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WO2021160154A1 - Cldn18.2抗体及其用途 - Google Patents

Cldn18.2抗体及其用途 Download PDF

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Publication number
WO2021160154A1
WO2021160154A1 PCT/CN2021/076481 CN2021076481W WO2021160154A1 WO 2021160154 A1 WO2021160154 A1 WO 2021160154A1 CN 2021076481 W CN2021076481 W CN 2021076481W WO 2021160154 A1 WO2021160154 A1 WO 2021160154A1
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antibody
seq
cancer
cells
antigen
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PCT/CN2021/076481
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English (en)
French (fr)
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周清
许传营
粘伟红
何向宇
张新敏
郑欣桐
何峰
肖婧
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上海诗健生物科技有限公司
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Priority to US17/798,863 priority Critical patent/US20230312704A1/en
Priority to JP2022573794A priority patent/JP7663883B2/ja
Priority to KR1020227030885A priority patent/KR20220139357A/ko
Priority to CA3167299A priority patent/CA3167299A1/en
Priority to AU2021220887A priority patent/AU2021220887B2/en
Priority to EP21753717.4A priority patent/EP4105237A4/en
Priority to CN202180013663.5A priority patent/CN115427453B/zh
Publication of WO2021160154A1 publication Critical patent/WO2021160154A1/zh
Priority to JP2024171863A priority patent/JP2025011153A/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • G01N33/57492—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 involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to the field of antibody technology, as well as to the use and preparation method of the antibody. Specifically, it relates to a CLDN18.2 antibody and its use.
  • Claudin is a type of transmembrane protein with a molecular weight of 20-30kDa. Its family has 24 members and is expressed in multiple tissues. It is an important molecule that forms tight junctions (TJ) between cells. . Epithelial cells or endothelial cells form highly ordered tissue associations through tightly binding molecules (including Claudin molecules) expressed on the cell surface, that is, tight junctions between cells, which control the flow of molecules in the epithelial cell gap. TJ plays a vital role in maintaining the stable state of epithelial cells or endothelial cells under normal physiological conditions and maintaining cell polarity.
  • claudin 18 (Claudin18, CLDN18), which includes two variants formed by splicing: CLDN18 splice variant 1 (Claudin18.1, CLDN18.1) and CLDN18 splice variant 2 (Claudin18.2, CLDN18.2).
  • CLDN18.2 (Genbank accession numbers NM_001002026, NP_001002026) is a transmembrane protein with a molecular weight of 27.8kDa, and both the N-terminal and C-terminal of the protein are located in the cell.
  • the main conformation of the protein contains 4 transmembrane domains (TMD) and 2 extracellular loops (ECL).
  • CLDN18.2 is highly conserved in a variety of mammals, with a total length of 261 amino acids, of which amino acids 1-6 are the N-terminal intracellular region, amino acids 7-27 are transmembrane region 1 (TMD1), and positions 28-78 Amino acids are extracellular loop 1 (ECL1), amino acids 79-99 are transmembrane region 2 (TMD2), amino acids 123-144 are transmembrane region 3 (TMD3), and amino acids 100-122 are the cell connecting TMD2 and TMD3.
  • amino acids 1-6 are the N-terminal intracellular region
  • amino acids 7-27 are transmembrane region 1 (TMD1)
  • positions 28-78 Amino acids are extracellular loop 1 (ECL1)
  • amino acids 79-99 are transmembrane region 2 (TMD2)
  • amino acids 123-144 are transmembrane region 3 (TMD3)
  • amino acids 100-122 are the cell connecting TMD2 and TMD3.
  • amino acids at positions 145-167 are extracellular loop 2 (ECL2)
  • amino acids at positions 168-190 are transmembrane region 4 (TMD4)
  • amino acids at positions 191-261 are the C-terminal intracellular region.
  • ECL2 extracellular loop 2
  • TMD4 transmembrane region 4
  • amino acids at positions 191-261 are the C-terminal intracellular region.
  • N-glycosylation motif on asparagine at position 116 There is a classical N-glycosylation motif on asparagine at position 116.
  • CLDN18.1 and CLDN18.2 differ only in the first 26 amino acid sequences of the N-terminus in the region of the protein N-terminus-transmembrane region 1-extracellular loop 1, and the remaining sequences are the same.
  • CLDN18.2 is expressed at a high level in tumor cells of stomach, pancreas, esophagus and other tissues, especially adenocarcinoma subtype tumor cells.
  • CLDN18.1 is selectively expressed in epithelial cells of lung and stomach tissues;
  • CLDN18.2 is only expressed in gastric glandular epithelial mucosal tissues (differentiated glandular epithelial cells) with a short life span, but not in It is expressed in gastric epithelial stem cells, and differentiated glandular epithelial cells can be continuously supplemented by gastric epithelial stem cells.
  • Companion Diagnostics methods to select tumor patients with high expression of CLDN18.2 in clinical trials and clinical applications has important guiding significance for guiding the applicable patients of anticancer drugs targeting CLDN18.2.
  • the patient’s tumor specimens are usually fixed-processed paraffin-embedded (FFPE) tissues, and the natural conformation of tumor-specific antigens will change during FFPE processing.
  • FFPE paraffin-embedded
  • special epitopes different from the natural conformation will be exposed (Biotech Histochem, 2009, 84(5): 207-215), so usually screening can be specific for cancer-specific antigens on tumor FFPE tissues.
  • Sexually bound antibodies for diagnosis are usually fixed-processed paraffin-embedded (FFPE) tissues, and the natural conformation of tumor-specific antigens will change during FFPE processing.
  • special epitopes different from the natural conformation will be exposed (Biotech Histochem, 2009, 84(5): 207-215), so usually screening can be specific for cancer-specific
  • the invention relates to a preparation method, screening, sequence and application of an antibody that specifically recognizes the CLDN18.2 antigen in a tumor and its FFPE tissue.
  • the present invention provides a CLDN18.2 antibody for diagnosing cancer, which can recognize CLDN18.2 antigen molecules in fixed tissue samples to diagnose cancer lesions including gastric cancer, pancreatic cancer, or esophageal cancer.
  • the CLDN18.2 antibody provided by the present invention can also effectively treat cancer lesions including gastric cancer, pancreatic cancer or esophageal cancer.
  • this application relates to:
  • a CLDN18.2 antibody comprising heavy chain CDRs and light chain CDRs selected from the group consisting of
  • heavy chain CDRs are:
  • the antibody according to item 1 comprising the heavy chain variable region shown in SEQ ID NO: 36 or SEQ ID NO: 37.
  • the antibody according to item 1 comprising the light chain variable region shown in SEQ ID NO: 38 or SEQ ID NO: 39.
  • the antibody of any one of items 1 to 3, comprising a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 39.
  • the antibody of item 9 which is Fab, Fab'-SH, Fv, scFv or (Fab')2.
  • the antibody according to any one of items 1-8 which is a multispecific antibody or a bispecific antibody.
  • the multispecific antibody or bispecific antibody comprises a binding domain that binds to a second biomolecule, wherein the second biomolecule is a cell surface antigen.
  • tumor antigen is selected from: CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET , Steap1 and TenB2.
  • An immunoconjugate comprising a therapeutic agent, an interferon gene stimulating factor (STING) receptor agonist, a cytokine, a radionuclide or an enzyme linked to the antibody of any one of items 1-10.
  • STING interferon gene stimulating factor
  • a fusion protein or polypeptide comprising the antibody or antigen-binding fragment of any one of items 1-10.
  • a pharmaceutical composition comprising the antibody of any one of items 1-14, the immunoconjugate of any one of items 15-17, the fusion protein or the polypeptide of item 18.
  • nucleic acid of item 20 comprising a polynucleotide sequence encoding the amino acid sequence of one of SEQ ID NOs: 36-39.
  • nucleic acid of item 21 comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment of any one of items 1-10.
  • a vector comprising the polynucleotide sequence of any one of items 20-22.
  • a host cell comprising the polynucleotide sequence of any one of items 20-22 or the vector of item 23.
  • a method for preparing the antibody of any one of items 1-10 comprising culturing the host cell of item 24 and isolating the antibody from the culture.
  • a cancer detection reagent comprising the antibody or antigen-binding fragment thereof according to any one of items 1-10.
  • a cancer diagnosis kit comprising the cancer detection reagent according to any one of items 29 to 31.
  • kits according to item 32 wherein the cancer is gastric cancer, pancreatic cancer or esophageal cancer.
  • a method for diagnosing cancer in a subject comprising combining the antibody of any one of items 1-14, the fusion protein or polypeptide of item 18, or the detection reagent of any one of items 29-31 with the subject Contact with the tissue sample.
  • tissue sample is a body fluid (for example, blood, urine) and a tissue section (for example, a tissue biopsy sample) of the subject.
  • body fluid for example, blood, urine
  • tissue section for example, a tissue biopsy sample
  • a method for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody of any one of items 1-14, the immunoconjugate of any one of items 15-17, and the fusion protein of item 18 Or a polypeptide, or the pharmaceutical composition of item 19.
  • FIG. 8 IHC staining of positive control antibody on HEK293-hCLDN18.2(A), HEK293-hCLDN18.1(B) and HEK293-WT(C) cell block paraffin-embedded (FFPE) tissue sections
  • Figure 10 Flow cytometric analysis of hybridoma monoclonal supernatant on HEK293-hCLDN18.2 cells
  • antibody herein is used in a broad sense to encompass various antibody structural molecules that bind to CLDN 18.2 and include one or more of the CDR domains disclosed herein, including but not limited to monoclonal antibodies, polyclonal antibodies, and polyclonal antibodies.
  • Specific antibodies such as bispecific antibodies
  • antibody fragments such as Fv, Fab, Fab', Fab'-SH, F(ab') 2
  • linear antibodies and single-chain antibody molecules such as scFv
  • Those skilled in the art can fuse one or more CDR domains disclosed in the present invention with one or more other polypeptide sequences to prepare functional fusion proteins or polypeptide molecules that bind to CLDN 18.2 molecules, such as vaccines, cell membrane receptors Antagonists, signal pathway regulators, chimeric antigen receptor molecules, etc.
  • CLDN18.2 CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy
  • the modifier "monoclonal” in the term “monoclonal antibody” means that the antibody is obtained from a substantially homogeneous antibody population and contains only traces of naturally occurring mutations or occurs during the preparation of monoclonal antibodies. Mutation. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody in the monoclonal antibody preparation is directed against a single epitope on the antigen.
  • the monoclonal antibodies of the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci.
  • full-length antibody and “whole antibody” refer to an antibody having a structure that is substantially similar to the structure of a natural antibody, and the terms are used interchangeably herein.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • a “chimeric antibody” is an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species and at least a portion of the constant region derived from another species.
  • the chimeric antibody may comprise murine variable regions and human constant regions.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVR and amino acid residues derived from human FR.
  • a humanized antibody will comprise at least one, and usually two, substantially the entire variable domain, wherein all or substantially all of the HVR (e.g., CDR) corresponds to those of the non-human antibody, and all or substantially All FRs correspond to those of human antibodies.
  • a humanized antibody may comprise at least a part of the constant region of an antibody derived from a human antibody.
  • a "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.
  • human common framework is a framework representing the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences.
  • human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences.
  • the sequence subgroup is as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volume 1-3.
  • the subgroup is subgroup ⁇ I as described by Kabat et al. (supra).
  • the subgroup is subgroup III as described by Kabat et al. (supra).
  • Human antibodies may also be referred to as “human antibodies”, “fully human antibodies” or “fully human antibodies”, antibodies whose amino acid sequence corresponds to those produced by humans or human cells. This human antibody definition specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • Human antibodies can be prepared using various techniques known in the art, including phage display library technology, such as those described in the following documents: Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J . Mol. Biol., 222:581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.
  • Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been disabled (e.g., immunized xenogeneic mice) (for XENOMOUSETM technology, see, e.g., U.S. Patent No. 6,075,181 and 6,150,584).
  • endogenous locus e.g., immunized xenogeneic mice
  • XENOMOUSETM technology see, e.g., U.S. Patent No. 6,075,181 and 6,150,584
  • human antibodies produced via human B-cell hybridoma technology see also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).
  • hypervariable region refers to an antibody variable domain having a sequence hypervariable region (also called a “complementarity determining region” or “CDR") and/or forming a structurally defined loop ("Hypervariable loop") and/or regions containing antigen contact residues ("antigen contacts”).
  • CDR sequence hypervariable region
  • Hypervariable loop structurally defined loop
  • antigen contacts regions containing antigen contact residues
  • an antibody contains 6 HVRs (CDR regions): 3 are in VH (H1, H2, H3) and 3 are in VL (L1, L2, L3).
  • Exemplary HVRs (CDR regions) herein include:
  • (d) A combination of (a), (b) and/or (c), including HVR (CDR region) amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).
  • HVR CDR region amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).
  • HVR CDR region residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra).
  • variable region refers to an antibody heavy chain or light chain domain involved in the binding of an antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains 4 conserved framework regions (FR) and 3 complementarity determining regions (CDR regions).
  • FR conserved framework regions
  • CDR regions complementarity determining regions
  • Antibody fragment refers to a molecule that contains a part of the intact antibody that binds to the antigen to which the intact antibody binds, in addition to the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; bifunctional antibodies; linear antibodies; single-chain antibody molecules (such as scFv); Specific antibodies.
  • cytotoxic agent refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioisotopes (such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents or drugs (such as methotrexate, sub Derimicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other inserts Agents); growth inhibitors; enzymes and fragments thereof, such as nuclear degrading enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; Known various anti-tumor drugs or anti-
  • an “immunoconjugate” is a conjugate of an antibody and one or more heterologous molecules (including but not limited to cytotoxic agents).
  • Interferon gene stimulator stimulate of interferon genes, STING
  • STING receptor agonists activate STING-dependent signaling pathways to promote the secretion of type I interferon and the expression of proteins related to antiviral and antitumor immunity, and block virus replication , Molecules that promote the immune response to cancer cells.
  • Such molecules such as cyclic dinucleotides, aminobenzimidazoles, xanthones and acridinones, benzothiophenes and benzodiones and other structural types of STING agonists.
  • Subject or “individual” is a mammal. Mammals include, but are not limited to, domesticated animals (such as cattle, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and large animals). mouse). In certain embodiments, the subject or individual is a human.
  • package insert is used to refer to the instructions usually included in the commercial packaging of therapeutic products, which contain information about indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings about the use of such therapeutic products information.
  • Bind refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise indicated, as used herein, "binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between binding partner members (eg, antibody and antigen). The affinity of a molecule X to its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • the "percentage of amino acid sequence homology (%)" with respect to the reference polypeptide sequence is defined as after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps if necessary to achieve the maximum sequence homology percentage and without any conservative substitutions
  • the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence is defined as after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps if necessary to achieve the maximum sequence homology percentage and without any conservative substitutions
  • the determination of the percentage of amino acid sequence homology can be achieved in various ways in the art, for example, software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) can be used for homology comparison. Those skilled in the art can determine the parameters suitable for the alignment of the sequences, including any algorithm required to achieve the maximum alignment over the entire length of the sequence being compared.
  • amino acid sequence homology% of the designated amino acid sequence A is the same as, with or with respect to the designated amino acid sequence B is calculated as follows:
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 when A and B are compared
  • Y is the total number of amino acid residues in B. It should be understood that in the case where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the amino acid sequence homology% of A and B will not be equal to the amino acid sequence homology% of B and A. Unless otherwise specified, all amino acid sequence homology% values used herein are obtained using the ALIGN-2 computer program.
  • the present invention relates to anti-CLDN18.2 antibodies.
