CLDN18.2 antibody and use thereof

Abstract

To provide an antibody that specifically recognizes claudin 18.2 (CLDN18.2) antigen in tumor and its FFPE tissue.SOLUTION: A CLDN18.2 antibody includes heavy chain CDRs and light chain CDRs, where the heavy chain CDRs include CDR1, CDR2, and CDR3 with specific sequences, and the light chain CDRs include CDR1, CDR2, and CDR3 with specific sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the technical field of antibodies and to methods for the use and production of said antibodies. The present invention relates to CLDN18.2 antibodies and uses thereof. [Background technology]

[0002] Claudins are transmembrane proteins with molecular weights of 20-30 kDa. The family contains a total of 24 members, which are expressed in many tissues and mediate cell-cell interactions. An important molecule for forming tight junctions (TJ) structure Epithelial cells and endothelial cells express tight junction molecules (Claudin molecules) on the cell surface. The epithelial cells form tight junctions, which are highly ordered tissue junctions, through the intercellular membrane. TJs regulate the movement of molecules at the junctions of epithelial and endothelial cells under normal physiological conditions. They play an important role in maintaining cell integrity and cell polarity. is Claudin 18 (CLDN18), and CLDN18 supramolecular Is variant 1 (Claudin18.1, CLDN18.1) and CLDN18 splice There are two types of splice variants called Claudin18.2 (CLDN18.2) There are variants of

[0003] Among these, CLDN18.2 (Genbank accession number NM_001002026 , NP_001002026) is a transmembrane protein with a molecular weight of 27.8 kDa. Both the N-terminus and C-terminus of the protein are intracellular. It contains four transmembrane domains (TMDs) and two extracellular loops (ECLs). CLDN18.2 is highly conserved across multiple mammalian species and contains a total length of 261 amino acids. The amino acids 1 to 6 are the N-terminal intracellular domain, and the amino acids 7 to 27 are the transmembrane domain. The amino acids 28 to 78 of the extracellular loop 1 (ECL1) are The amino acids 79 to 99 are transmembrane domain 2 (TMD2), and the amino acids 123 to 144 are Transmembrane domain 3 (TMD3), with amino acids 100-122 connecting TMD2 and TMD3 The amino acids 145 to 167 of the extracellular loop 2 (ECL2) are The amino acids 168 to 190 are transmembrane domain 4 (TMD4), and the amino acids 191 to 261 are The amino acid is the C-terminal intracellular region. A sylation motif is present.

[0004] Compared with CLDN18.2, CLDN18.1 has a smaller N-terminal transmembrane domain 1 of the protein. -There is a difference in the first 26 amino acids of the N-terminus in the region called extracellular loop 1. The arrangement of the ribs is the same.

[0005] As evidence suggests, tumor cells in tissues such as the stomach, pancreas, and esophagus, especially adenocarcinoma subtypes, CLDN18.2 is highly expressed in type 2 tumor cells. CLDN18.1 is selectively expressed in epithelial cells of lung and stomach tissues, whereas CLDN18.2 is expressed in the longevity It is expressed only in the short-lived gastric glandular epithelial mucosa (differentiated glandular epithelial cells) and in gastric epithelial stem cells. These non-expressing differentiated glandular epithelial cells are constantly replenished by gastric epithelial stem cells. Molecular expression profile reveals potential for antibody-based therapy of CLDN18.2-associated cancers provide.

[0006] Companion diagnostics (CDx) Therefore, it is important to select tumor patients with high CLDN18.2 expression in clinical trials and clinical applications. It is important to guide patients on the appropriate use of antitumor drugs targeting LDN18.2. In clinical practice, tumor specimens from patients are usually used for diagnosis of tumor-specific antigens. Fixed paraffin-embedded (FFPE) in embedded tissue, the natural components of tumor-specific antigens are preserved during FFPE processing. The conformation of the protein changes, and during subsequent antigen retrieval, a special conformation different from the native conformation is formed. epitope exposure (Biotech Histochem, 2009, 84(5):207-215), which is usually Screening FPE tissue for antibodies that can specifically bind to cancer-specific antigens for diagnosis .

[0007] The present invention provides an antibody that specifically recognizes the CLDN18.2 antigen in tumors and their FFPE tissues. The present invention relates to methods for producing, screening, sequencing and using. Summary of the Invention

[0008] The present invention is directed to identifying CLDN18.2 antigen molecules in fixed tissue samples. CLDN18 for cancer diagnosis, which can diagnose cancer lesions including gastric cancer, pancreatic cancer, esophageal cancer, etc. The present invention also provides a CLDN18.2 antibody that can be used to treat gastric cancer, pancreatic cancer, or can effectively treat cancer lesions, including esophageal cancer, etc.

