，所述claudin18.2的氨基酸序列如SEQ ID NO：2所示，SEQ ID NO：2为
    MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV。
  9. 如权利要求8所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段特异性结合胃组织；
    优选的，所述抗体或抗原结合片段特异性结合胃组织，不结合肺组织。
  10. 一种缀合物，其特征在于，所述缀合物包含权利要求1-9任一项所述的抗体或抗原结合片段，以及至少一个可检测标记物；
    优选的，所述的可检测标记物为荧光标记物、显色标记物、发光标记物、发色团标记物、放射性同位素标记物、同位素标记物、同量异位素标记物(isobaric label)、酶标记物、颗粒标记物、通过使用结合剂检测的核酸或蛋白。
  11. 一种杂交瘤，其能够产生权利要求1-9任一所述的抗体。
  12. 如权利要求11所述的杂交瘤，所述杂交瘤的保藏登记号为CCTCCNO.C202007或CCTCCNO.C202008。
  13. 一种检测待测样品claudin18.2表达情况的方法，其特征在于，所述方法包括：使待测样品与权利要求1-9任一所述的抗体或抗原结合片段，或者与权利要求10所述的缀合物相接触；检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的情况。
  14. 一种用于诊断、检测或监测癌症的方法，其特征在于，所述方法包括：
    将待检测样品与权利要求1-9任一所述的抗体或抗原结合片段，或者与权利要求10所述的缀合物相接触；检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量。
  15. 一种用于分析肿瘤是否能够通过靶向claudin18.2的肿瘤疗法来治疗的方法，其特征在于，所述方法包括：
    (i)使包含肿瘤细胞的样品与权利要求1-9任一所述的抗体或抗原结合片段，或者与权利要求10所述的缀合物相接触；
    (ii)检测所述抗体或抗原结合片段、或者所述缀合物与claudin18.2形成的复合物的量；
    优选的，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、肝癌细胞、头颈癌细胞、或胆道系统肿瘤；
    更优选的，所述肿瘤细胞为胃癌细胞、食管癌细胞、胰腺癌细胞、或胆道系统肿瘤。
  16. 一种检测试剂盒，其包含权利要求1-9任一所述的抗体或抗原结合片段，或者与权利要求10所述的缀合物。
  17. 权利要求13至15中任一项所述的方法，其中所述claudin18.2包含序列表的SEQ ID NO：2的氨基酸序列或其变体。
  18. 产生靶向claudin18.2的单克隆抗体的方法，其特征在于，所述方法包括采用含有SEQ ID NO：6、7、8、9或10所示的序列的多肽为免疫原，或者用与SEQ ID NO：6、7、8、9或10所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性的多肽为免疫原，对动物进行免疫后，取动物的B细胞与骨髓瘤细胞进行融合，得到杂交瘤细胞，以产生单克隆抗体；
    优选的，所述动物为鼠；
    优选的，所述B细胞来源于脾细胞。
  19. 如权利要求18所述的方法，其特征在于，还包括从所述杂交瘤细胞中分离所述单克隆抗体。
  20. 如权利要求18的方法，其特征在于，用SEQ ID NO：3(MMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV)所示的多肽为免疫原。
  21. 权利要求18的方法，其中所述单克隆抗体特异性结合所述抗原。
  22. 权利要求19的方法，还包括产生保留与所述抗原特异性结合的所述单克隆抗体的修饰形式。
  23. 权利要求20的方法，其中所述单克隆抗体的修饰形式是人源化抗体。
PCT/CN2021/070139 2020-01-03 2021-01-04 抗Claudin18.2的抗体及其应用 WO2021136550A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
KR1020227026732A KR20220121873A (ko) 2020-01-03 2021-01-04 항-클라우딘 18.2 항체 및 그 용도
EP21736163.3A EP4086287A4 (en) 2020-01-03 2021-01-04 ANTI-CLAUDIN 18.2 ANTIBODIES AND USE THEREOF
AU2021204861A AU2021204861B2 (en) 2020-01-03 2021-01-04 Anti-claudin 18.2 antibody and use thereof
CA3166498A CA3166498A1 (en) 2020-01-03 2021-01-04 Anti-claudin 18.2 antibody and use thereof
CN202211674726.4A CN117343186A (zh) 2020-01-03 2021-01-04 抗Claudin18.2的抗体及其应用
US17/790,657 US20230061005A1 (en) 2020-01-03 2021-01-04 Anti-claudin 18.2 antibody and use thereof
JP2022540843A JP7543411B2 (ja) 2020-01-03 2021-01-04 抗クロ―ディン１８．２の抗体及びその使用
CN202180001577.2A CN113272332B (zh) 2020-01-03 2021-01-04 抗Claudin18.2的抗体及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010006872.4 2020-01-03
CN202010006872 2020-01-03