  • the present invention provides an anti-CLDN18.2 antibody comprising at least 1, 2, 3, 4, 5 or 6 hypervariable regions (HVR) or complementarity determining regions selected from (CDR) binding domain:
  • HVR-H1 which contains the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 21 or is at least 90%, 95%, 96%, 97%, 98% with the sequence , An amino acid sequence with 99% homology
  • HVR-H2 which contains the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 22 or is at least 90%, 95%, 96%, 97% with the sequence , 98%, 99% homology of amino acid sequence
  • HVR-H3 which contains the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23 or is at least 90%, 95%, 96% with the sequence , 97%, 98%, 99% homology of amino acid sequences
  • HVR-L1 hypervariable regions
  • CDR complement
  • HVR-L2 which contains the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 28 or at least 90% of the sequence , 95%, 96%, 97%, 98%, 99% homology of amino acid sequences
  • HVR-L3 which includes the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 29 or is similar to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 29 Amino acid sequences with at least 90%, 95%, 96%, 97%, 98%, 99% homology in sequence.
  • variable heavy (VH) domain (region) possessed by the anti-CLDN18.2 antibody may include at least 90% sequence homology with the sequence SEQ ID NO: 36 or SEQ ID NO: 37 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology) amino acid sequence or sequence SEQ ID NO: 36 or SEQ ID NO: 37 amino acid sequence , And/or its light chain variable (VL) domain (region) comprising at least 90% sequence homology with the sequence SEQ ID NO: 38 or SEQ ID NO: 39 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology) of the amino acid sequence or the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.
  • the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following amino acid sequence:
  • the antibodies provided herein are antibody fragments.
  • Antibody fragments include but are not limited to Fab, Fab', Fab'-SH, (Fab') 2 , Fv and scFv fragments and other fragments described below.
  • Fab fragment antigen binding protein
  • Fab' fragment antigen binding protein
  • Fv fragment antigen binding protein
  • scFv fragments fragment antigen binding protein fragments
  • other fragments described below For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). See, for example, WO 93/16185 for scFv fragments.
  • Bifunctional antibodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, for example, EP 404,097; WO1993/01161. For trifunctional antibodies and tetrafunctional antibodies, see, for example, Hudson et al., Nat. Med. 9:129-134 (2003).
  • Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.
  • the single domain antibody is a human single domain antibody (see, for example, US Patent 6,248,516 B1).
  • Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of intact antibodies and recombinant host cell (such as E. coli or phage) production.
  • the antibodies provided herein are chimeric antibodies.
  • chimeric antibodies see, for example, U.S. Patent No. 4,816,567.
  • the chimeric antibody is a humanized antibody.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody contains one or more variable domains, in which all HVR (CDR) regions, or parts thereof, are derived from non-human antibodies, and FR (or parts thereof) are derived from human antibody sequences.
  • the humanized antibody optionally comprises at least a part of a human constant region.
  • some FR residues in a humanized antibody can be replaced with corresponding residues from a non-human antibody to repair or improve the affinity of the antibody.
  • the antibodies provided herein are human antibodies.
  • Human antibodies can be produced using various techniques known in the art.
  • Human antibodies can be prepared by administering immunogens to modified transgenic animals and then attacking with antigens to prepare complete human antibodies or complete antibodies with human variable regions.
  • Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or exists outside the chromosomes or is randomly integrated into the animal chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated.
  • U.S. Patent Nos. 6,075,181 and 6,150,584 description of XENOMOUSETM technology
  • U.S. Patent No. 5,770,429 U.S. Patent No. 7,041,870
  • U.S. Application Publication No. US No. 2007/0061900 The human variable regions derived from intact antibodies produced by such animals can be further modified, for example by combining them with different human constant regions.
  • Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described, see, for example, Boerner et al., J. Immunol., 147:86 (1991). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include, for example, U.S. Patent No.
  • Human antibodies can also be prepared by isolating Fv clone variable domain sequences selected from a phage display library of human origin. Such variable domain sequences can then be combined with the desired human constant domains.
  • the antibodies of the present invention with high affinity can be isolated by screening combinatorial libraries for antibodies with the activity of binding to CLDN18.2.
  • various methods for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art.
  • Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001), Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Edit Lo, Human Press, Totowa, NJ, 2003) and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).
  • VH and VL gene lineages are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then screened for antigen-binding phage.
  • Phages typically present antibody fragments as single chain Fv (scFv) fragments or Fab fragments.
  • Patents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication No. 2005/0079574, No. 2005/0119455, No. 2005/0266000, No. 2007/0117126, No. 2007/0160598, No. 2007/0237764, 2007/0292936 and 2009/0002360.
  • Antibodies or antibody fragments isolated from human antibody repertoires are referred to herein as human antibodies or human antibody fragments.
  • the anti-CLDN18.2 antibody provided herein is a multispecific antibody, such as a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
  • one binding specificity is for CLDN 18.2
  • the other binding specificity is for any other antigen (e.g., a second biomolecule, such as a cell surface antigen, such as a tumor antigen).
  • the bispecific anti-CLDN18.2 antibody can target CLDN18.2 and tumor antigens, such as CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1 , MRP4, RET, Steap1 or TenB2 have binding specificity.
  • Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • Techniques for manufacturing multispecific antibodies include but are not limited to the recombinant co-expression of two immunoglobulin heavy chain-light chain pairings with different specificities, see WO 93/08829, WO2009/08025, WO2009/089004A1, etc.
  • the antibody of the present invention encompasses amino acid sequence variants of the anti-CLDN18.2 antibody of the present invention.
  • antibody variants prepared to further improve the binding affinity and/or other biological properties of the antibody may be required.
  • Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the required characteristics, such as binding properties to the CLDN18.2 antigen.
  • antibody variants with one or more amino acid substitutions are provided.
  • Substitution mutants (including conservative substitution mutants or non-conservative substitution mutants) can be obtained by substitution at one or more positions in the HVR (CDR) region and/or FR region.
  • Amino acids can be grouped according to the properties of the common side chain:
  • amino acid substitutions can be introduced into the antibody of the present invention and the product can be screened for the desired activity (for example, retention/improvement of antigen binding or improvement of ADCC or CDC) to obtain the antibody variant of the present invention.
  • the present invention covers antibody variants containing non-conservative mutations and/or conservative mutations obtained according to the antibodies disclosed in the present invention, as long as the variants still have the required CLDN18.2 binding activity.
  • substitutional variant involves an antibody variant that replaces one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody).
  • a parent antibody e.g., a humanized antibody or a human antibody
  • the resulting variants selected for further research will be modified (e.g. improved) relative to the parent antibody in terms of certain biological properties (e.g. increased affinity) and/or will substantially retain certain biological properties of the parent antibody. nature.
  • Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced using, for example, phage display-based affinity maturation techniques such as those described herein.
  • one or more HVR (CDR) residues are mutated and the mutated antibody is displayed on the phage, and the mutated antibody is screened for a specific biological activity (such as binding affinity).
  • substitutions, insertions, or deletions may occur within one or more HVRs (CDRs), as long as such changes do not substantially impair the ability of the antibody to bind CLDN 18.2.
  • CDRs HVRs
  • conservative changes that do not substantially reduce binding affinity can be made in HVR (CDR).
  • such changes can be outside the antigen contact residues in the HVR, for example, conservative or non-conservative amino acid substitutions can occur at 1, 2, 3, 4, and 5 amino acid residues in the FR region.
  • the anti-CLDN18.2 antibody of the present invention can be prepared using recombinant methods, for example, as described in U.S. Patent No. 4,816,567.
  • an isolated nucleic acid encoding an anti-CLDN18.2 antibody described herein is provided.
  • Such nucleic acid may encode the VL amino acid sequence of the antibody and/or the VH amino acid sequence.
  • one or more vectors (e.g., expression vectors) containing such nucleic acids are provided.
  • a host cell containing such nucleic acid is provided.
  • the host cell comprises (e.g., transformed to have): (1) a vector comprising a nucleic acid that encodes the amino acid sequence of the VL of the antibody and the amino acid sequence of the VH of the antibody; or (2 ) A first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody.
  • the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of manufacturing an anti-CLDN18.2 antibody comprises culturing the host cell comprising the nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally from the The host cell (or host cell culture medium) recovers the antibody.
  • nucleic acid encoding the antibody is isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in host cells.
  • nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic cells or eukaryotic cells described herein.
  • prokaryotic cells or eukaryotic cells described herein antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, the antibody in the soluble fraction can be isolated from the bacterial cytoplasm and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors, including glycosylation pathways that have been "humanized” to produce partially or fully human glycosylation patterns.
  • Antibodies to fungal and yeast strains See Li et al., Nat. Biotech. 24:210-215 (2006).
  • Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used to bind insect cells, especially for transfecting Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (description of PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells can also be used as hosts.
  • mammalian cell lines suitable for growth in suspension may be suitable.
  • suitable mammalian host cell lines are monkey kidney CV1 cell lines (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 cells); baby hamster kidney cells (BHK); mouse Serto Li cells (such as TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and Myeloma cell lines, such as Y0, NS0, and Sp2/0.
  • COS-7 monkey kidney CV1 cell lines
  • human embryonic kidney cell lines such as 293 cells
  • BHK baby hamster kidney cells
  • the present invention also provides immunoconjugates comprising the anti-CLDN18.2 antibody herein combined with one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins ( For example, protein toxins, bacterial, fungal, plant or animal-derived enzymatically active toxins or fragments thereof) or radioisotopes.
  • cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins ( For example, protein toxins, bacterial, fungal, plant or animal-derived enzymatically active toxins or fragments thereof) or radioisotopes.
  • the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody binds to one or more drugs, including but not limited to maytansine, orlistatin, dolastatin, Methotrexate, vindesine, taxane, trichothecene and CC1065.
  • ADC antibody-drug conjugate
  • the immunoconjugate comprises a conjugate of an anti-CLDN18.2 antibody as described herein and an enzyme-active toxin or a fragment thereof, the enzyme-active toxin includes, but is not limited to, diphtheria A chain, diphtheria toxin Non-binding active fragments, exotoxin A chain and trichothecene, etc.
  • the immunoconjugate comprises a radioconjugate formed by combining an anti-CLDN18.2 antibody and a radioactive atom as described herein.
  • a radioconjugate formed by combining an anti-CLDN18.2 antibody and a radioactive atom as described herein.
  • a variety of radioisotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu.
  • Conjugates of antibodies and cytotoxic agents can be manufactured using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinyl Imino-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imide ester Bifunctional derivatives (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis( (P-azidobenzoyl) hexamethylene diamine), dual nitrogen derivatives (such as bis(p-diazobenzoyl) ethylene diamine), diisocyanates (such as toluene 2,6-diisocyanate), and double-reactive fluorine compounds (Such as 1,5-difluoro-2,4
  • the pharmaceutical preparation of the anti-CLDN18.2 antibody of the present invention is prepared by mixing the antibody with the desired purity and one or more optional pharmaceutically acceptable carriers in the form of a lyophilized preparation or an aqueous solution.
  • the pharmaceutically acceptable carrier is generally non-toxic to the receptor at the dose and concentration used, and includes but not limited to: buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methyl sulfide Acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexaalkyl quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; p-hydroxybenzoic acid Alkyl esters, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-
  • Exemplary freeze-dried antibody formulations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908.
  • the formulation herein may also contain more than one active ingredient that must be present for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other.
  • active ingredients with complementary activities that do not adversely affect each other.
  • additional therapeutic agents e.g., chemotherapeutics, cytotoxic agents, growth inhibitors, and/or antihormonal agents.
  • Such active ingredients are suitably present in combination in an amount effective for the intended purpose.
  • an article containing the antibody or pharmaceutical composition of the present invention includes a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Such containers can be formed of various materials, such as glass or plastic.
  • the container contains the composition of the present invention itself or a combination of the composition and another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a bottle with a stopper pierceable by a hypodermic injection needle). Vial).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used to treat the selected tumor.
  • the product may comprise (a) a first container containing a composition, wherein the composition contains the antibody of the present invention; and (b) a second container containing a composition, wherein the composition contains another tumor treatment Drug or another antibody.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that such a composition can be used to treat tumors.
  • the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution And dextrose solution. It may further include other materials that are desirable from the standpoint of business and users, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • the present invention also provides a cancer detection reagent comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof herein.
  • Cancer detection reagents can be used to test tumors or blood tissues of actual patients. For example, PCR, microarray or chip technology can be used for testing. Once cancer detection reagents are found in the patient, those reagents can be selected to treat the patient or guide treatment.
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof is chemically labeled, and specifically may be enzyme labels, such as alkaline phosphatase and glucose oxidase; fluorescent labels, such as luciferin and rhodamine; isotope Labels, such as iodine (125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99mTc); or chemiluminescent substance labels, such as luminol and fluorescein Enzyme.
  • enzyme labels such as alkaline phosphatase and glucose oxidase
  • fluorescent labels such as luciferin and rhodamine
  • isotope Labels such as iodine (125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99mTc)
  • chemiluminescent substance labels such as luminol
  • the present invention also provides a cancer detection kit comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof herein.
  • the kit may also contain a labeled anti-CLDN18.2 antibody or antigen-binding fragment thereof.
  • the cancer detection kit can be used to detect gastric cancer, pancreatic cancer or esophageal cancer.
  • the hCLDN18.2 cDNA (SEQ ID NO:1) was cloned and a T-cell epitope peptide was fused to the C-terminus to obtain hCLDN18.2-TCE.
  • T-cell epitope peptides can enable antigens to break through immune tolerance and promote antibody production (Percival-Alwyn J. et al, mAbs, 2015, 7(1), 129-137).
  • the hCLDN18.2-TCE was cloned into the retroviral vector to obtain the hCLDN18.2-TCE retroviral expression plasmid.
  • the hCLDN18.2-TCE retrovirus expression plasmid was transiently transfected into HEK293-T cells, and the expression of hCLDN18.2 on the surface of HEK293-T cells was analyzed by flow cytometry 72 hours after transfection.
  • the primary antibody (1st Ab) is a self-prepared positive control (Benchmarker) chimeric antibody 163E12.
  • the hCLDN18.2-TCE retroviral expression plasmid and the lentiviral packaging plasmid were mixed and transfected into 293-T cells to prepare hCLDN18.2-TCE lentiviral particles.
  • the hCLDN18.2-TCE lentiviral particles were transfected into mouse tumor cell lines. After 72 hours, the mouse tumor cell pool with high expression of hCLDN18.2-TCE was detected and sorted by flow cytometry.
  • the primary antibody (1st Ab) is a self-prepared positive control (Benchmarker) chimeric antibody 163E12. The cells were first incubated with the primary antibody (1 ⁇ g/mL) at 4°C for 45 minutes.
  • HBV hCLDN18.2 extracellular loop type 1 virus particles are prepared in the refolding buffer. The preparation of the hCLDN18.2 extracellular loop type 1 virus particles will be used in the (3) virus-like particle multi-point repeated immunity-cellular immunity protocol and (4) virus-like particle attached joint immunity-cellular immunity protocol in Example 2. .
  • hCLDN18.2 (SEQ ID NO: 4), hCLDN18.1 (SEQ ID NO: 5), mouse mhCLDN18.2 (SEQ ID NO: 6), mCLDN18.1 (SEQ ID NO: 7) gene full length, respectively, And cloned into pcDNA3.4 vector plasmid respectively.
  • the plasmid was transfected into HEK293 cells and neomycin (G418) was added for pressurized selection. The limiting dilution method was used to select HEK293 stably transfected cell lines with high expression of hCLDN18.2, hCLDN18.1, mCLDN18.2, mCLDN18.1.