[0009] 1. A CLDN18.2 antibody comprising heavy chain CDRs and light chain CDRs, The heavy chain CDRs CDR1 as shown in SEQ ID NO:18, CDR2 as shown in SEQ ID NO:19 CDR2, CDR3 as shown in SEQ ID NO:20, or CDR1 as shown in SEQ ID NO:21, CDR2 as shown in SEQ ID NO:22 CDR2, CDR3 as shown in SEQ ID NO:23, The light chain CDRs CDR1 as shown in SEQ ID NO:24, CDR2 as shown in SEQ ID NO:25 CDR2, CDR3 as shown in SEQ ID NO:26, or CDR1 as shown in SEQ ID NO:27, CDR2 as shown in SEQ ID NO:28 CLDN18.2 antibody comprising CDR2, CDR3 as shown in SEQ ID NO:29. 2. The heavy chain variable region shown in SEQ ID NO:36 or SEQ ID NO:37 The antibody described in item 1, comprising a region. 3. The light chain variable region shown in SEQ ID NO:38 or SEQ ID NO:39 The antibody according to item 1, comprising a region. 4. A heavy chain variable region as shown in SEQ ID NO:36 and a heavy chain variable region as shown in SEQ ID NO:3 8. The antibody according to any one of items 1 to 3, comprising a light chain variable region as shown in 5. A heavy chain variable region as shown in SEQ ID NO:37 and a heavy chain variable region as shown in SEQ ID NO:3 9. The antibody according to any one of items 1 to 3, comprising a light chain variable region as shown in 6. The antibody according to any one of items 1 to 5, which is a chimeric antibody, a humanized antibody, or a fully human antibody. The antibodies listed are: 7. The antibody according to any one of items 1 to 6, which is a full-length antibody. 8. The antibody described in item 7, which is an IgG antibody. 9. The antibody fragment according to any one of items 1 to 6, which is an antibody fragment that binds to CLDN18.2. antibody. 10. The antibody according to item 9, which is Fab, Fab'-SH, Fv, scFv or (Fab')2. The antibody described. 11. The antibody according to any one of items 1 to 8, which is a multispecific antibody or a bispecific antibody. antibody. 12. The multispecific or bispecific antibody has a binding domain that binds to a second biological molecule. Item 12. The antibody of item 11, comprising the second biological molecule being a cell surface antigen. 13. The antibody described in item 12, wherein the cell surface antigen is a tumor antigen. 14. The tumor antigens are CD3, CD20, FcRH5, HER2, LYPD1, and LY 6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD From 79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2 The antibody of claim 13, selected from the group consisting of 15. A therapeutic agent linked to the antibody according to any one of items 1 to 10, an interferon Stimulator of Inflammatory Responses (STING) receptor agonists, cytokines, radionuclides or enzymes , an immune complex. 16. The immunoconjugate of claim 15, wherein the therapeutic agent is a chemotherapeutic agent. 17. The immunoconjugate according to item 15, wherein the therapeutic agent is a cytotoxic agent. . 18. A fusion protein comprising the antibody or antigen-binding fragment according to any one of items 1 to 10. Proteins or polypeptides. 19. The antibody according to any one of items 1 to 14, or the antibody according to any one of items 15 to 17. Item 19. A pharmaceutical composition comprising the immunoconjugate of item 18, or the fusion protein or polypeptide of item 18. 20. Encodes one of the amino acid sequences of SEQ ID NOs: 18 to 29 An isolated nucleic acid comprising a polynucleotide sequence. 21. Encodes one of the amino acid sequences of SEQ ID NOs: 36 to 39 21. The nucleic acid of claim 20, comprising a polynucleotide sequence. 22. A polypeptide encoding the antibody or antigen-binding fragment according to any one of items 1 to 10. 22. The nucleic acid according to claim 21, comprising a nucleotide sequence. 23. A vector comprising the polynucleotide sequence according to any one of items 20 to 22. 24. The polynucleotide sequence according to any one of items 20 to 22 or the polynucleotide sequence according to item 23. A host cell comprising the vector. 25. Culturing the host cell according to item 24 and isolating the antibody from the culture. A method for producing the antibody according to any one of Items 1 to 10. 26. The antibody according to any one of items 1 to 14, or the antibody according to any one of items 15 to 17. Item 19. A pharmaceutical composition comprising the immunoconjugate of item 18 or the fusion protein or polypeptide of item 18, for use in treating cancer. Use in the manufacture of reagents for diagnosing 27. The antibody according to any one of items 1 to 14, or the antibody according to any one of items 15 to 17. Item 19. A pharmaceutical composition comprising the immunoconjugate of item 18 or the fusion protein or polypeptide of item 18, for use in treating cancer. Use in the manufacture of therapeutic drugs. 28. The use according to item 26 or 27, wherein the cancer is gastric cancer, pancreatic cancer or esophageal cancer. 29. A cancer detection test comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 10. medicine. 30. The antibody or antigen-binding fragment thereof according to claim 29, wherein the antibody or antigen-binding fragment is chemically labeled. Cancer detection reagents listed above. 31. The label is an enzyme label, a fluorescent label, an isotope label, or a chemiluminescent label, 10. A cancer detection reagent according to claim 0. 32. A cancer diagnostic kit comprising the cancer detection reagent according to any one of items 29 to 31. 33. The kit according to item 32, wherein the cancer is gastric cancer, pancreatic cancer, or esophageal cancer. 34. The antibody according to any one of items 1 to 14, the fusion protein according to item 18, or A polypeptide or a detection reagent according to any one of items 29 to 31 is administered to a tissue sample derived from a subject. A method for diagnosing cancer in a subject, comprising contacting the subject with a protein. 35. The tissue samples include body fluids (e.g., blood, urine) and tissue sections (e.g., tissue Item 35. The diagnostic method according to Item 34, wherein the sample is a tissue biopsy sample. 36. The method according to claim 34 or 35, wherein the cancer is selected from gastric cancer, pancreatic cancer, and esophageal cancer. Cutting method. 37. A therapeutically effective amount of the antibody according to any one of items 1 to 14 or any one of items 15 to 17. Item 18. The immunoconjugate according to Item 1 or 2, the fusion protein or polypeptide according to Item 1 or 2. 10. A method for treating cancer in a subject, comprising administering to the subject the pharmaceutical composition according to 9. 38. The method of treatment according to item 37, wherein the cancer is selected from gastric cancer, pancreatic cancer and esophageal cancer. [Brief description of the drawings]