Publications (1)

Publication Number Publication Date
WO2021136550A1 true WO2021136550A1 (zh) 2021-07-08

Family

ID=76687340

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/070139 WO2021136550A1 (zh) 2020-01-03 2021-01-04 抗Claudin18.2的抗体及其应用

Country Status (8)

Country Link
US (1) US20230061005A1 (zh)
EP (1) EP4086287A4 (zh)
JP (1) JP7543411B2 (zh)
KR (1) KR20220121873A (zh)
CN (2) CN113272332B (zh)
AU (1) AU2021204861B2 (zh)
CA (1) CA3166498A1 (zh)
WO (1) WO2021136550A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途
WO2019173420A1 (en) * 2018-03-08 2019-09-12 Phanes Therapeutics, Inc. Anti-claudin 18.2 antibodies and uses thereof
WO2019219089A1 (en) * 2018-05-18 2019-11-21 Bridge Health Bio-Tech Co., Ltd Anti-claudin 18.2 antibodies and uses thereof

Family Cites Families (2)

* 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
CN110606891B (zh) * 2018-06-17 2022-12-06 上海健信生物医药科技有限公司 一种针对人cldn18.2的抗体分子，抗原结合片段及其医药用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108047331A (zh) * 2012-05-09 2018-05-18 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
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
WO2019173420A1 (en) * 2018-03-08 2019-09-12 Phanes Therapeutics, Inc. Anti-claudin 18.2 antibodies and uses thereof
WO2019219089A1 (en) * 2018-05-18 2019-11-21 Bridge Health Bio-Tech Co., Ltd Anti-claudin 18.2 antibodies and uses thereof
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. XP _001114708.1
"Molecular cloning: A Laboratory Manual", 2002, SCIENCE PRESS
FISCHER, R. ET AL., BIOL. CHEM., vol. 380, 1999, pages 825 - 839
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KOZAK, J. BIOL. CHEM., vol. 266, 1991, pages 19867 - 19870
MORRISON, S., SCIENCE, vol. 229, 1985, pages 1202
NEDDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
PEARSONLIPMAN, PROC .NATL ACAD.SCI.USA, vol. 85, 1988, pages 2444
POLLOCK, J. IMMUNOL. METH., vol. 231, 1999, pages 147 - 157
See also references of EP4086287A4
SMITHWATERMAN, ADS APP. MATH., vol. 2, 1981, pages 482
SPIEKER-POLET ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 92, 1995, pages 9348
VERMA, R. ET AL., J. IMMUNOL. METH., vol. 216, 1998, pages 165 - 181

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用

Also Published As

Publication number Publication date
AU2021204861A1 (en) 2022-07-28
EP4086287A4 (en) 2024-03-06
AU2021204861B2 (en) 2024-10-17
CA3166498A1 (en) 2021-07-08
CN113272332B (zh) 2022-11-18
JP2023510209A (ja) 2023-03-13
CN113272332A (zh) 2021-08-17
EP4086287A1 (en) 2022-11-09
US20230061005A1 (en) 2023-03-02
KR20220121873A (ko) 2022-09-01
CN117343186A (zh) 2024-01-05
JP7543411B2 (ja) 2024-09-02

Similar Documents

Publication Publication Date Title
US20240309108A1 (en) Antibodies against claudin 18.2 useful in cancer diagnosis
US11919951B2 (en) Antibodies useful in cancer diagnosis
WO2021136550A1 (zh) 抗Claudin18.2的抗体及其应用
RU2815883C1 (ru) Антитела, пригодные в диагностике рака
JP2023540452A (ja) 抗体を使用した細胞表面ｍｉｃａ及びｍｉｃｂの検出
NZ787569A (en) Antibodies useful in cancer diagnosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21736163

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3166498

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022540843

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2021204861

Country of ref document: AU

Date of ref document: 20210104

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20227026732

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021736163

Country of ref document: EP

Effective date: 20220803