  • HEK293 stably transfected cell lines transfected with hCLDN18.1 and mCLDN18.1 (HEK293-hCLDN18.1, HEK293-mCLDN18.1), paraformaldehyde-fixed and non-fixed cells can not be self-made anti-CLDN18.2 positive control antibody 163E12 or 175D10 specifically recognizes ( Figure 3.B, D), but it can be recognized by the commercial broad anti-CLDN18 antibody (34H14L15) ( Figure 3.B, D).
  • the above-mentioned HEK293 stably transfected cell line will be used in the hybridoma screening in Example 3.
  • the human-mouse chimeric positive control antibody 43A11 heavy chain (SEQ ID NO: 8) and light chain (SEQ ID NO: 9), 175D10 heavy chain (SEQ ID NO: 10) and light chain (SEQ ID NO: 11) were synthesized respectively
  • the 163E12 heavy chain (SEQ ID NO: 12) and light chain (SEQ ID NO: 13) antibody genes are full length with signal peptide added to the N-terminus respectively, and then cloned into the pcDNA3.4 eukaryotic expression vector.
  • the 43A11, 175D10, and 163E12 heavy chain and light chain expression plasmids were mixed and co-transfected into CHOS cells for transient protein expression. The supernatant was collected and affinity purified with Protien A and tested by SDS-PAGE. The results are shown in Figure 4. Show.
  • the hybridomas from three batches were cloned and cultured by the limiting dilution method, and the clone supernatant was collected and used for three rounds of binding or reverse binding screening by flow cytometry.
  • Antibody typing and antibody-dependent cell toxicity (Antibody-dependent cell- mediated cytoxity (ADCC) assay, screened out 128 hybridoma clones that can specifically bind to CLDN18.2, but not or weakly bind to CLDN18.1, and have ADCC function.
  • Table 3 shows the summary of the screening steps and the hybridoma clones obtained at each stage of the screening.
  • 3 batches of hybridomas each batch was cloned into 10 384-well plates by limiting dilution method. After primary screening, 341 (MFI>100,000), 336 (MFI>75,000 or MFI>80,000) and 89 (MFI>90,000) mouse tumor cells with CLDN18.2-TCE were obtained from three batches of hybridomas. Line positive binding hybridoma clones.
  • Verification screening The supernatant expression products derived from the 306 (165 + 120 + 21) positive hybridomas obtained in the three batches of hybridoma confirmation screening were respectively compared with HEK293-hCLDN18.2 cells and HEK293- Incubate hCLDN18.1 cells, HEK293-mCLDN18.2 cells, HEK293-mCLDN18.1 cells and HEK293 wild-type cells (20,000 cells/well, 4°C, 45 minutes), and use Alexa Fluor-488-goat anti-mouse IgG (ThermoFisher Catalog No. A28175) was used as a secondary antibody for analysis by flow cytometry.
  • the flow cytometry analysis results of some clones on 5 cell lines are shown in Figure 6, showing that the average fluorescence intensity (MFI) of positive clones in HEK293-hCLDN18.2 cells and HEK293-mCLDN18.2 cells both exceeded 500,000, while The average fluorescence intensity on HEK293-hCLDN18.1 cells, HEK293-mCLDN18.1 cells and HEK293 wild-type cell lines are all below 50,000, and most clones (16/22) are in HEK293-hCLDN18.2 cells and HEK293-mCLDN18. The average fluorescence intensity (MFI) of the 2 cells both exceeded the average fluorescence intensity (MFI) of HEK293-hCLDN18.1 cells and HEK293-mCLDN18.1 cells by 50 times.
  • MFI average fluorescence intensity
  • Antibody typing Because different subtypes of IgG molecules also have different abilities to mediate ADCC effects. Use mouse antibody subtype detection kit to confirm the supernatant of 306 (165+120+21) positive hybridomas obtained in three batches of hybridomas before performing antibody-dependent cell killing detection. The antibody expression products are subtyped into mouse antibodies. Most of the 306 clones belonged to mouse IgG2a or IgG2b or IgG3 with ADCC function, and only a few (26/306) were IgG1 subtypes without ADCC function (corresponding to human antibody IgG4 subtype). Some of the clones are of mixed subtypes, which may be caused by non-monoclonal hybridoma clones. The light chains are all mouse kappa subtypes.
  • the saturated supernatants of 128 positive clones were selected for antibody-dependent cytotoxicity function detection.
  • the effector cells are human peripheral blood mononuclear cells (PBMCs), sourced from two donors (No. 45 and 46). The donors were drawn the day before the test. The blood collected was stored at room temperature. The PBMCs were fresh through the Ficoll gradient. Separated. The target cells were HEK293 cells expressing CLDN18.2 (HEK293-hCLDN18.2). Hybridoma expression products are uniformly diluted by 4 times. Freshly obtained PBMCs and target cells were incubated with each sample for 4 hours at an effective-to-target ratio of 25:1.
  • the ADCC effect of antibodies was characterized by measuring lactate dehydrogenase (LDH) associated with cytotoxicity.
  • LDH lactate dehydrogenase
  • the absorbance value detected under the spontaneous lysis of the target cell is defined as 0%, and the absorbance value detected under the condition of all the target cells is defined as 100%.
  • the relative percentage activity of each test sample is used to characterize the ADCC effect.
  • the ADCC effect results of some clones are shown in Figure 7.
  • the ADCC killing effect of the clone supernatant is higher than 20% on average, and the ADCC effect of some clones (9D-1, 8-O11) is higher than 50%, and for mouse IgG1 Types of 6-G11 and 4-O2, ADCC effect is less than 10%.
  • FFPE paraffin-embedded
  • the aforementioned preferred 128 positive hybridoma clone supernatants have the ability to specifically bind to the natural CLDN18.2 conformation of the cell surface and ADCC function, but as a companion diagnostic antibody, IHC screening for antigen-specific binding must be performed on FFPE tissue sections.
  • the immunohistochemistry (Immunohistology chemistry, IHC) method of PPFE tissue sections was used to detect 128 positive hybridoma supernatants in 3 engineered cell lines (HEK293-hCLDN18.2 cells, HEK293-hCLDN18.1 cells and HEK293 wild Type cell) cell block (cell block) paraffin-embedded (FFPE) tissue sections.
  • the obtained positive hybridoma supernatant was verified by IHC staining on FFPE tissue sections of human gastric cancer tissue.
  • the verified IHC positive staining hybridoma supernatant was IHC stained on human normal stomach and gastric cancer FFPE tissue microarray (Tissue Micro-Array, TMA), and the final IHC positive staining clone was determined according to the IHC staining intensity and staining ratio.
  • Figure 8 shows the IHC staining results of the positive control commercial antibody EPR19202 on the HEK293 cell block FFPE tissue section.
  • the antibody can specifically develop color on the HEK293-hCLDN18.2 cell block FFPE tissue section of the HEK293-hCLDN18.2 stable expression cell line, and the cell membrane
  • the positive color development is obvious ( Figure 8.A, arrow), which is consistent with the conclusion that CLDN18.2 is a membrane protein ( Figure 8.A).
  • the antibody showed no obvious color on the FFPE tissue section of the cell block of the HEK293-hCLDN18.1 stably expressing cell line ( Figure 8.B), while there was no obvious color on the HEK293 wild cell line ( Figure 8.C). ).
  • the incubation time of a single hybridoma clone supernatant and the positive control antibody is 30 minutes, and the incubation time of the 4 hybridoma supernatant mixture is 120 minutes.
  • Table 4 summarizes the tissue sites, tissue types, and corresponding positive staining clones that have positive cell membrane coloration and cell cytoplasmic coloration.
  • the positive cell membrane coloration rates of 5-I10, 5-G17, 5-I5, 10-D3 and 4 clone supernatant mixtures on all 76 tissue sites were 28.9%, 17.1%, 14.5%, 11.8% and 28.9%, and the positive rate of the cell membrane of the positive control antibody (EPR19202) was 23.7%.
  • the positive staining rate of each hybridoma supernatant on gastric cancer tissue is huge, and the positive staining rate of cell membrane on gastric cancer tissue is 5-110 23.5%, 5-G17 10%, 5- I5 4.4%, 10-D3 2.9% and 4 hybridoma supernatant mixture (Mix. Of 4) 22%, which may be related to the high heterogeneity of gastric cancer tissue.
  • the 5-I10 hybridoma clone supernatant has the highest positive coloration rate in the cell membrane of gastric cancer tissues, reaching 23.5%, which is significantly higher than the 14.7% of the positive control antibody EPR19202, and is comparable to Zhu G. et al. (Zhu G. et al, Sci Rep. , 2019, 9: 8420-8431) reported 16 to 23% positive rate is basically the same.
  • the limiting dilution method was used to subclone and sequence the variable region sequence of the antibody molecule on four IHC-positive hybridoma clones (5-I10, 5-G17, 5-I5 and 10-D3) on FFPE tissue sections.
  • the cryopreserved and resuscitated hybridoma clones were subjected to limiting dilution, planted into a 192-well plate at a density of 0.8 cells/well, single clones were selected by microscopy, and the supernatant was collected after 14 days of culture to detect subclones using flow cytometry analysis The supernatant specifically binds to HEK293-hCLDN18.2 cells, and the single clone is amplified and cultured at the same time.
  • the flow cytometric analysis method is to incubate the monoclonal supernatant with HEK293-CLDN18.2 cells (20,000 cells/well) at 4°C for 45 minutes, wash the plate, and use Alexa Fluor-488-goat anti-mouse IgG (ThermoFisher Catalog No. A28175) as a secondary antibody for flow cytometry detection.
  • HEK293-CLDN18.2 cells 20,000 cells/well
  • Alexa Fluor-488-goat anti-mouse IgG ThermoFisher Catalog No. A28175
  • FIG. 10 The flow cytometric analysis of HEK293-hCLDN18.2 cells with the monoclonal supernatants of 4 hybridomas is shown in Figure 10, showing that the monoclonal supernatants maintain the specific binding of HEK293-hCLDN18.2 cells, and some of the monoclonal supernatants maintain the specific binding of HEK293-hCLDN18.2 cells.
  • the binding strength of the supernatant to HEK293-hCLDN18.2 cells is equivalent to the binding strength of the corresponding hybridoma supernatant (Parental Supe.).
  • the supernatants of the top 5 monoclonals (shown in the blue box in Figure 9) with the highest binding strength to HEK293-hCLDN18.2 cells were selected for IHC verification of FFPE tissue sections, and all the monoclonal supernatants All showed positive IHC color development.
  • select the 2 monoclonals with the highest IHC color intensity on FFPE tissue sections (5-I5-31 and 5-I5-25, 10-D3-22 and 10-D3-21, 5-G17-30 And 5-G17-6, 5-I10-33 and 5-I10-18) for heavy chain and light chain variable region gene cloning and sequencing.
  • the RACE (rapid amplification of cDNA ends) method was used to amplify the DNA sequences of hybridoma monoclonal VH and VL.
  • RNA was extracted from the amplified hybridoma monoclonal cells, reverse transcribed into cDNA and used 5'-RACE reaction using 5'-universal primers and 3'-H, L( ⁇ ) or L( ⁇ ) FR1 region
  • the primer combination is used to perform PCR on the heavy chain or light chain V region, and the TOPO clone method connects the PCR amplified fragments to the sequencing vector for sequencing.
  • the V regions of the heavy and light chains of 5-I10 and 5-G17 were respectively compared with the mouse IgG2a type heavy chain constant region (Table 7, Mouse IGHC2A, SEQ ID NO: 30) and the mouse ⁇ type light chain constant region ( Table 7, Mouse IGKC, SEQ ID NO: 31) was fused, and a signal peptide was added to the N-terminus of the heavy chain and light chain sequences, respectively.
  • the mouse heavy chain full-length gene (SEQ ID NO: 32, 33) and light chain full-length gene (SEQ ID NO: 34, 35) of 5-I10 and 5-G17 were synthesized and cloned into the expression vector, and sequenced verification ( Jinweizhi Biological Technology Co., Ltd., Suzhou).
  • the expression plasmid constructed for each candidate molecule is amplified and plasmid extracted, verified by agarose gel electrophoresis, and used as a transfection material.
  • HEK293 cells were inoculated into shake flasks for suspension seed culture, using serum-free medium with clear chemical composition.
  • the expanded HEK293 cells were inoculated into a new shake flask and replaced with fresh medium.
  • the heavy chain and light chain expression plasmids of 5-I10 and 5-G17 were co-transfected into HEK293 cells, and cultured in a shake flask until the cell viability decreased, and the supernatant was collected for product purification. Firstly, the supernatant is centrifuged and filtered to remove most of the cell debris and insoluble particles, and then the feed solution is loaded on the Protein A column.
  • the antibody molecules are combined with the Protein A on the packing, and most of the impurities are removed after the equilibration step. , Use a low pH eluent to elute the antibody protein and collect the fractions. The pH is adjusted and replaced by ultrafiltration to the formulation buffer. Finally, the OD280 was measured to calculate the protein concentration. As shown in Figure 11, the electrophoresis of 5-I10 and 5-G17 monoclonal antibodies on reduced SDS-PAGE showed that the positions of the heavy and light chain bands were consistent with those of the positive control IgG heavy and light chains, and the molecular weights were in line with expectations. There is no obvious miscellaneous band, and the protein purity is >95%.
  • Example 7 HEK293-hCLDN18.2 binding activity of recombinant mouse monoclonal antibody
  • Flow cytometry was used to verify the binding of 5-I10 and 5-G17 purified monoclonal antibodies on HEK293-hCLDN18.2.
  • HEK293-hCLDN18.2 was planted in a 96-well U-shaped plate (1.5 ⁇ 105 cells/well), which contained 1 The plate was washed once with %FBS in PBS buffer.
  • 5-I10 and 5-G17 purified monoclonal antibodies were diluted with PBS (1%FBS) respectively, starting from 10 ⁇ g/mL and performing 3-fold dilutions to obtain a total of 8 concentration points.
  • FIG. 12 shows the binding curves of 5-I10 and 5-G17 purified monoclonal antibodies on HEK293-hCLDN18.2, both of which can specifically bind to HEK293-hCLDN18.2 in a dose-dependent manner, and the binding capacity is equivalent.
  • Example 8 Recombinant mouse monoclonal antibody stained positively on FFPE tissue sections of gastric cancer
  • the CLDN18.2 IHC positive staining of 5-I10 and 5-G17 purified monoclonal antibodies was verified on 6 FFPE tissue sections of gastric cancer, and EPR19202 was used as a positive control.
  • the IHC staining steps are the same as those described in Example 4.
  • the working concentration of the positive control antibody EPR19202 is 1:100 dilution
  • the working concentration of the 5-I10 and 5-G17 purified antibodies is 5 ⁇ g/mL.
  • the color development results are shown in Figure 13.
  • the recombinant and purified mouse IgG2a type 5-I10 and 5-G17 monoclonal antibodies can stain positively on multiple FFPE tissue sections of gastric cancer tissues.
  • the positive staining ratios of 5-G17 and 5-I10 reached 83.3% (5/6) and 66.7% (4/6) respectively, which were significantly higher than the 50% (3/6) of the positive control antibody EPR19202.
  • gastric cancer cells formed a relatively obvious gastric gland epithelial structure.
  • 5-I10, 5-G17 and the positive control antibody EPR19202 all showed strong Glandular epithelial cells are stained specifically, but there is no staining on stromal cells.
  • the gastric cancer cells formed a gland-like structure, but it was obviously different from the gland structure on the aforementioned section; the positive control SPR19202 had no obvious IHC staining, while 5-I10 and 5-G17 had Strong positive staining, staining into a diffuse type, glandular epithelium and stromal cells are stained.