[0010] [Figure 1] Transient transfection of human hCLDN18.2-TCE (T-cell epitope peptide) retroviral expression plasmid into HEK293-T cells. [Diagram 2] Transfection of mice with hCLDN18.2-TCE lentiviral particles into mouse tumor cell lines and selection of mouse tumor cells expressing high levels of hCLDN18.2-TCE by flow cytometry. [Diagram 3] This is a flow cytometric assay of cell lines stably transfected with hCLDN18.2, hCLDN18.1, mCLDN18.2, and mCLDN18.1. [Figure 4] SDS-PAGE detection of positive control antibodies 43A11, 175D10 and 163E12. [Diagram 5] Binding of positive hybridoma clone supernatants to HEK293-hCLDN18.2 cells and HEK293-hCLDN18.1 cells in confirmation screening. [Figure 6] Flow cytometry analysis of supernatants of some positive hybridoma clones in validation screening in five cell lines. [Figure 7] This shows the ADCC effect of some of the positive hybridoma clone supernatants. [Figure 8] IHC staining on paraffin-embedded (FFPE) tissue sections of positive control antibody HEK293-hCLDN18.2 (A), HEK293-hCLDN18.1 (B), and HEK293-WT (C) cell blocks. [Figure 9] IHC staining of 5-I5 hybridoma clone supernatant and positive control antibody in paraffin-embedded (FFPE) tissue sections of gastric cancer tissue. [Figure 10] Flow cytometry analysis of hybridoma monoclonal supernatants into HEK293-hCLDN18.2 cells. [Figure 11] Reducing SDS-PAGE electrophoresis of recombinant purified 5-I10 and 5-G17 mouse monoclonal antibodies. [Figure 12] Flow cytometry analysis of recombinant purified 5-I10 and 5-G17 monoclonal antibodies in HEK293-hCLDN18.2. [Figure 13] Positive staining of recombinant purified mouse 5-I10 and 5-G17 monoclonal antibodies in gastric cancer FFPE tissue sections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1.Definition The term "antibody" as used herein is used broadly to refer to any antibody that binds to CLDN18.2. The present invention relates to a method for the production of a monoclonal antibody comprising administering to a subject the invention ... monoclonal antibody comprising administering to a subject the invention a Clonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and anti- antibody fragments (e.g. Fv, Fab, Fab', Fab'-SH, F(ab')2), linear antibodies and single chain antibody molecules (e.g., scFv), but are not limited to these. Any antibody that exhibits the desired binding activity to CLDN18.2 may be used.