  • the tumor cells on the S09-15785-2FS and S07-7939-C slices had no obvious glandular structure formation.
  • the positive controls SPR19202, 5-I10 and 5-G17 had no positive IHC staining. Only 5-G17 on S07-7939-C showed weak IHC positive staining.

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Abstract

提供了一种密蛋白18.2(CLDN18.2)的抗体，及其在诊断和治疗胃、胰腺、食管癌症中的用途。

Description

CLDN18.2抗体及其用途 技术领域
本发明涉及抗体技术领域，同时涉及该抗体的用途和制备方法。具体涉及一种CLDN18.2抗体及其用途。
背景技术
密蛋白(Claudin)是一类分子量为20～30kDa的跨膜蛋白，其家族共有24个成员，在多个组织中均有表达，是形成细胞间紧密连接(Tight Junctions，TJ)结构的重要分子。上皮细胞或者内皮细胞通过表达于细胞表面的紧密结合分子(包括Claudin分子)形成高度有序组织的结合，即细胞间紧密连接，控制上皮细胞间隙中分子的流动。TJ对于保持正常生理条件下的上皮细胞或者内皮细胞的稳定状态，维持细胞极性有着至关重要的作用。这其中，比较引人注目的是密蛋白18(Claudin18，CLDN18)，它包括2种剪接形成的变异体：CLDN18剪切变异体1(Claudin18.1，CLDN18.1)和CLDN18剪切变异体2(Claudin18.2，CLDN18.2)。
这其中，CLDN18.2(Genbank登记号NM_001002026、NP_001002026)是一种分子量为27.8kDa的跨膜蛋白，蛋白N端和C端均位于胞内。该蛋白的主要构象包含4个跨膜结构域(TMD)和2个胞外环(ECL)。CLDN18.2在多种哺乳类动物中高度保守，全长261个氨基酸，其中1-6位氨基酸为N端胞内区，7-27位氨基酸为跨膜区1(TMD1)，28-78位氨基酸为胞外环1(ECL1)，79-99位氨基酸为跨膜区2(TMD2)，123-144位氨基酸为跨膜区3(TMD3)，100-122位氨基酸为连接TMD2与TMD3的胞内区，145-167位氨基酸为胞外环2(ECL2)，168-190位氨基酸为跨膜区4(TMD4),191-261位氨基酸为C端胞内区。在第116位的天冬酰胺上存在一个经典N糖基化基序。
CLDN18.1与CLDN18.2仅在蛋白N端-跨膜区1-胞外环1这一段区域内的N端前26个氨基酸序列存在差异，其余序列均相同。
有相当多的证据表明，在胃、胰腺、食管等组织的肿瘤细胞，特别是腺癌亚型肿瘤细胞中，CLDN18.2高水平表达。在正常组织中，CLDN18.1选择性表达于肺及胃组织的上皮细胞；CLDN18.2则仅在寿命很短的胃腺状上皮粘膜组织(已分化的腺状上皮细胞)中表达，但并不在胃上皮干细胞中表达，而已分化的腺状上皮细胞可由胃上皮干细胞不断补充。这些分子表达特性为基于抗体治疗CLDN18.2相关的癌症提供了潜在可能。
采用伴随诊断(Companion diagnostics,CDx)方法在临床试验及临床应用中挑选CLDN18.2高表达的肿瘤患者，对指导靶向CLDN18.2靶点的抗肿瘤药物的适用患者具有重要的指导意义。临床中针对肿瘤特异性抗原的诊断中，患者肿瘤标本通常为经过固定处理的石蜡包埋(Formalin Fixed Paraffin Embedded,FFPE)的组织，而肿瘤特异性抗原在FFPE处理过程中其天然构象会改变，后续抗原修复过程中又会出现有别于天然构象的特殊表位暴露(Biotech Histochem,2009,84(5):207-215)，所以通常筛选能够在肿瘤FFPE组织上针对癌症特异性抗原具有特异性结合的抗体来进行诊断。
本发明中涉及了针对肿瘤及其FFPE组织中特异性识别CLDN18.2抗原抗体的制备方法、筛选、序列及用途。
发明内容
本发明提供了一种用于诊断癌症的CLDN18.2抗体，该抗体能够识别经过固定处理的组织样本中的CLDN18.2抗原分子从而诊断包括胃癌、胰腺癌或食管癌等的癌症病变。同时本发明提供的CLDN18.2抗体还能够有效治疗包括胃癌、胰腺癌或食管癌等的癌症病变。具体地，本申请涉及：
1.一种CLDN18.2抗体，包含选自如下的重链CDRs和轻链CDRs，
其中所述重链CDRs为：
包含SEQ ID NO:18所示的CDR1、SEQ ID NO:19所示的CDR2、SEQ ID NO:20所示的CDR3；或
包含SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、SEQ ID NO:23所示的CDR3，
其中所述轻链CDRs为：
包含SEQ ID NO:24所示的CDR1、SEQ ID NO:25所示的CDR2、SEQ ID  NO:26所示的CDR3；或
包含SEQ ID NO:27所示的CDR1、SEQ ID NO:28所示的CDR2、SEQ ID NO:29所示的CDR3。
2.如项1所述的抗体，包含如SEQ ID NO:36或SEQ ID NO:37所示的重链可变区。
3.如项1所述的抗体，包含如SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区。
4.项1-3任一项的抗体，包含如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:38所示的轻链可变区。
5.项1-3任一项的抗体，包含如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:39所示的轻链可变区。
6.项1-5任一项的抗体，其为嵌合抗体、人源化抗体或全人抗体。
7.项1-6任一项所述的抗体，其为全长抗体。
8.项7所述的抗体，其为IgG抗体。
9.项1-6任一项所述的抗体，其为结合CLDN18.2的抗体片段。
10.项9的抗体，其为Fab、Fab'-SH、Fv、scFv或(Fab')2。
11.项1-8任一项的抗体，其为多特异性抗体或双特异性抗体。
12.项11的抗体，其中所述多特异性抗体或双特异性抗体包含结合第二生物分子的结合域，其中所述第二生物分子为细胞表面抗原。
13.项12所述的抗体，其中所述细胞表面抗原为肿瘤抗原。
14.项13所述的抗体，其中所述肿瘤抗原选自：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1和TenB2。
15.一种免疫缀合物，包括与项1-10任一项的所述的抗体连接的治疗剂、干扰素基因刺激因子(STING)受体激动剂、细胞因子、放射性核素或酶。
16.项15的免疫缀合物，其中所述治疗剂为化疗药物。
17.项15的免疫缀合物，其中所述治疗剂为细胞毒性剂。
18.包含项1-10任一项所述的抗体或抗原结合片段的融合蛋白或多肽。
19.包含项1-14任一项的抗体、项15-17任一项的免疫缀合物、项18 的融合蛋白或多肽的药物组合物。
20.一种分离的核酸，包含编码SEQ ID NOs：18-29之一的氨基酸序列的多核苷酸序列。
21.项20的核酸，包含编码SEQ ID NOs：36-39之一的氨基酸序列的多核苷酸序列。
22.项21的核酸，包含编码项1-10任一项所述的抗体或抗原结合片段的多核苷酸序列。
23.一种载体，其包含项20-22任一项所述的多核苷酸序列。
24.一种宿主细胞，其包含项20-22任一项所述的多核苷酸序列或项23所述的载体。
25.一种制备项1-10任一项所述抗体的方法，包括培养项24的宿主细胞并从培养物中分离获得所述抗体。
26.包含项1-14任一项的抗体、项15-17任一项的免疫缀合物、项18的融合蛋白或多肽的药物组合物在制备用于诊断癌症的试剂中的用途。
27.包含项1-14任一项的抗体、项15-17任一项的免疫缀合物、项18的融合蛋白或多肽的药物组合物在制备用于治疗癌症的药物中的用途。
28.项26或27的用途，其中所述癌症为胃癌、胰腺癌或食管癌。
29.包含项1-10任一项所述的抗体或其抗原结合片段的癌症检测试剂。
30.项29的癌症检测试剂，其中所述抗体或其抗原结合片段是经过化学标记的。
31.项30的癌症检测试剂，其中所述标记为酶标记、荧光标记、同位素标记或化学发光物标记。
32.包含项29-31任一项的癌症检测试剂的癌症诊断试剂盒。
33.如项32所述的试剂盒，所述癌症为胃癌、胰腺癌或食管癌。
34.一种诊断受试者癌症的方法，包括使项1-14中任一项的抗体、项18的融合蛋白或多肽或项29-31中任一项的检测试剂与来源于受试者的组织样本接触。
35.项34的诊断方法，所述组织样本为受试者体液(例如血液、尿液)和组织切片(例如组织活检样本)。
36.项34或35的诊断方法，其中所述癌症选自胃癌、胰腺癌或食管癌。
37.一种治疗受试者癌症的方法，包括给药受试者治疗有效量的项1-14任一项的抗体、项15-17任一项的免疫缀合物、项18的融合蛋白或多肽、或项19的药物组合物。
38.项37的治疗方法，其中所述癌症选自胃癌、胰腺癌或食管癌。
附图说明
图1.人hCLDN18.2-TCE(T-细胞表位肽)逆转录病毒表达质粒瞬时转染HEK293-T细胞
图2.hCLDN18.2-TCE慢病毒颗粒转染小鼠肿瘤细胞系并通过流式细胞术分选高水平表达hCLDN18.2-TCE的小鼠肿瘤细胞
图3.hCLDN18.2、hCLDN18.1和mCLDN18.2、mCLDN18.1稳转细胞株的流式细胞分析
图4.阳性对照抗体43A11、175D10和163E12的SDS-PAGE检测
图5.确认筛选中阳性杂交瘤克隆上清与HEK293-hCLDN18.2细胞和HEK293-hCLDN18.1细胞的结合
图6.验证筛选中部分阳性杂交瘤克隆上清在5个细胞株上的流式细胞术分析
图7.部分阳性杂交瘤克隆上清的ADCC效应
图8.阳性对照抗体在HEK293-hCLDN18.2(A)、HEK293-hCLDN18.1(B)和HEK293-WT(C)细胞块石蜡包埋(FFPE)组织切片上的IHC染色
图9.5-I5杂交瘤克隆上清及阳性对照抗体在胃癌组织石蜡包埋(FFPE)组织切片上的IHC显色
图10.杂交瘤单克隆上清对HEK293-hCLDN18.2细胞的流式细胞术分析
图11.重组纯化5-I10和5-G17小鼠单克隆抗体的还原SDS-PAGE电泳
图12.重组纯化5-I10和5-G17单克隆抗体在HEK293-hCLDN18.2流式细胞术分析
图13.重组纯化的小鼠5-I10和5-G17单克隆抗体在胃癌FFPE组织切片上阳性染色
具体实施方式
1.定义
本文中的术语“抗体”以广义使用，涵盖结合CLDN18.2的、包含本文所公开的一个或多个CDR结构域的各种抗体结构分子，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)以及抗体片段(例如Fv、Fab、Fab'、Fab'-SH、F(ab') 2)、线性抗体和单链抗体分子(例如scFv)等，只要其展现所需要的与CLDN18.2的结合活性即可。
本领域技术人员可以将本发明公开的一个或多个CDR结构域与一种或多种其它多肽序列融合，制备成结合CLDN18.2分子的功能性融合蛋白或多肽分子，例如疫苗、细胞膜受体拮抗剂、信号通路调节剂和嵌合抗原受体分子等等。例如，可以利用本发明公开的一个或多个CDR结构域制备成CLDN18.2 CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法)分子。这些基于本发明公开的序列内容衍生、制备的融合蛋白分子也涵盖在本发明的保护范围内。
本文中所使用的术语“单克隆抗体”中的修饰语“单克隆”是指该抗体获自实质上均质的抗体群体，仅含有微量的天然存在的突变或在单克隆抗体制备过程中出现的突变。与典型地包括针对不同表位的不同抗体的多克隆抗体制剂相比，单克隆抗体制剂中的各单克隆抗体针对抗原上的单一表位。本发明的单克隆抗体可通过多种技术制造，包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法及利用含有所有或部分人类免疫球蛋白基因座的转基因动物的方法制造。
术语“全长抗体”、“完整抗体”指具有基本上与天然抗体结构相似的结构的抗体，该术语在本文中可互换使用。
抗体的“类”指其重链拥有的恒定域或恒定区的类型。抗体有5大类：IgA、IgD、IgE、IgG、和IgM，并且这些中的几种可以进一步分成亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1、和IgA2。与不同类免疫球蛋白对应的重链恒定域分别称作α、δ、ε、γ、和μ。
“嵌合抗体”是具有来源于一种物种的至少一部分重链可变区和至少一部分轻链可变区以及来源于另一物种的至少一部分恒定区的抗体。例如，在一个实施方案中，嵌合抗体可包含鼠可变区和人恒定区。
“人源化”抗体指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中，人源化抗体会包含至少一个，通常两个基本上整个可变域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的那些，且所有或基本上所有FR对应于人抗体的那些。任选地，人源化抗体可以至少包含自人抗体衍生的抗体恒定区的一部分。抗体(例如非人抗体)的“人源化形式”指已经经历人源化的抗体。
“人类共同框架”为代表在选择人类免疫球蛋白VL或VH框架序列中最常存在的氨基酸残基的框架。一般而言，人类免疫球蛋白VL或VH序列选自于可变域序列的亚组。一般而言，序列亚组为如Kabat等人，Sequences of Proteins of Immunological Interest，第5版，NIH Publication 91-3242，Bethesda MD(1991)，第1-3卷中的亚组。在一个实施方案中，对于VL而言，所述亚组为如Kabat等人(同上)描述的亚组κI。在一个实施方案中，对于VH而言，所述亚组为如Kabat等人(同上)描述的亚组III。
“人类抗体”也可称为“人抗体”、“全人源抗体”或“全人抗体”，为其氨基酸序列对应于由人类产生的或人类细胞产生的氨基酸序列的抗体。此人类抗体定义特定地排除了包含非人类抗原结合残基的人源化抗体。人类抗体可使用本领域中已知的各种技术制备，包括噬菌体展示库技术，如下文献描述的技术：Hoogenboom及Winter,J.Mol.Biol.,227:381(1991)；Marks等人，J.Mol.Biol.,222:581(1991)。Cole等，Monoclonal Antibodies and Cancer Therapy,Alan R.Liss，第77页(1985)；Boerner等，J.Immunol.,147(1):86-95(1991)。人类抗体可通过向经修饰以响应于抗原攻击而产生此类抗体但其内源基因座已失能的转基因动物(例如免疫异种小鼠)施用抗原来制备(关于XENOMOUSETM技术，参见例如美国专利第6,075,181号及第6,150,584号)。关于经由人类B细胞杂交瘤技术产生的人类抗体，也参见例如Li等人，Proc.Natl.Acad.Sci.USA，103:3557-3562(2006)。
如本文中所使用的术语“高变区”或“HVR”是指抗体可变域中具有序列高变区(也称为“互补决定区”或”CDR”)和/或形成结构确定的环(“高变环”)和/或含有抗原接触残基(“抗原触点”)的各区。一般而言，抗体包含6个HVR(CDR区)：3个处于VH中(H1、H2、H3)且3个处于VL中(L1、L2、L3)。本文中的例示性HVR(CDR区)包括：
(a)在氨基酸残基26-32(L1)、50-52(L2)、91-96(L3)、26-32(H1)、53-55(H2)及96-101(H3)处出现的高变环(Chothia及Lesk,J.Mol.Biol.196:901-917(1987))；