[0012] Those skilled in the art will appreciate that one or more of the CDR domains disclosed in the present invention can be used to synthesize other polypeptides. A functional fusion protein that binds to the CLDN18.2 molecule and is fused to one or more of the above sequences. and polypeptide molecules, such as vaccines, cell membrane receptor antagonists, signal pathway regulators, and Chimeric antigen receptor molecules can be produced, for example, by using one or more of the antigen receptor molecules disclosed in the present invention. We developed CLDN18.2 CAR-T (Chimeric A) using multiple CDR domains. Antigen Receptor T-Cell Immunotherapy, Chimera Antigen receptor T cell immunotherapy molecules may be produced based on the sequences disclosed in the present invention. The derived and manufactured fusion protein molecules are also within the scope of the present invention.

[0013] The modifier "monoclonal" in the term "monoclonal antibody" as used herein means that the antibody is obtained from a substantially homogeneous antibody population and does not contain naturally occurring mutations Or, it refers to a monoclonal antibody that contains only a small amount of mutations that occur during the production of the antibody. Compared to representative polyclonal antibody preparations that contain different antibodies against different epitopes, Each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. The monoclonal antibodies of the present invention may be produced by a number of different techniques, including hybridization. Methods for the detection of human immunoglobulins, recombinant DNA methods, phage display methods, and human immunoglobulin genetics These methods include, but are not limited to, methods that utilize genetically modified animals that contain all or part of the gene locus. Not determined.

[0014] The terms "full length antibody" and "complete antibody" refer to a molecule having a structure substantially similar to that of a natural antibody. and these terms are used interchangeably herein.

[0015] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of the antibodies can be further classified as, for example, IgG1, IgG2, IgG3, IgG4, IgA may be classified into subclasses (isotypes), such as IgA1, IgA2, and IgA3. The heavy chain constant domains corresponding to the immunoglobulins of each group are α, δ, ε, γ, and μ, respectively. Called.

[0016] A "chimeric antibody" is an antibody that comprises at least a portion of a heavy chain variable region derived from one species and at least a portion of a An antibody that comprises a light chain variable region and at least a portion of a constant region from another species. For example, In one embodiment, a chimeric antibody may comprise a murine variable region and a human constant region.

[0017] A "humanized" antibody is one that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In some embodiments, a humanized antibody comprises at least one of: Typically, the two or nearly all variable domains are included, where all or nearly all of the HVRs (e.g., C DR) corresponds to a non-human antibody, and all or almost all of the FRs correspond to human antibodies. Optionally, a humanized antibody has at least one antibody derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) may include a portion of a human constant region. It is an antibody that has been modified.

[0018] A "human consensus framework" is a human immunoglobulin VL or VH framework sequence. The frameworks are selected from the following, representing the most commonly occurring amino acid residues: Generally, human immunoglobulin VL or VH sequences are derived from a subgroup of variable domain sequences. Generally, the sequence subgroups are selected according to the methods described in Kabat et al., Sequences of Proteins of Immunology. In: Biological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is a subgroup of In one embodiment, for VH, the subgroup is This group is subgroup III according to Kabat et al. (ibid.).

[0019] "Human antibodies" are also called "human antibodies," "fully humanized antibodies," or "fully human antibodies." The amino acid sequence of the antibody corresponds to an amino acid sequence derived from a human or human cell. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. may be produced by a variety of techniques known in the art, such as phage display. Play library technology, Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Mark S et al., J. Mol. Biol., 222: 581 (1991). Cole et al., Monoclonal Antibodies and Cancer Ther. apy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1): 86-95 (1991) Human antibodies can be modified to respond to antigen challenge. Genetically modified animals that produce such antibodies but have lost the ability to do so at their endogenous loci ( For example, it may be produced by administering the antigen to an immunized xenogeneic mouse (XENOMOU). For information on SETM technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584. (See also the issue of human antibodies produced by human B cell hybridoma technology. See Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006).

[0020] As used herein, the term "hypervariable region" or "HVR" refers to a region located in an antibody variable domain. and / or forming structured loops ("hypervariable loops") and / or antigen contact residues ("antigen contact residues"). Generally, an antibody contains six HVRs (CDR regions). Of these, three (H1, H2, and H3) are in the VH and three (L1, L2, and L3) are in the VH. In the VL, examples of HVRs (CDR regions) herein include: (a) amino acid residues 26-32 (L1), 50-52 (L2), and 91-96 (L3); Hypervariable loops appearing in 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3); HVRs appearing in 31-35b (H1), 50-65 (H2) and 95-102 (H3) CDR regions) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Publ ic Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) amino acid residues 27c-36 (L1), 46-55 (L2), and 89-96 (L3) , 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and (d) HVR (CDR region) amino acid residues 46 to 56 (L2), 47 to 56 (L2), 48~56(L2), 49~56(L2), 26~35(H1), 26~35b(H1) , 49-65 (H2), 93-102 (H3) and 94-102 (H3) (a); (b) and / or (c).