(b)在氨基酸残基24-34(L1)、50-56(L2)、89-97(L3)、31-35b(H1)、50-65(H2)及95-102(H3)处出现的HVR(CDR区)(Kabat等人,Sequences of Proteins of Immunological Interest,第5版，Public Health Service,National Institutes of Health,Bethesda,MD(1991))；
(c)在氨基酸残基27c-36(L1)、46-55(L2)、89-96(L3)、30-35b(H1)、47-58(H2)及93-101(H3)处出现的抗原触点(MacCallum等人,J.Mol.Biol.262:732-745(1996))；及
(d)(a)、(b)和/或(c)的组合，包括HVR(CDR区)氨基酸残基46-56(L2)、47-56(L2)、48-56(L2)、49-56(L2)、26-35(H1)、26-35b(H1)、49-65(H2)、93-102(H3)及94-102(H3)。
除非另外指出，否则HVR(CDR区)残基及可变域中的其他残基(例如FR残基)在本文中根据Kabat等人(同上)进行编号。
术语“可变区”或“可变域”是指与抗体与抗原结合有关的抗体重链或轻链结构域。天然抗体的重链及轻链(分别为VH及VL)的可变域一般具有类似结构，其中各域包含4个保守框架区(FR)及3个互补决定区(CDR区)。(参见例如Kindt等人，Kuby Immunology，第6版。单一VH或VL域即可足以赋予抗原结合特异性。
“抗体片段”是指除完整抗体以外包含完整抗体中结合该完整抗体所结合的抗原的一部分的分子。抗体片段的实例包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab') 2；双功能抗体；线性抗体；单链抗体分子(例如scFv)；及由抗体片段形成的多特异性抗体。
如本文中所使用的术语“细胞毒性剂”是指抑制或预防细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂包括但不限于放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212及Lu的放射性同位素)；化学治疗剂或药物(例如氨甲蝶呤、亚德里米星、长春花生物碱(长春新碱、长春花碱、依托泊苷)、阿霉素、美法仑、丝裂霉素C、苯丁酸氮芥、道诺红菌素或其他插入剂)；生长抑制剂；酶及其片段， 诸如核分解酶；抗生素；毒素，诸如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体；本领域已知的各种抗肿瘤药物或抗癌剂。
“免疫缀合物”为抗体与一个或多个异源分子(包括但不限于细胞毒性剂)的缀合物。
干扰素基因刺激因子(stimulator of interferon genes,STING)受体激动剂是激活STING依赖的信号通路从而促进I型干扰素分泌和促进与抗病毒和抗肿瘤免疫相关的蛋白的表达，阻断病毒复制、促进对癌细胞的免疫反应的分子。这类分子例如环二核苷酸类、氨基苯并咪唑类、呫吨酮类和吖啶酮类、苯并噻吩类及苯并二茂类等结构类型的STING激动剂。
“受试者”或“个体”为哺乳动物。哺乳动物包括但不限于驯化动物(例如牛、羊、猫、狗及马)、灵长类动物(例如人类及非人类灵长类动物，诸如猴)、兔及啮齿动物(例如小鼠及大鼠)。在某些实施方案中，所述受试者或个体为人类。
术语“包装插页”用于指通常包括在治疗产品的商业包装中的说明书，其含有关于适应症、用法、剂量、给药、组合疗法、禁忌症和/或关于使用此类治疗产品的警告的信息。
“亲和力”是指分子(例如抗体)的单一结合位点与其结合伴侣(例如抗原)之间的非共价相互作用总和的强度。除非另有指示，否则如本文中所使用，“结合亲和力”是指固有结合亲和力，其反映出结合伴侣成员(例如抗体与抗原)之间的1:1相互作用。分子X对其伴侣Y的亲和力一般可由解离常数(Kd)表示。亲和力可通过本领域中已知的常用方法加以测量，包括本文中所描述的那些方法。用于测量结合亲和力的特定说明性及例示性实施方案描述于下文中。
关于参考多肽序列的“氨基酸序列同源性百分比(％)”定义为在将候选序列与参考多肽序列对准且必要时引入间隙以达成最大序列同源性百分比后且在任何保守取代均不被视为序列同源性的一部分的情况下，候选序列中与参考多肽序列中的氨基酸残基一致的氨基酸残基的百分比。用于确定氨基酸序列同源性百分比可用本领域多种方式实现，例如可使用诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件进行同源性比对。 本领域技术人员可确定适用于比对序列的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
例如，在采用ALIGN-2进行氨基酸序列比较的情形下，指定氨基酸序列A同、与或相对于指定氨基酸序列B的氨基酸序列同源性％计算如下：
100×分数X/Y
其中X为在对A与B进行程序比对时由序列比对程序ALIGN-2评分为相同匹配的氨基酸残基数目，且其中Y为B中的氨基酸残基总数。应了解，在氨基酸序列A的长度不等于氨基酸序列B的长度的情况下，A与B的氨基酸序列同源性％将不等于B与A的氨基酸序列同源性％。除非另有说明，否则本文中所使用的所有氨基酸序列同源性％值使用ALIGN-2计算机程序获得。
2.抗体、制备方法、组合物及制品
1)抗体
本发明涉及抗CLDN18.2抗体。在某些实施方案中，本发明提供一种抗CLDN18.2抗体，其包含含有至少1、2、3、4、5或6个选自以下的高变区(HVR)或称为互补决定区(CDR)的结合域：(a)HVR-H1，其包含序列SEQ ID NO:18或SEQ ID NO:21的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(b)HVR-H2，其包含序列SEQ ID NO:19或SEQ ID NO:22的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(c)HVR-H3，其包含序列SEQ ID NO:20或SEQ ID NO:23的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(d)HVR-L1，其包含序列SEQ ID NO:24或SEQ ID NO:27的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(e)HVR-L2，其包含序列SEQ ID NO:25或SEQ ID NO:28的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；及(f)HVR-L3，其包含序列SEQ ID NO:26或SEQ ID NO:29的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列。在一些情况下，抗CLDN18.2抗体所具有的重链可变(VH)域(区)可包括与序列SEQ ID NO:36或SEQ ID NO:37具有至少90％序列同源性(例如至少91％、92％、93％、94％、95％、 96％、97％、98％或99％序列同源性)的氨基酸序列或序列SEQ ID NO:36或SEQ ID NO:37的氨基酸序列，和/或其轻链可变(VL)域(区)包含与序列SEQ ID NO:38或SEQ ID NO:39具有至少90％序列同源性(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性)的氨基酸序列或序列SEQ ID NO:38或SEQ ID NO:39的氨基酸序列。
在一些实施方案中，抗CLDN18.2抗体包含重链可变区和轻链可变区，其中重链可变区包含如下的氨基酸序列：
2)抗体片段
在某些实施方案中，本文中所提供的抗体为抗体片段。抗体片段包括但不限于Fab、Fab’、Fab’-SH、(Fab’) 2、Fv及scFv片段及下文所描述的其他片段。关于某些抗体片段的综述，参见Hudson等人，Nat.Med.9:129-134(2003)。关于scFv片段参见例如WO 93/16185。
双功能抗体为具有两个抗原结合位点的抗体片段，其可为二价或双特异性的。参见例如EP 404,097；WO1993/01161。三功能抗体及四功能抗体可参见例如Hudson等人，Nat.Med.9:129-134(2003)中。
单域抗体为包含抗体的所有或部分重链可变域或所有或部分轻链可变域的抗体片段。在某些实施方案中，单域抗体为人类单域抗体(参见例如美国专利6,248,516B1)。
抗体片段可通过各种技术产生，包括但不限于完整抗体的蛋白水解消化以及重组宿主细胞(例如大肠杆菌或噬菌体)产生。
3)嵌合抗体及人源化抗体
在某些实施方案中，本文中所提供的抗体为嵌合抗体。嵌合抗体的制备可参见例如美国专利4,816,567。
在某些实施方案中，嵌合抗体为人源化抗体。典型地，非人类抗体经人源化以降低对人类的免疫原性，同时保留亲本非人类抗体的特异性及亲和力。一般而言，人源化抗体包含一个或多个可变域，其中全部HVR(CDR)区，或其部分源自于非人类抗体，且FR(或其部分)源自于人类抗体序列。人源化抗体任选地包含人类恒定区的至少一部分。在一些实施方案中，可用来自于非人类抗体的相应残基取代人源化抗体中的一些FR残基，以修复或改善抗体的亲和力。
人源化抗体及其制造方法可参照美国专利第5,821,337号、第7,527,791号、第6,982,321号及第7,087,409号。
4)人类抗体
在某些实施方案中，本文中所提供的抗体为人类抗体。人类抗体可使用本领域中已知的各种技术产生。
人类抗体可通过将免疫原施用给经修饰的转基因动物，然后用抗原攻击制备完整人类抗体或具有人类可变区的完整抗体。此类动物典型地含有所有或部分人类免疫球蛋白基因座，其置换内源免疫球蛋白基因座或存在于染色体外或随机整合于动物染色体中。在此类转基因小鼠中，内源免疫球蛋白基因座一般已灭活。关于由转基因动物获得人类抗体的方法，参见例如美国专利第6,075,181号及第6,150,584号(描述XENOMOUSETM技术)；美国专利第5,770,429号；美国专利第7,041,870号(描述K-M技术)；及美国申请公布第US 2007/0061900号。得自于由此类动物产生的完整抗体的人类可变区可经进一步修饰，例如将其与不同的人类恒定区进行组合。
人类抗体也可通过基于杂交瘤的方法制造。已描述用于产生人类单克隆抗体的人类骨髓瘤及小鼠-人类杂交骨髓瘤细胞株，参见例如Boerner等人，J.Immunol.,147:86(1991)。经由人类B细胞杂交瘤技术产生的人类抗体也描述于Li等人，Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)中。其他方法包括例如美国专利第7,189,826号(描述由杂交瘤细胞株产生单克隆人类IgM抗体)及Ni，Xiandai Mianyixue,26(4):265-268(2006)(描述人类-人类杂交瘤)中所描述的方法。
人类抗体也可通过分离选自人类来源噬菌体展示库的Fv克隆可变域序列来制备。接着可将此类可变域序列与所要的人类恒定域组合。
具体地，可通过针对具有结合CLDN18.2活性的抗体筛选组合库来分离具有高亲和力的本发明抗体。例如，本领域中已知用于产生噬菌体展示库及针对具有所要结合特征的抗体筛选此类库的多种方法。此类方法可查阅例如Hoogenboom等人，Methods in Molecular Biology 178:1-37(O’Brien等人编，Human Press,Totowa，NJ，2001)，Marks及Bradbury，Methods in Molecular Biology 248:161-175(Lo编，Human Press,Totowa,NJ,2003)及Lee等人，J.Immunol.Methods 284(1-2):119-132(2004)。
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)单独克隆VH及VL基因谱系，且随机重组于噬菌体库中，接着针对抗原-结合噬菌体进行筛选。噬菌体典型地将抗体片段呈现为单链Fv(scFv)片段或Fab片段。描述人类抗体噬菌体库的专利包括例如：美国专利第5,750,373号及美国专利公布第2005/0079574号、第2005/0119455号、第2005/0266000号、第2007/0117126号、第2007/0160598号、第2007/0237764号、第2007/0292936号及第2009/0002360号。
自人类抗体库分离的抗体或抗体片段在本文中被视为人类抗体或人类抗体片段。
5)多特异性抗体
在任何上述方面中，本文中所提供的抗CLDN18.2抗体为多特异性抗体，例如双特异性抗体。多特异性抗体为对至少两个不同的位点具有结合特异性的单克隆抗体。在某些实施方案中，一个结合特异性针对CLDN18.2，而另一结合特异性针对任何其他抗原(例如第二生物分子，例如细胞表面抗原，例如肿瘤抗原)。相应地，双特异性抗CLDN18.2抗体可对CLDN18.2及肿瘤抗原，例如CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1或TenB2有结合特异性。双特异性抗体可制备为全长抗体或抗体片段。
用于制造多特异性抗体的技术包括但不限于具有不同特异性的两个免疫球蛋白重链-轻链配对的重组共表达，参见WO 93/08829、WO2009/08025及WO 2009/089004A1等。
6)抗体变体
本发明的抗体涵盖本发明抗CLDN18.2抗体的氨基酸序列变体。例如，可能需要进一步改善抗体的结合亲和力和/或其他生物学性质而制备的抗体变体。抗体的氨基酸序列变体可通过向编码该抗体的核苷酸序列中引入适当修饰来制备。此类修饰包括例如抗体氨基酸序列内的残基缺失和/或插入和/或取代。可对缺失、插入及取代进行任何组合以获得最终的构建体，条件是最终的构建体具有所需要的特征，例如与CLDN18.2抗原的结合特性。
在某些实施方案中，提供具有一个或多个氨基酸取代的抗体变体。可以在HVR(CDR)区和/或FR区的一个或多个位点进行取代获得取代型突变体(包括保守取代突变体或非保守取代突变体)。
可根据共同侧链性质对氨基酸进行分组：
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；
(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链取向的残基：Gly、Pro；
(6)芳族：Trp、Tyr、Phe。
保守取代定义为同一组氨基酸之间的取代，非保守取代定义为不同类别之一的氨基酸被另一类别的氨基酸的取代。可将氨基酸取代引入本发明的抗体中且针对所希望获得的活性(例如保留/改善抗原结合或改善ADCC或CDC)来筛选产物，获得本发明的抗体变体。
本发明涵盖根据本发明公开的抗体获得的含有非保守突变和/或保守突变的抗体变体，只要该变体仍然具有所需要的CLDN18.2结合活性。
一种类型的取代型变体涉及取代亲本抗体(例如人源化抗体或人类抗体)的一个或多个高变区残基的抗体变体。一般而言，选择用于进一步研究的所得变体将在某些生物学性质(例如增加亲和力)方面相对于亲本抗体得以修饰(例如改善)和/或将实质上保留亲本抗体的某些生物学性质。例示性取代型变体为亲和力成熟抗体，其可使用例如基于噬菌体展示的亲和力成熟技术(诸如本文中所描述的那些)便利地产生。简言之，使一个或多个HVR(CDR)残基突变且将该突变的抗体展现于噬菌体上，且针对特定生物活性(例如结合亲和力)对突变的抗体进行筛选。
在某些实施方案中，一个或多个HVR(CDR)内可发生取代、插入或缺失，只要此类变化实质上不削弱抗体结合CLDN18.2的能力即可。例如，可在HVR(CDR)中进行实质上不降低结合亲和力的保守变化。例如，此类变化可在HVR中的抗原接触残基以外，例如可在FR区的1个1、2个、3个、4个、5个氨基酸残基处发生保守或非保守氨基酸取代。
7)重组方法
本发明的抗CLDN18.2抗体可使用重组方法来制备，例如，如美国专利第4,816,567号中所描述。在一个实施方案中，提供编码本文中所描述的抗CLDN18.2抗体的经分离核酸。此类核酸可编码抗体的VL氨基酸序列和/或VH的氨基酸序列。在另一实施方案中，提供包含此类核酸的一种或多种载体(例如表达载体)。在另一实施方案种，提供包含此类核酸的宿主细胞。在一个此类实施方案中，宿主细胞包含(例如，经转化而具有)：(1)包含编码包含该抗体的VL的氨基酸序列及包含该抗体的VH的氨基酸序列的核酸的载体；或(2)包含编码包含该抗体的VL的氨基酸序列的核酸的第一载体及包含编码包含该抗体的VH的氨基酸序列的核酸的第二载体。在一个实施方案中，该宿主细胞为真核细胞，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如Y0、NS0、Sp20细胞)。在一个实施方案中，提供制造抗CLDN18.2抗体的方法，其中该方法包括在适合表达该抗体的条件下培养如上文所提供的包含编码该抗体的核酸的宿主细胞，及任选地自该宿主细胞(或宿主细胞培养基)回收该抗体。
为了重组产生抗CLDN18.2抗体，分离编码抗体的核酸(例如，如上所描述)且插入一个或多个载体中以便在宿主细胞中进行进一步克隆和/或表达。此类核酸可使用常规程序(例如，通过使用能够特异性结合编码该抗体的重链及轻链的基因的寡核苷酸探针)容易地分离并测序。
适用于克隆或表达抗体编码载体的宿主细胞包括本文中所描述的原核细胞或真核细胞。例如，可在细菌中产生抗体，尤其在不需要糖基化及Fc效应功能时。对于在细菌中表达抗体片段及多肽，参见例如美国专利第5,648,237号、第5,789,199号及第5,840,523号。在表达之后，可自细菌细胞浆中分离处于可溶性部分中的抗体且可进一步纯化。
除原核生物以外，诸如丝状真菌或酵母的真核微生物也为抗体编码载体的合适克隆或表达宿主，包括糖基化途径已“人源化”从而产生具有部分或完全人类糖基化模式的抗体的真菌及酵母菌株。参见Li等人,Nat.Biotech.24:210-215(2006)。
适用于表达糖基化抗体的宿主细胞也可来源于多细胞生物体(无脊椎动物及脊椎动物)。无脊椎动物细胞的实例包括植物及昆虫细胞。已鉴别许多 可用于结合昆虫细胞，尤其用于转染草地贪夜蛾(Spodoptera frugiperda)细胞的杆状病毒菌株。
植物细胞培养物也可用作宿主。参见例如美国专利第5,959,177号、第6,040,498号、第6,420,548号、第7,125,978号及第6,417,429号(描述用于在转基因植物中产生抗体的PLANTIBODIESTM技术)。
脊椎动物细胞也可用作宿主。例如，适于在悬浮液中生长的哺乳动物细胞株可能适用。适用哺乳动物宿主细胞株的其他实例为经SV40转化的猴类肾CV1细胞株(COS-7)；人类胚肾细胞株(例如293细胞)；幼仓鼠肾细胞(BHK)；小鼠塞尔托利细胞(例如TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人类子宫颈癌细胞(HELA)；犬类肾细胞(MDCK)；布法罗大鼠肝细胞(BRL3A)；人类肺细胞(W138)；人类肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；及骨髓瘤细胞株，诸如Y0、NS0及Sp2/0。
8)免疫缀合物
本发明也提供包含本文中的抗CLDN18.2抗体与一种或多种细胞毒性剂结合的免疫缀合物，此类细胞毒性剂为诸如化学治疗剂或化学治疗药物、生长抑制剂、毒素(例如蛋白毒素、细菌、真菌、植物或动物来源的酶活性毒素或其片段)或放射性同位素。
在一个实施方案中，免疫缀合物为抗体-药物缀合物(ADC)，其中抗体与一种或多种药物结合，包括但不限于美登素、奥利司他汀、多拉司他汀、氨甲蝶呤、长春地辛、紫杉烷、单端孢霉毒素(trichothecene)及CC1065。
在另一实施方案中，免疫缀合物包含如本文中所描述的抗CLDN18.2抗体与酶活性毒素或其片段的缀合物，该酶活性毒素包括但不限于白喉A链、白喉毒素的非结合活性片段、外毒素A链及单端孢霉毒素等。
在另一实施方案中，免疫缀合物包含如本文中所描述的抗CLDN18.2抗体与放射性原子结合形成的放射性缀合物。多种放射性同位素可用于产生放射性缀合物。实例包括At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212及Lu的放射性同位素。
抗体与细胞毒性剂的缀合物可使用多种双官能蛋白偶合剂制造，诸如N-丁二酰亚胺基-3-(2-吡啶基二硫)丙酸酯(SPDP)、丁二酰亚胺基-4-(N-顺丁 烯二酰亚胺基甲基)环己烷-1-甲酸酯(SMCC)、亚胺基硫杂环戊烷(IT)、酰亚胺酯的双官能衍生物(诸如己二酸二甲酯盐酸盐)、活性酯(诸如辛二酸二丁二酰亚胺酯)、醛(诸如戊二醛)、双叠氮基化合物(诸如双(对叠氮基苯甲酰)己二胺)、双重氮衍生物(诸如双(对重氮苯甲酰)乙二胺)、二异氰酸酯(诸如甲苯2,6-二异氰酸酯)及双活性氟化合物(诸如1,5-二氟-2,4-二硝基苯)。
9)药物制剂
本发明的抗CLDN18.2抗体的药物制剂通过将具有所要纯度的抗体与一种或多种任选的药学上可接受的载体混合呈冻干制剂或水溶液形式来制备。药学上可接受的载体在所采用的剂量及浓度下一般对受体无毒，且包括但不限于：缓冲剂，诸如磷酸盐、柠檬酸盐及其他有机酸；抗氧化剂，包括抗坏血酸及甲硫氨酸；防腐剂(诸如十八烷基二甲基苯甲基氯化铵；氯化六烃季铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸烷基酯，诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；及间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，诸如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯啶酮；氨基酸，诸如甘氨酸、谷酰氨酸、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、岩藻糖或山梨糖醇；成盐相对离子，诸如钠；金属配合物(例如锌-蛋白配合物)；和/或非离子表面活性剂，诸如聚乙二醇(PEG)。
例示性冻干抗体制剂描述于美国专利第6,267,958号中。水性抗体制剂包括美国专利第6,171,586号及WO2006/044908中所描述的那些。
本文中的制剂也可含有超过一种对于所治疗的特定适应症而言必需存在的活性成分，优选具有不会对彼此造成不利影响的互补活性的活性成分。例如，可能需要进一步提供额外治疗剂(例如化疗剂、细胞毒性剂、生长抑制剂和/或抗激素剂)。此类活性成分适合以组合形式以对预定目的有效的量存在。
10)制品
在本发明的另一方面中，提供含有本发明抗体或药物组合物的制品。该制品包含容器及处于该容器上或与该容器相关联的标签或包装插页。合 适的容器包括例如瓶、小瓶、注射器、IV溶液袋等。此类容器可由各种材料形成，诸如玻璃或塑料。该容器容纳本发明组合物自身或该组合物与另一组合物的组合，且可具有无菌进入口(例如，该容器可为静脉内溶液袋或具有可由皮下注射针刺穿的瓶塞的小瓶)。该组合物中的至少一种活性剂为本发明的抗体。该标签或包装插页指示该组合物用于治疗选定肿瘤。此外，该制品可包含(a)其中含有组合物的第一容器，其中该组合物包含本发明的抗体；及(b)其中含有组合物的第二容器，其中该组合物包含另一肿瘤治疗药物或另一抗体。本发明的此实施方案中的制品可进一步包含指示此类组合物可用于治疗肿瘤的包装插页。可选地，或另外，该制品可进一步包括第二(或第三)容器，该容器包含药学上可接受的缓冲剂，诸如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液及右旋糖溶液。其可进一步包括自商业及使用者的立场来看合乎需要的其他材料，包括其他缓冲剂、稀释剂、过滤器、针及注射器。
11)检测试剂和试剂盒
本发明也提供包含本文中的抗CLDN18.2抗体或其抗原结合片段的癌症检测试剂。癌症检测试剂可以用于测试实际患者的肿瘤或血液组织。例如，可以用PCR、微阵列或芯片技术进行测试。一旦发现癌症检测试剂存在于患者中，可以选择那些试剂，用于治疗患者或指导治疗。
在一个实施方案中，抗CLDN18.2抗体或其抗原结合片段是经过化学标记的，具体的可以为酶标记，如碱性磷酸酶和葡萄糖氧化酶；荧光标记，如荧光素和罗丹明；同位素标记，如碘(125I、131I)、碳(14C)、硫(35S)、氚(3H)、铟(121In)、以及锝(99mTc)；或化学发光物物标记，如鲁米诺和荧光素酶。
本发明还提供包含本文中的抗CLDN18.2抗体或其抗原结合片段的癌症检测试剂盒。试剂盒也可包含经过标记的抗CLDN18.2抗体或其抗原结合片段。具体的，癌症检测试剂盒可用于检测胃癌、胰腺癌或食管癌。
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
实施例
实施例1免疫及筛选材料的制备
(1)人hCLDN18.2-TCE(T-细胞表位肽)逆转录病毒表达质粒的构建
将hCLDN18.2 cDNA(SEQ ID NO:1)进行克隆并在C-末端融合T-细胞表位肽，得到hCLDN18.2-TCE。T-细胞表位肽能够使抗原突破免疫耐受，促使抗体产生(Percival-Alwyn J.et al,mAbs,2015,7(1),129-137)。将hCLDN18.2-TCE克隆到逆转录病毒载体，得到hCLDN18.2-TCE逆转录病毒表达质粒。将hCLDN18.2-TCE逆转录病毒表达质粒瞬时转染HEK293-T细胞，并于转染72小时后流式细胞术分析hCLDN18.2在HEK293-T细胞表面的表达。一抗(1st Ab)为自主制备的阳性对照(Benchmarker)嵌合抗体163E12。细胞首先与一抗(1μg/mL)在4℃条件下孵育45min，PBS洗涤后与Alexa Fluor 488标记的羊抗人Fc二抗(Thermofisher，货号A-11013))(2nd Ab，1：200稀释)在4℃条件下孵育45分钟。PBS洗涤2次后流式细胞术(FACS)分析，阴性对照细胞只孵育二抗。结果如图1所示，显示野生HEK293-T细胞表面无hCLDN18.2表达；瞬时转染hCLDN18.2-TCE逆转录病毒表达质粒的HEK293-T细胞(HEK293T CLDN18.2 TCE)有80.9％的阳性细胞表达hCLDN18.2。该载体质粒将被用于实施例2中的(1)DNA免疫方案。
(2)高水平表达hCLDN18.2-TCE的小鼠肿瘤细胞
将hCLDN18.2-TCE逆转录病毒表达质粒与慢病毒包装质粒混合并转染293-T细胞，制备hCLDN18.2-TCE慢病毒颗粒。将hCLDN18.2-TCE慢病毒颗粒转染小鼠肿瘤细胞系，72小时后通过流式细胞术检测并分选高表达hCLDN18.2-TCE的小鼠肿瘤细胞池。一抗(1st Ab)为自主制备的阳性对照(Benchmarker)嵌合抗体163E12。细胞首先与一抗(1μg/mL)在4℃条件下孵育45分钟，PBS洗涤后与Alexa Fluor 488标记的羊抗人Fc二抗(2nd Ab，1：200稀释)在4℃条件下孵育45分钟。PBS洗涤2次后流式细胞术(FACS)分析，阴性对照细胞只孵育二抗。结果如图2所示，显示野生小鼠肿瘤细胞(UBER parental)表面只有16.8％的阳性信号；hCLDN18.2-TCE逆转录病毒转染的小鼠肿瘤细胞(UBER CLDN18.2 TCE)有82.7％的阳性细胞。通过流式细胞术分析分选后，高达95.7％的小鼠肿瘤细胞具有高水平的 hCLDN18.2-TCE表达。该hCLDN18.2-TCE的小鼠肿瘤细胞将被用于实施例2中的(2)细胞免疫方案。
(3)hCLDN18.2胞外环1类病毒颗粒的制备
按照Thorsten K.发表的方法(Thorsten K.,et al,Cancer Res,2011,71(2),515-527)，将hCLDN18.2的胞外环1(Extra Cellular Loop1,ECL1)(SEQ ID NO:2)插入乙肝病毒核心抗原(Hepatitis B virus core antigen,HBcAg)的主要免疫显性区(Major immunodominant region,MIR)并在ECL1N-末端和C-末端各引入G4SG4连接子，在全长融合蛋白的C-末端添加6xHis标签(SEQ ID NO:3)。合成融合蛋白的基因全长，并克隆至pET24a(+)质粒，之后转染至BL21(DE3)大肠杆菌表达系统进行融合蛋白表达。融合蛋白纯化后在复性缓冲液中制备HBV hCLDN18.2胞外环1类病毒颗粒。该hCLDN18.2胞外环1类病毒颗粒的制备将被用于实施例2中的(3)病毒样颗粒多点重复免疫-细胞免疫方案和(4)病毒样颗粒附关节免疫-细胞免疫方案。
(4)CLDN18稳转细胞株的构建
分别合成hCLDN18.2(SEQ ID NO:4)，hCLDN18.1(SEQ ID NO:5)，鼠mhCLDN18.2(SEQ ID NO:6)，mCLDN18.1(SEQ ID NO:7)基因全长，并分别克隆至pcDNA3.4载体质粒。质粒转染HEK293细胞并加新霉素(G418)进行加压筛选。采用有限稀释法分别挑选高表达hCLDN18.2，hCLDN18.1，m hCLDN18.2，mCLDN18.1的HEK293稳定转染细胞株。流式细胞术检测结果表明转染hCLDN18.2和mCLDN18.2的HEK293稳转细胞株(HEK293-hCLDN18.2，HEK293-mCLDN18.2)，多聚甲醛固定和非固定细胞均能够被自制抗CLDN18.2阳性对照抗体163E12或175D10特异性识别(图3.A,C)，同时能够被商业化广泛抗CLDN18抗体34H14L15，(Abcam货号ab203563)，Abcam,ab203563))识别(图3.A,C)。转染hCLDN18.1和mCLDN18.1的HEK293稳转细胞株(HEK293-hCLDN18.1，HEK293-mCLDN18.1)，多聚甲醛固定和非固定细胞均不能被自制抗CLDN18.2阳性对照抗体163E12或175D10特异性识别(图3.B,D)，但能够被商业化广泛抗CLDN18抗体(34H14L15)识别(图3.B,D)。上述HEK293稳定转染细胞株将被用于实施例3中的杂交瘤筛选。
(5)阳性对照抗体43A11、175D10和163E12的制备
分别合成人鼠嵌合阳性对照抗体43A11重链(SEQ ID NO:8)和轻链(SEQ ID NO:9)、175D10重链(SEQ ID NO:10)和轻链(SEQ ID NO:11)以及163E12重链(SEQ ID NO:12)和轻链(SEQ ID NO:13)抗体基因全长并分别在N-末端添加信号肽，然后克隆至pcDNA3.4真核表达载体。分别将43A11、175D10和163E12重链和轻链表达质粒混合共转染CHOS细胞进行瞬时转染蛋白表达，收集上清并采用Protien A进行亲和纯化并进行SDS-PAGE检测，结果如图4所示。
实施例2小鼠免疫程序及杂交瘤制备
一共采取了4种免疫方案(表1)，且每种免疫方案采用了至少两个不同种(strain)的小鼠进行免疫(表2)。每种免疫方案中都有部分小鼠个体出现较高水平的血清免疫滴度。选择各免疫方案中出现高水平免疫滴度的小鼠个体处死并取脾脏，分离B淋巴细胞进行混合后与小鼠骨髓瘤细胞系进行电融合，制备杂交瘤。一共进行了三个批次的杂交瘤制备(表2)。
表1
Figure PCTCN2021076481-appb-000001
Figure PCTCN2021076481-appb-000002
表2
Figure PCTCN2021076481-appb-000003
实施例3杂交瘤筛选
对来自三个批次的杂交瘤采用有限稀释法克隆化培养，取克隆上清采用流式细胞术进行三轮结合或反向结合筛选，抗体分型和抗体依赖性细胞毒性(Antibody dependent cell-mediated cytoxity,ADCC)测定，筛选出128个能够和CLDN18.2特异性强结合而与CLDN18.1不结合或微弱结合，且具有ADCC功能的杂交瘤克隆。筛选步骤及各阶段筛选所得杂交瘤克隆总结如表3所示。
表3
Figure PCTCN2021076481-appb-000004
Figure PCTCN2021076481-appb-000005