[0021] Unless otherwise specified, HVR (CDR region) residues and other residues in the variable region (e.g., FR Residues) are numbered herein according to Kabat et al., supra.

[0022] The term "variable region" or "variable domain" refers to the antibody heavy metal domains that are involved in binding to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies The main domains generally have a similar structure, with each domain containing four conserved framework regions (F R) and three complementarity determining regions (CDR regions) (see, e.g., Kindt et al., Kuby Immunology, (See 6th ed.). A single VH or VL domain is sufficient to confer antigen-binding specificity. do.

[0023] An "antibody fragment" is a fragment of an intact antibody that binds to an antigen to which the intact antibody binds, but is not an intact antibody. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and anti Examples of antibodies include, but are not limited to, multispecific antibodies formed with antibody fragments.

[0024] As used herein, the term "cytotoxic agent" refers to an agent that inhibits or prevents the function of cells, and Cytotoxic agents are substances that cause cell death or destruction. At211, I131, I125, Y90, Re186, Re188, Sm153, B radioactive isotopes of I212, P32, Pb212 and Lu; chemotherapeutic agents or drugs (e.g. Methotrexate, adriamycin, vinca alkaloids (vincristine, vinbra statin, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil siloxane, daunorubicin or other intercalating agents; growth inhibitors; enzymes and fragments thereof, e.g., nucleic acid molecules Antibiotics; Toxins, e.g. small molecule toxins of bacterial, fungal, plant or animal origin, or enzymatic activity toxins, such as fragments and / or variants thereof; any anti-tumor or anti-cancer drug known in the art; This includes, but is not limited to:

[0025] An "immunoconjugate" refers to a compound that contains an antibody and one or more heterologous molecules (including, but not limited to, a cytotoxic agent). It is a complex of the two.

[0026] Stimulator of interferon genes (STING) ERFERON genes receptor agonists inhibit STING-dependent signaling pathways Activation of the IL-1 receptor agonist, which is involved in the secretion of type I interferon and in antiviral and antitumor immunity It promotes the expression of proteins that block viral replication and stimulate an immune response to cancer cells. Such molecules include, for example, cyclic dinucleotides, aminobenzimidazoles, Dazoles, xanthones, acridones, benzothiophenes and benzodioxoles It is a STING agonist with a structural type such as

[0027] A "subject" or "individual" is a mammal. Mammals include domesticated animals (e.g., cattle, sheep, etc.). , cats, dogs and horses), primates (e.g. humans and non-human primates, e.g. monkeys), rabbits and rats. Rodents (e.g., mice and rats) are included, but are not limited to these. In some embodiments, the subject or individual is a human.

[0028] The term "protocol" refers to the instructions normally included in the commercial packaging of a therapeutic product. This instruction manual includes the indications, usage, dosage, administration, concomitant therapy, contraindications and / or or contains information regarding warnings regarding the use of such therapeutic products.

[0029] "Affinity" refers to the ability of a single binding site of a molecule (e.g., an antibody) to bind to its binding partner (e.g., an antigen Unless otherwise specified, as used herein, the term "non-covalent bond" refers to the total strength of non-covalent interactions between a molecule and a target molecule. When used herein, "binding affinity" refers to the intrinsic binding affinity and not to the binding affinity of the binding partner members (e.g. It reflects the 1:1 interaction between two molecules (e.g., an antibody and an antigen) of molecule X with its partner Y. Affinity may generally be expressed as a dissociation constant (Kd). Affinity may be determined by the methods described herein. Binding affinity may be measured by any method known in the art. And exemplary specific embodiments are described below.

[0030] The "percentage of amino acid sequence identity (%)" of a reference polypeptide sequence is the percentage of identity between a candidate sequence and a reference polypeptide sequence. The peptide sequences are aligned and gaps are introduced, if necessary, to find the maximum sequence homology. If the percent identity is achieved and any conservative substitutions are not considered part of the sequence homology, the candidate the percentage of amino acid residues that are identical between the sequence and the reference polypeptide sequence; Percent homology of amino acid sequences can be determined by several means known in the art. For example, the homology may be determined using methods such as BLAST, BLAST-2, ALIGN or The alignment was performed using software such as Megalign (DNASTAR). Those skilled in the art may determine appropriate parameters for alignment of sequences, It includes any algorithm required to achieve maximal alignment over the entire length of a sequence. do.