1)初级筛选：利用流式细胞术分析各克隆上清表达产物与高水平表达CLDN18.2-TCE的小鼠肿瘤细胞系(UBER CLDN18.2 TCE)的结合。采用有限稀释法将融合后的杂交瘤种植到384孔板，培养十天后取上清采用流式细胞术进行阳性杂交瘤克隆筛选。将上清与UBER CLDN18.2 TCE细胞(20000个细胞/孔)在4℃下共孵育45分钟后，洗板，以Alexa Fluor-488-羊抗鼠IgG(ThermoFisher货号A28175)作为二抗进行流式细胞术检测，筛选平均荧光强度在100,000以上的克隆进行扩增，并收集饱和上清进行确认筛选。
3个批次的杂交瘤，每个批次有限稀释法克隆化10个384孔板。经过初级筛选，三个批次杂交瘤分别获得341个(MFI>100,000)、336个(MFI>75,000或MFI>80,000)和89个(MFI>90,000)与CLDN18.2-TCE的小鼠肿瘤细胞系阳性结合杂交瘤克隆。
2)确认筛选：将上一步初级筛选到的阳性克隆的上清表达产物分别与 HEK293-hCLDN18.2细胞、HEK293-hCLDN18.1细胞和HEK293野生型细胞孵育(20000个细胞/孔，4℃，45分钟)，以Alexa Fluor-488-羊抗鼠IgG(ThermoFisher货号A28175)作为二抗采用流式细胞术筛选与HEK293-hCLDN18.2细胞结合呈阳性，但是与HEK293-hCLDN18.1细胞和HEK293野生型细胞结合呈阴性的克隆进行扩增，并收集饱和上清进行验证筛选。
经过确认筛选，三个批次杂交瘤分别获得165个(HEK293-hCLDN18.2MFI>200,000，HEK293-hCLDN18.1MFI<100,000)、120个(HEK293-hCLDN18.2MFI>50,000，HEK293-hCLDN18.1MFI<40,000)和21个(HEK293-hCLDN18.2MFI>70,000，HEK293-hCLDN18.1MFI<30,000)克隆能够与HEK293-hCLDN18.2细胞特异性强结合但是与HEK293-hCLDN18.1细胞弱结合及HEK293野生型细胞不结合的克隆。部分阳性克隆FACS分析如图5所示。
3)验证筛选：将来源于三个批次杂交瘤确认筛选中获得的306个(165个+120个+21个)阳性杂交瘤的上清表达产物分别与HEK293-hCLDN18.2细胞、HEK293-hCLDN18.1细胞、HEK293-mCLDN18.2细胞、HEK293-mCLDN18.1细胞和HEK293野生型细胞孵育(20000个细胞/孔，4℃，45分钟)，以Alexa Fluor-488-羊抗鼠IgG(ThermoFisher货号A28175)作为二抗采用流式细胞术进行分析。部分克隆在5个细胞株上的流式细胞术分析结果如图6所示，显示阳性克隆在HEK293-hCLDN18.2细胞和HEK293-mCLDN18.2细胞的平均荧光强度(MFI)均超过500,000，而在HEK293-hCLDN18.1细胞、HEK293-mCLDN18.1细胞和HEK293野生型细胞细胞株上的平均荧光强度均低于50,000，多数克隆(16/22)在HEK293-hCLDN18.2细胞和HEK293-mCLDN18.2细胞的平均荧光强度(MFI)均超过在HEK293-hCLDN18.1细胞和HEK293-mCLDN18.1细胞平均荧光强度(MFI)的50倍。
4)抗体分型：因为不同亚型的IgG分子也有着不同的介导ADCC效应的能力。在进行抗体依赖性细胞杀伤检测之前采用小鼠抗体亚型检测试剂盒对来源于三个批次杂交瘤确认筛选中获得的306个(165个+120个+21个)阳性杂交瘤的上清抗体表达产物进行小鼠抗体亚型分型。306个克隆中大部 分亚型属于具有ADCC功能的小鼠IgG2a或IgG2b或IgG3，仅有少数(26/306)为不具有ADCC功能的IgG1亚型(对应人的抗体IgG4亚型)。部分克隆为混合亚型，可能因为杂交瘤克隆非单克隆所致。轻链均为小鼠κ亚型。
5)ADCC效应测定
综合考虑前述确认筛选、验证筛选以及抗体分型结果，优选了128个阳性克隆的饱和上清进行抗体依赖性细胞毒性功能检测。效应细胞为人外周血单个核细胞(PBMCs)，来源自两名捐赠者(45和46号)，在试验前一天对捐献者进行抽血，采集到的血液保存在室温下，PBMCs经Ficoll梯度新鲜分离得到。靶细胞为表达CLDN18.2的HEK293细胞(HEK293-hCLDN18.2)。杂交瘤表达产物统一按照4倍稀释。新鲜获取的PBMCs细胞和靶细胞以效靶比为25:1的比例与各样品共孵育4小时。通过测定与细胞毒相关的乳酸脱氢酶(LDH)来表征抗体ADCC效应。以靶细胞自发裂解情况下检测到的吸光度值定义为0％，以靶细胞全部裂解情况下检测到的吸光度值定义为100％，各测试样品以相对百分活性来表征ADCC效应。部分克隆的ADCC效应结果如图7所示，克隆上清的ADCC杀伤效果平均高于20％，部分克隆(9D-1、8-O11)的ADCC效应高于50％，而对于小鼠IgG1亚型的6-G11和4-O2，ADCC效应小于10％。
实施例4 PPFE组织切片上hCLDN18.2免疫组织化学阳性染色杂交瘤筛选
在伴随诊断过程中，患者的肿瘤组织活检后进行石蜡包埋(FFPE)组织切片病理检测是通用流程。癌症抗原(Tumor associated antigen,TAA)在进行FFPE组织切片过程中，抗原的天然结构丢失，即使进行抗原复性(Antigen Retrieval)，抗原也只能在一定程度上只恢复部分构象结构，或者只暴露某些线性肽段。前述优选的128个阳性杂交瘤克隆上清具有特异性结合细胞表面天然CLDN18.2构象的能力和ADCC功能，但是作为伴随诊断抗体，必须应在FFPE组织切片上进行抗原特异性结合的IHC筛选。本实施例采用PPFE组织切片的免疫组织化学(Immunohistology chemistry,IHC)方法检测了128个阳性杂交瘤上清在3个工程细胞株(HEK293-hCLDN18.2细胞、HEK293-hCLDN18.1细胞和HEK293野生型细胞)细胞块(cell block)石蜡包埋(FFPE)组织切片上的结合。
首先在CLDN18稳定表达的HEK293细胞块FFPE组织切片上进行IHC染色，包括培养HEK293-hCLDN18.2细胞、HEK293-hCLDN18.1细胞和HEK293野生型细胞，收集细胞，离心形成细胞块，然后进行脱水、透明、石蜡包埋步骤后进行FFPE组织切片和IHC染色。筛选能够在HEK293-hCLDN18.2细胞块FFPE组织切片上阳性显色而在HEK293-hCLDN18.1细胞和HEK293野生型细胞块FFPE组织切片上成阴性染色的杂交瘤克隆上清。然后对获得的阳性杂交瘤上清在人胃癌组织FFPE组织切片上进行IHC染色验证。最后对验证的IHC阳性染色杂交瘤上清在人正常胃及胃癌FFPE组织微阵列(Tissue Micro-Array,TMA)上进行IHC染色，根据IHC染色强度及染色比例确定最终的IHC阳性染色克隆。
杂交瘤克隆针对细胞块FFPE组织切片、胃癌FFPE组织切片以及正常胃及胃癌FFPE组织微阵列(TMA)的IHC方法包括以下步骤：1)脱蜡烤片：60℃，30分钟；2)脱蜡：三级(I、II、III)二甲苯中浸泡，每级5分钟；3)水化：梯度乙醇浸泡100％×2次，95％×1次，75％×1次，每次3分钟，蒸馏水浸泡5分钟；4)抗原修复：将抗原修复液(Tris-EDTA，pH＝9.0)置100℃水浴中，FFPE组织切片放入抗原修复液中孵育30分钟，室温冷却30分钟，PBS缓冲液洗涤3次，每次5分钟；5)封闭：3％H2O2-PBS室温避光孵育10分钟，PBS洗涤3次，然后5％BSA(PBS-BSA)中室温孵育60分钟，PBS缓冲液洗涤3次；6)一抗孵育：兔抗CLDN18.2商业化阳性抗体(EPR19202,Abcam,货号ab222512)以1：100稀释，或者小鼠杂交瘤上清，4℃孵育过夜；7)二抗孵育：HRP-conjugated羊抗兔(Abcam,货号ab6721，1：1000稀释)和HRP-conjugated羊抗鼠二抗(ThermoFisher,货号62-6520，1：2000稀释)；8)显色：二氨基联苯胺(DAB)显色3分钟；9)苏木素复染1分钟，0.5％HCl-75％乙醇分化数秒，流水反蓝20分钟；10)脱水透明：70％乙醇、80％乙醇、95％乙醇、无水乙醇I、无水乙醇II各2分钟脱水；二甲苯I和二甲苯II各5分钟透明；11)封片：中性封片剂封片。
图8所示为阳性对照商业化抗体EPR19202在HEK293细胞块FFPE组织切片上的IHC染色结果，抗体能够在HEK293-hCLDN18.2稳定表达细胞株的细胞块FFPE组织切片上特异性显色，且细胞膜位置阳性显色明显(图8.A，箭头所示)，这与CLDN18.2是膜蛋白结论一致(图8.A)。相对而言， 抗体在HEK293-hCLDN18.1稳定表达细胞株的细胞块FFPE组织切片上的显色不明显(图8.B)，而在HEK293野生细胞株上无明显显色(图8.C)。
通过对120个杂交瘤克隆上清在3个细胞株(HEK293-hCLDN18.2细胞、HEK293-hCLDN18.1细胞和HEK293野生型细胞)细胞块FFPE组织切片上的IHC染色，获得了4个杂交瘤克隆(5-I5、5-I10、5-G17和10-D3)。它们的克隆上清都能够在HEK293-hCLDN18.2细胞块FFPE组织切片上出现阳性IHC显色，而对HEK293-hCLDN18.1和HEK293-WT细胞块FFPE组织切片不显色或弱显色。
对在HEK293-hCLDN18.2细胞块FFPE组织切片上筛选的4个IHC阳性杂交瘤克隆上清在来源于人胃癌组织FFPE组织切片上进行IHC显色验证，其中5-I5杂交瘤克隆上清在3例人胃癌组织FFPE组织切片上的IHC显色结果如图9(A,B,C)，抗体的阳性显色只出现在腺体上皮细胞位置(箭头所示)，对于基质细胞无明显阳性显色反应或者弱染色(三角箭头所示)。5-I5在3例胃癌组织FFPE组织切片上的阳性着色位置，阳性染色组织结构上与阳性对照抗体(EPR19202)的IHC显色结果(图9D，E和F)相对一致。
作为伴随诊断的抗体，能够高比例的识别出肿瘤相关抗原高表达的患者进行治疗具有重要的临床意义。因为癌症患者本身的差异以及患者肿瘤组织的异质性，往往导致针对同一肿瘤相关抗原的不同的诊断抗体的IHC显色结果会出现差异。我们对4个IHC阳性候选杂交瘤表达上清以及4个上清的混合物(Cocktail)在人正常胃及胃癌FFPE组织微阵列(TMA)芯片(Biomax,ST807)上进行了IHC显色。在抗体孵育过程中，单个杂交瘤克隆上清以及阳性对照抗体的孵育时间为30分钟，4个杂交瘤上清混合物的孵育时间为120分钟。表4总结了细胞膜阳性显色和细胞胞浆阳性显色的组织位点，组织类型以及对应的阳性染色克隆。5-I10、5-G17、5-I5、10-D3和4个克隆上清混合物在所有76个组织位点上的细胞膜阳性显色率分别为28.9％、17.1％、14.5％、11.8％和28.9％，阳性对照抗体(EPR19202)的细胞膜阳性率为23.7％。值得注意的是所有克隆上清及阳性抗体在正常胃组织上的染色几乎均为阳性(5-I10 87.5％、5-G17 87.5％、5-I5 100％、10-D3 87.5％、Mix.Of 4 100％、EPR19202 100％)，这与文献报道的CLDN18.2在正常胃黏膜上皮细胞特异性表达一致(Sahin.U.et al,Human Cancer Biology,2008, 14(23),7624-7634)。与正常胃组织的高阳性率染色相比，各个杂交瘤上清在胃癌组织上的阳性染色差异巨大，胃癌组织上细胞膜阳性染色率分别为5-I10 23.5％、5-G17 10％、5-I5 4.4％、10-D3 2.9％和4个杂交瘤上清混合物(Mix.Of 4)22％，这可能与胃癌组织的高异质性有关。其中5-I10杂交瘤克隆上清在胃癌组织细胞膜阳性显色率最高，达到23.5％，显著高于阳性对照抗体EPR19202的14.7％，且与Zhu G.等(Zhu G.et al,Sci Rep.,2019,9:8420-8431)报道的16～23％阳性率基本一致。
特别注意的是5-I10和5-G17在胃癌FFPE组织的阳性显色率较高，且两者的复合(5-I10+5-G17)阳性率最高可达26％，阳性显色胃癌FFPE组织位点基本上包含了5-I5和10-D3上出现的阳性位点。因此，将重组表达纯化的5-I10和5-G17抗体按照一定比例组成抗体混合物用于伴随诊断能够最大限度的挑选CLDN18.2高表达的胃癌患者，这对指导靶向CLDN18.2药物的临床给药具有重要意义。
表4
Figure PCTCN2021076481-appb-000006
注：1)表统计为出现阳性染色的组织位点；2)TMA共80个组织位点，其中4个组织位点组织形态差，排除在外；3)M表示为细胞膜阳性染色，C表示为细胞浆阳性染色，/表示阴性染色。
实施例5、FFPE组织切片hCLDN18.2阳性染色杂交瘤克隆的亚克隆及抗体分子序列测序
本实施例采用有限稀释法对4个在FFPE组织切片上IHC阳性染色杂交瘤克隆(5-I10,5-G17,5-I5和10-D3)进行亚克隆及抗体分子可变区序列测序。将冻存复苏的杂交瘤克隆进行有限稀释，按照0.8个细胞/孔的密度种植到192孔板，镜检挑选单克隆，并在培养14天后收集上清采用流式细胞术分析方法检测亚克隆上清与HEK293-hCLDN18.2细胞的特异性结合，同时对单克隆进行扩增培养。流式细胞分析方法为将单克隆上清与HEK293-CLDN18.2细胞(20000个细胞/孔)在4℃下共孵育45分钟后，洗板，以Alexa Fluor-488-羊抗鼠IgG(ThermoFisher货号A28175)作为二抗进行流式细胞术检测。对每个杂交瘤选择10个(5-I5，9个)单克隆进行扩增冻存，并收集上清进行FFPE组织切片IHC显色验证。4个杂交瘤的单克隆上清对HEK293-hCLDN18.2细胞的流式细胞术分析如图10所示，显示单克隆上清保持了HEK293-hCLDN18.2细胞的特异性结合，且部分单克隆上清与HEK293-hCLDN18.2细胞的结合强度与对应的杂交瘤上清(Parental supe.)的结合强度相当。从每个杂交瘤克隆中选择与HEK293-hCLDN18.2细胞的结合强度最高的前5个单克隆(图9中蓝色框所示)上清进行FFPE组织切片的IHC验证，所有单克隆上清均表现出IHC阳性显色。对于每个杂交瘤选择FFPE组织切片上IHC显色强度最高的2个单克隆(5-I5-31和5-I5-25，10-D3-22和10-D3-21，5-G17-30和5-G17-6，5-I10-33和5-I10-18)进行重链和轻链可变区基因克隆测序。
使用RACE(cDNA末端快速扩增技术)方法对杂交瘤单克隆的VH和VL的DNA序列进行扩增。从扩增杂交瘤单克隆细胞中抽提RNA，逆转录为cDNA并通过5’-RACE反应使用5’-的通用引物和3’-H，L(κ)或L(λ)FR1区的简并引物组合，对重链或者轻链V区进行PCR，TOPO clone方法将PCR扩增片段连接到测序载体上进行测序。测序结果表明5-I5-31和5-I5-25具有相同的VH但是VL序列不同，说明5-I5-31和5-I5-25是不同的单克隆。除5-I5外，10-D3、5-G17和5-I10各自所测序的两个单克隆均具有一致的VH和VL，其中5-I10和5-G17的VH和VL DNA序列见表5，对应的氨基酸序列见表6，其中轻、重链中的CDR区域根据Chothia编号系统划分。
表5
Figure PCTCN2021076481-appb-000007
表6 CDR的氨基酸序列
重链的CDR序列(Chothia编号)
Figure PCTCN2021076481-appb-000008
Figure PCTCN2021076481-appb-000009
实施例6重组小鼠单克隆抗体的表达
将5-I10和5-G17的重链和轻链的V区分别与小鼠IgG2a型重链恒定区(表7，Mouse IGHC2A，SEQ ID NO:30)及小鼠κ型轻链恒定区(表7，Mouse IGKC，SEQ ID NO:31)融合，并分别在重链和轻链序列N-端添加信号肽。对5-I10和5-G17的小鼠重链全长基因(SEQ ID NO:32,33)和轻链全长基因(SEQ ID NO:34,35)合成并克隆至表达载体，测序验证(金唯智生物科技有限公司，苏州)。对每个候选分子构建好的表达质粒进行扩增和质粒抽提，通过琼脂糖凝胶电泳进行验证，并作为转染材料使用。