[0031] For example, when comparing amino acid sequences using ALIGN-2, a given amino acid sequence A and a given amino acid sequence B, or an amino acid sequence homology to a given amino acid sequence B The percent efficacy is calculated as follows: 100 x fraction X / Y In the formula, X is the sequence alignment when performing a program alignment for A and B. is the number of amino acid residues evaluated as matching by the program ALIGN-2, Y is the total number of amino acid residues in B. As can be understood, if the length of amino acid sequence A is If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent identity of amino acid sequences A and B is the percent identity of amino acid sequences B and A. Unless otherwise specified, all amino acid sequences used herein are not equivalent to percent homology. The percent homology value for amino acid sequences is obtained by the ALIGN-2 computer program.

[0032] 2. Antibodies, manufacturing methods, compositions and products 1) Antibody The present invention relates to anti-CLDN18.2 antibodies. In some embodiments, the present invention relates to anti-CLDN18.2 antibodies. The present invention provides an LDN18.2 antibody, which is an antibody that inhibits at least one or two of the following: Three, four, five or six hypervariable regions (HVRs) (also called complementarity determining regions (CDRs) (a) HVR-H1, comprising the sequence SEQ ID NO: or an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:21, or a sequence having at least 90 amino acids identical thereto %, 95%, 96%, 97%, 98%, and 99% homology to the amino acid sequence; b) HVR-H2, having sequence SEQ ID NO:19 or SEQ ID NO: 22 amino acid sequences or sequences with at least 90%, 95%, 96%, 97%, 98%, (c) HVR-H3, comprising an amino acid sequence having 99% homology thereto; The amino acid sequence of SEQ ID NO:20 or SEQ ID NO:23, or a sequence at least similar thereto The amino acid sequences with 90%, 95%, 96%, 97%, 98%, and 99% homology with each other were (d) HVR-L1, comprising sequence SEQ ID NO:24 or SEQ ID NO: or a sequence identical to said sequence with at least 90%, 95%, 96%, 97%, (e) HVR-L2, comprising an amino acid sequence having 98%, 99% homology thereto; The amino acid sequence of SEQ ID NO:25 or SEQ ID NO:28 or the sequence Amino acids having at least 90%, 95%, 96%, 97%, 98%, or 99% homology to and (f) HVR-L3 comprising the sequence SEQ ID NO:26 or The amino acid sequence of SEQ ID NO:29 or a sequence identical to said sequence at least 90%, 95%, 96%, or %, 97%, 98%, or 99% amino acid sequence identity. The heavy chain variable (VH) domain of the 18.2 antibody has the sequence SEQ ID NO: 36 or SEQ ID NO: 37. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or the sequence SEQ ID NO:36 or SEQ ID NO 37 and / or its light chain variable (VL) domain (region) comprises the amino acid sequence of At least 90% sequence identity with SEQ ID NO:38 or SEQ ID NO:39 Same-sex (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8% or 99% sequence identity) with the amino acid sequence SEQ ID NO:3 8 or the amino acid sequence of SEQ ID NO:39.

[0033] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region. and the heavy chain variable region comprises the amino acid sequence:

[0034] 2) Antibody fragment In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include: Fab, Fab', Fab'-SH, (Fab')2, Fv and scFv fragments, and Examples of antibody fragments include, but are not limited to, those listed above. Regarding scFv fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). See, for example, WO 93 / 16185.

[0035] Bifunctional antibodies are antibody fragments that have two antigen-binding sites and are bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161. For antibodies and tetrafunctional antibodies, see, e.g., Hudson et al., Nat. Med. 9: 129-134 (2003).

[0036] Single domain antibodies may comprise all or a portion of the heavy chain variable region of an antibody. In some embodiments, a single domain antibody is an antibody fragment that comprises a light chain variable region. It is a human single domain antibody (see, for example, US Pat. No. 6,248,516 B1).

[0037] Antibody fragments may be produced by a variety of techniques, including deproteinization and dehydrogenation of intact antibodies. and recombinant host cell (e.g., E. coli or phage) production. It is not something that can be achieved.

[0038] 3) Chimeric and humanized antibodies In some embodiments, the antibodies provided herein are chimeric antibodies. Chimeric antibodies The preparation of may be seen, for example, in US Pat. No. 4,816,567.

[0039] In some embodiments, the chimeric antibody is a humanized antibody. Typically, the non-human antibody is a human This reduces immunogenicity in humans and preserves the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, among which The whole or part of the HVR (CDR) region is derived from a non-human antibody, and the FR (or its A humanized antibody may optionally contain at least one human constant region. In some embodiments, the humanized antibody is modified with the corresponding residue from the non-human antibody. Substitution of some FR residues can restore or improve the affinity of the antibody. do.

[0040] Humanized antibodies and methods for producing them are described in U.S. Patents 5,821,337 and 7,527,791. See, for example, US Pat. Nos. 6,982,321 and 7,087,409.

[0041] 4) Human antibodies In some embodiments, the antibodies provided herein are human antibodies. It may be produced by a variety of techniques known in the art.

[0042] For human antibodies, the modified genetically altered animals are administered an immunogen and then challenged with an antigen. By using such an animal, a completely human antibody or a complete antibody having a human variable region can be produced. Typically, they replace endogenous immunoglobulin loci, are present extrachromosomally, or The method comprises the steps of: randomly integrating all or part of the human immunoglobulin locus into the animal's chromosome; In such genetically modified mice, the endogenous immunoglobulin loci are generally Regarding methods for obtaining human antibodies from genetically modified animals, see, for example, US Patent No. Patent Nos. 6,075,181 and 6,150,584 (including XENOMOUSETM technology) U.S. Patent No. 5,770,429; U.S. Patent No. 7,041,870 (KM Technology and U.S. Patent Application Publication No. US 2007 / 0061900. The human variable regions obtained from whole antibodies from animals such as The polypeptide may be combined with a human constant region.

[0043] Human antibodies may be produced by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines producing human antibodies have been reported. See, e.g., Boerner et al., J. Immunol., 147:86 (1991). Human antibodies produced by the mouse technique have also been reported by Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 ( Other methods are described, for example, in U.S. Pat. No. 7,189,826 (Hybrid The production of monoclonal human IgM antibodies from Ni,X cells has been described. iandai Mianyixue, 26(4): 265-268(2006) (human-human hybridomas are described) The method includes the method described in.

[0044] Human antibodies are generated by Fv clones selected from a phage display library of human origin. Alternatively, the antibody may be prepared by isolating the variable region sequences. It may be combined with any desired human constant domain.

[0045] Specifically, the high affinity antibody of the present invention has an antibody having CLDN18.2 binding activity. The antibodies may be isolated by screening combinatorial libraries against the antibodies. For example, generating a phage display library and screening for antibodies with the desired binding characteristics; Several methods for screening such libraries are known in the art. Such methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'B (Eds., Rien et al., Human Press, Totowa, NJ, 2001), Marks and Bradbury, Methods in Molecular Biology ology 248: 161-175 (ed. Lo, Human Press, Totowa, NJ, 2003) and Lee et al., J. Immunol. Met. hods 284(1-2):119-132 (2004).

[0046] Some phage display methods use the polymerase chain reaction (PCR) to VH and VL gene families were cloned independently and randomly recombined into a phage library. The phage are then screened for antigen-binding phages. The antibody fragments are displayed as single chain Fv (scFv) fragments or Fab fragments. Patents describing libraries include, for example, U.S. Pat. No. 5,750,373 and U.S. Pat. Opening No. 2005 / 0079574, No. 2005 / 0119455, No. 2005 / 026 No. 6000, No. 2007 / 0117126, No. 2007 / 0160598, No. 200 Nos. 7 / 0237764, 2007 / 0292936 and 2009 / 0002360 The number is included.

[0047] As used herein, an antibody or antibody fragment isolated from a human antibody library is referred to as a human antibody or human antibody fragment. It is considered an inter-specific antibody fragment.

[0048] 5) Multispecific antibodies In any of the above aspects, the anti-CLDN18.2 antibodies provided herein are multispecific. A multispecific antibody is an antibody that binds to at least two different sites, such as a bispecific antibody. In some embodiments, the antibody is a monoclonal antibody having binding specificity for The binding specificity of one is for CLDN18.2, and the binding specificity of the other is for any other antibody. to a second biological molecule, such as a cell surface antigen, such as a tumor antigen. The bispecific anti-CLDN18.2 antibody binds both CLDN18.2 and a tumor antigen, e.g., CD3, C D20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MR It may have binding specificity for P4, RET, Steap1 or TenB2. Heteroantibodies may be produced as full length antibodies or antibody fragments.

[0049] The technology for producing multispecific antibodies involves combining two immunoglobulin heavy-chain-light chain pairs with different specificities. These include, but are not limited to, recombinant co-expression of 9, WO2009 / 08025 and WO 2009 / 089004A1, etc. stomach.