将HEK293细胞接种入摇瓶中进行悬浮式的种子培养，使用无血清且化学成分明确的培养基。在转染前一天，将扩增的HEK293细胞接种入新的摇瓶中，并更换新鲜的培养基。将5-I10和5-G17的重链和轻链表达质粒分别共转染至HEK293细胞中，摇瓶培养直至细胞活力下降，收集上清进行产物纯化。首选通过对上清液的离心和过滤，去除掉大部分细胞碎片和 不溶性微粒，然后将料液加载到Protein A柱上，抗体分子与填料上的Protein A结合，经过平衡步骤后去除大部分杂质，使用低pH洗脱液洗脱抗体蛋白并收集组分。调节pH并通过超滤置换至制剂缓冲液。最后测定OD280以计算蛋白浓度。如图11所示，5-I10和5-G17单克隆抗体在还原SDS-PAGE上的电泳图显示重链和轻链条带位置与阳性对照IgG重链和轻链的位置一致，分子量符合预期，无明显杂带，蛋白纯度>95％。
表7
Figure PCTCN2021076481-appb-000010
实施例7、重组小鼠单克隆抗体的HEK293-hCLDN18.2结合活性
采用流式细胞术验证5-I10和5-G17纯化单克隆抗体在HEK293-hCLDN18.2上的结合，HEK293-hCLDN18.2种植96孔U型板(1.5×105个/孔)，用含有1％FBS的PBS缓冲液洗板1次，5-I10和5-G17纯化单克隆抗体分别采用PBS(1％FBS)稀释，从10μg/mL浓度开始进行3倍梯度稀释，共获得8个浓度点，加入96孔U型板100μL/孔，4℃孵育1小时。PBS(1％FBS)洗涤一次，加入1：1000稀释的Alexa488标记的山羊抗鼠二抗(eBioscience,A11001)100μL/孔，4℃孵育1小时。PBS(1％FBS)洗涤2次后200μL PBS(1％FBS)重悬，FACS检测。图12显示了5-I10和5-G17纯化单克隆抗体在HEK293-hCLDN18.2上的结合曲线，两者能够剂量依赖性与HEK293-hCLDN18.2特异性结合，结合能力相当。
实施例8、重组小鼠单克隆抗体在胃癌FFPE组织切片上阳性染色
在6例胃癌FFPE组织切片上对5-I10和5-G17纯化单克隆抗体的CLDN18.2 IHC阳性染色进行验证，采用EPR19202作为阳性对照。IHC染 色步骤与实施例4中描述相同，其中一抗孵育步骤中阳性对照抗体EPR19202工作浓度为1：100稀释，5-I10和5-G17纯化抗体工作浓度为5μg/mL。显色结果如图13所示，重组纯化的小鼠IgG2a型5-I10和5-G17单克隆抗体能够在多例胃癌组织FFPE组织切片上阳性染色。5-G17和5-I10的阳性染色比率分别达到83.3％(5/6)和66.7％(4/6)，显著高于阳性对照抗体EPR19202的50％(3/6)。在组织S07-37358-2A、S08-19346和S10-36009-3上，胃癌细胞形成了比较明显的胃腺体上皮结构，5-I10、5-G17和阳性对照抗体EPR19202均表现出较强的腺体上皮细胞特异性染色，而在基质细胞上无着色。在S11-4725-1组织上，胃癌细胞形成了类腺体样结构，但是与前述切片上的腺体结构明显有所区别；阳性对照SPR19202无明显IHC染色，而5-I10和5-G17具有较强的阳性染色，染色成弥漫型，类腺上皮及基质细胞都有着色。S09-15785-2FS和S07-7939-C切片上的肿瘤细胞无明显腺体结构形成，其中S09-15785-2FS上，阳性对照SPR19202、5-I10和5-G17均无IHC阳性染色。S07-7939-C上只有5-G17表现出IHC弱阳性染色。
序列表
SEQ ID NO:1
Figure PCTCN2021076481-appb-000011
SEQ ID NO:2
Figure PCTCN2021076481-appb-000012
SEQ ID NO:3
Figure PCTCN2021076481-appb-000013
SEQ ID NO:4
Figure PCTCN2021076481-appb-000014
Figure PCTCN2021076481-appb-000015
SEQ ID NO: 5
Figure PCTCN2021076481-appb-000016
SEQ ID NO: 6
Figure PCTCN2021076481-appb-000017
SEQ ID NO: 7
Figure PCTCN2021076481-appb-000018
SEQ ID NO: 8
Figure PCTCN2021076481-appb-000019
SEQ ID NO: 9
Figure PCTCN2021076481-appb-000020
Figure PCTCN2021076481-appb-000021
SEQ ID NO: 10
Figure PCTCN2021076481-appb-000022
SEQ ID NO: 11
Figure PCTCN2021076481-appb-000023
SEQ ID NO: 12
Figure PCTCN2021076481-appb-000024
SEQ ID NO: 13
Figure PCTCN2021076481-appb-000025
SEQ ID NO: 14
Figure PCTCN2021076481-appb-000026
Figure PCTCN2021076481-appb-000027
SEQ ID NO: 15
Figure PCTCN2021076481-appb-000028
SEQ ID NO: 16
Figure PCTCN2021076481-appb-000029
SEQ ID NO: 17
Figure PCTCN2021076481-appb-000030
SEQ ID NO: 18
Figure PCTCN2021076481-appb-000031
SEQ ID NO: 19
Figure PCTCN2021076481-appb-000032
SEQ ID NO: 20
Figure PCTCN2021076481-appb-000033
SEQ ID NO: 21
Figure PCTCN2021076481-appb-000034
SEQ ID NO: 22
Figure PCTCN2021076481-appb-000035
SEQ ID NO: 23
Figure PCTCN2021076481-appb-000036
SEQ ID NO: 24
Figure PCTCN2021076481-appb-000037
SEQ ID NO: 25
Figure PCTCN2021076481-appb-000038
SEQ ID NO: 26
Figure PCTCN2021076481-appb-000039
SEQ ID NO: 27
Figure PCTCN2021076481-appb-000040
SEQ ID NO: 28
Figure PCTCN2021076481-appb-000041
SEQ ID NO: 29
Figure PCTCN2021076481-appb-000042
SEQ ID NO: 30
Figure PCTCN2021076481-appb-000043
SEQ ID NO: 31
Figure PCTCN2021076481-appb-000044
SEQ ID NO: 32
Figure PCTCN2021076481-appb-000045
SEQ ID NO: 33
Figure PCTCN2021076481-appb-000046
SEQ ID NO: 34
Figure PCTCN2021076481-appb-000047
Figure PCTCN2021076481-appb-000048
SEQ ID NO: 35
Figure PCTCN2021076481-appb-000049
SEQ ID NO: 36
Figure PCTCN2021076481-appb-000050
SEQ ID NO: 37
Figure PCTCN2021076481-appb-000051
SEQ ID NO: 38
Figure PCTCN2021076481-appb-000052
SEQ ID NO: 39
Figure PCTCN2021076481-appb-000053
SEQ ID NO: 40
Figure PCTCN2021076481-appb-000054
SEQ ID NO: 41
Figure PCTCN2021076481-appb-000055
SEQ ID NO: 42
Figure PCTCN2021076481-appb-000056
SEQ ID NO: 43
Figure PCTCN2021076481-appb-000057
SEQ ID NO: 44
Figure PCTCN2021076481-appb-000058
SEQ ID NO: 45
Figure PCTCN2021076481-appb-000059
SEQ ID NO: 46
Figure PCTCN2021076481-appb-000060
SEQ ID NO: 47
Figure PCTCN2021076481-appb-000061
SEQ ID NO: 48
Figure PCTCN2021076481-appb-000062
SEQ ID NO: 49
Figure PCTCN2021076481-appb-000063
SEQ ID NO: 50
Figure PCTCN2021076481-appb-000064
SEQ ID NO: 51
Figure PCTCN2021076481-appb-000065
SEQ ID NO: 52
Figure PCTCN2021076481-appb-000066
SEQ ID NO: 53
Figure PCTCN2021076481-appb-000067
SEQ ID NO: 54
Figure PCTCN2021076481-appb-000068
SEQ ID NO: 55
Figure PCTCN2021076481-appb-000069

Claims (33)

  1. 一种CLDN18.2抗体，包含选自如下的重链CDRs和轻链CDRs，
    其中所述重链CDRs为：
    包含SEQ ID NO:18所示的CDR1、SEQ ID NO:19所示的CDR2、SEQ ID NO:20所示的CDR3；或
    包含SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、SEQ ID NO:23所示的CDR3，
    其中所述轻链CDRs为：
    包含SEQ ID NO:24所示的CDR1、SEQ ID NO:25所示的CDR2、SEQ ID NO:26所示的CDR3；或
    包含SEQ ID NO:27所示的CDR1、SEQ ID NO:28所示的CDR2、SEQ ID NO:29所示的CDR3。
  2. 如权利要求1所述的抗体，包含如SEQ ID NO:36或SEQ ID NO:37所示的重链可变区。
  3. 如权利要求1所述的抗体，包含如SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区。
  4. 如权利要求1-3任一项所述的抗体，包含如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:38所示的轻链可变区。
  5. 如权利要求1-3任一项所述的抗体，包含如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:39所示的轻链可变区。
  6. 如权利要求1-5任一项所述的抗体，其为嵌合抗体、人源化抗体或全人抗体。
  7. 如权利要求1-6任一项所述的抗体，其为全长抗体。
  8. 如权利要求7所述的抗体，其为IgG抗体。
  9. 如权利要求1-6任一项所述的抗体，其为结合CLDN18.2的抗体片段。
  10. 如权利要求9所述的抗体，其为Fab、Fab'-SH、Fv、scFv或(Fab') 2。
  11. 如权利要求1-8任一项所述的抗体，其为多特异性抗体或双特异性抗体。
  12. 如权利要求11所述的抗体，其中所述多特异性抗体或双特异性抗 体包含结合第二生物分子的结合域，其中所述第二生物分子为细胞表面抗原。
  13. 如权利要求12所述的抗体，其中所述细胞表面抗原为肿瘤抗原。
  14. 如权利要求13所述的抗体，其中所述肿瘤抗原选自：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1和TenB2。
  15. 一种免疫缀合物，包括与权利要求1-10任一项所述的抗体连接的治疗剂、干扰素基因刺激因子(STING)受体激动剂、细胞因子、放射性核素或酶。
  16. 如权利要求15所述的免疫缀合物，其中所述治疗剂为化疗药物。
  17. 如权利要求15所述的免疫缀合物，其中所述治疗剂为细胞毒性剂。
  18. 包含权利要求1-10任一项所述的抗体或抗原结合片段的融合蛋白或多肽。
  19. 包含权利要求1-14任一项所述的抗体、权利要求15-17任一项所述的免疫缀合物、权利要求18所述的融合蛋白或多肽的药物组合物。
  20. 一种分离的核酸，包含编码SEQ ID NOs：18-29之一的氨基酸序列的多核苷酸序列。
  21. 如权利要求20所述的核酸，包含编码SEQ ID NOs：36-39之一的氨基酸序列的多核苷酸序列。
  22. 如权利要求21所述的核酸，包含编码权利要求1-10任一项所述的抗体或抗原结合片段的多核苷酸序列。
  23. 一种载体，其包含权利要求20-22任一项所述的多核苷酸序列。
  24. 一种宿主细胞，其包含权利要求20-22任一项所述的多核苷酸序列或权利要求23所述的载体。
  25. 一种制备权利要求1-10任一项所述抗体的方法，包括培养权利要求24所述的宿主细胞并从培养物中分离获得所述抗体。
  26. 包含权利要求1-14任一项所述的抗体、权利要求15-17任一项所述的免疫缀合物、权利要求18所述的融合蛋白或多肽的药物组合物在制备用于诊断癌症的试剂中的用途。
  27. 包含权利要求1-14任一项所述的抗体、权利要求15-17任一项所述的免疫缀合物、权利要求18所述的融合蛋白或多肽的药物组合物在制备 用于治疗癌症的药物中的用途。
  28. 如权利要求26或27所述的用途，其中所述癌症为胃癌、胰腺癌或食管癌。
  29. 包含权利要求1-10任一项所述的抗体或其抗原结合片段的癌症检测试剂。
  30. 如权利要求29所述的癌症检测试剂，其中所述抗体或其抗原结合片段是经过化学标记的。
  31. 如权利要求30所述的癌症检测试剂，其中所述标记为酶标记、荧光标记、同位素标记或化学发光物标记。
  32. 包含权利要求29-31任一项所述的癌症检测试剂的癌症诊断试剂盒。
  33. 如权利要求32所述的试剂盒，所述癌症为胃癌、胰腺癌或食管癌。
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WO2022253284A1 (zh) 2021-06-02 2022-12-08 百奥泰生物制药股份有限公司 药物偶联物及其用途
WO2023196882A1 (en) * 2022-04-06 2023-10-12 Zai Lab (Us) Llc Claudin 18.2 immunohistochemistry assay and use thereof
WO2024211459A1 (en) * 2023-04-04 2024-10-10 Zai Lab (Shanghai) Co., Ltd Use of anti-claudin antibody for cancer treatment based on certain biomarkers
WO2025060679A1 (zh) * 2023-09-19 2025-03-27 上海交通大学医学院附属仁济医院 Cldn18.2单域抗体探针的制备方法及应用

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