[0050] 6) Antibody variants Antibodies of the invention include amino acid sequence variants of the anti-CLDN18.2 antibodies of the invention. For example, antibodies may be engineered to further improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody are derived from the nucleotide sequence encoding the antibody. These modifications may be made by introducing appropriate modifications into the nucleotide sequence. For example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. and substitutions are combined in any combination to obtain the final construct, with the condition that the final construct The body has desirable characteristics, such as binding properties with the CLDN18.2 antigen.

[0051] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Substitutions are made at one or more sites in the HVR (CDR) and / or FR regions to produce substituted type Mutants (including conservative substitution mutants or non-conservative substitution mutants) may be obtained. Amino acids may be grouped according to common side chain properties. (1) Hydrophobic: n-Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0052] Conservative substitutions are defined as substitutions between amino acids of the same family, whereas non-conservative substitutions are substitutions between amino acids of different classes. A cyclic amino acid is defined as a substitution of one amino acid from one group for an amino acid from another group. Amino acid substitutions can be introduced into the antibodies of the invention to retain / improve a desired activity (e.g., antigen binding or By screening the products based on the improvement of DCC or CDC, Antibody variants of the above can be obtained.

[0053] The present invention relates to non-conservative mutations and / or conservative mutations derived from the antibodies disclosed in the present invention. The antibody variants include those containing natural mutations, which variants have the desired CLDN18.2 binding activity. It is sufficient that the product has the above characteristics.

[0054] One type of substitutional variant is one or more of the amino acids of a parent antibody (e.g., a humanized or human antibody). The present invention relates to antibody variants in which hypervariable region residues of The variants selected for this purpose have some biological property (e.g., improved affinity) relative to the parent antibody. , modified (e.g., improved), and / or some biological property of the parent antibody is substantially Exemplary substitutional variants are affinity matured antibodies, e.g., phage dissociated antibodies. can be readily produced by prey-based affinity maturation techniques (such as those described herein) Briefly, one or more HVR (CDR) residues are mutated, and the mutated Antibodies are displayed on phage and identified based on a particular biological activity (e.g., binding affinity). The mutated antibodies are screened.

[0055] In some embodiments, substitutions, insertions or deletions are present in one or more HVRs (CDRs). Such alterations may result in substantial loss of the ability of the antibody to bind CLDN18.2. For example, the binding affinity in the HVR (CDR) may be substantially reduced. Conservative changes that do not alter the antigenic bond of the HVR may be made. For example, 1, 2, 3, 4, or 5 amino acid residues in the FR region are conserved or Non-conservative amino acid substitutions may occur.

[0056] 7) Recombination method The anti-CLDN18.2 antibodies of the present invention can be used, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, the anti-C antigens described herein may be produced by recombinant methods. An isolated nucleic acid encoding an LDN18.2 antibody is provided. Such a nucleic acid may comprise: The VL amino acid sequence and / or the VH amino acid sequence of the antibody may be encoded. Also provided are one or more vectors (e.g., expression vectors) that contain such nucleic acids. In another embodiment, a host cell is provided that contains such nucleic acid. In such embodiments, the host cell comprises (1) an amino acid sequence comprising the VL of the antibody and (2) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; A second vector containing the nucleic acid encoding the sequence is included (eg, carried by transformation). In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO ) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). A method for producing an anti-CLDN18.2 antibody is provided, the method comprising: Cultivating the host cell containing the nucleic acid encoding the antibody under the conditions described above, and optionally and recovering the antibody from the host cell (or host cell medium) by incubation at 4° C. for 24 hours.

[0057] To recombinantly produce an anti-CLDN18.2 antibody, a nucleic acid encoding the antibody (e.g., (as described above) and inserted into one or more vectors for further expression in a host cell. Such nucleic acids can be cloned and / or expressed using conventional procedures (e.g., the heavy and The method uses oligonucleotide probes capable of specifically binding to genes encoding the light and light chains. ) and subjected to sequencing.

[0058] Host cells suitable for cloning or expressing the antibody-encoding vectors include those described herein. For example, specific glycosylation and Fc effector functions are not required. In the absence of a specific antibody fragment or polypeptide, the antibody is produced in the bacteria. See, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,5 After expression, the antibody in the soluble portion is isolated from the bacterial cytoplasm and then subjected to It may be further purified.

[0059] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, also can be adapted for use with antibody-encoding vectors. The glycosylation pathway is "humanized" and partially or fungal and yeast strains which produce antibodies with fully human glycosylation patterns. See Li et al., Nat. Biotech. 24: 210-215 (2006).

[0060] Suitable host cells for the expression of glycosylated antibodies include multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Combined, especially with Spodoptera frugiperda A number of baculovirus strains capable of transfecting cells have been identified.

[0061] Plant cell cultures are also available as hosts. See, e.g., U.S. Patent No. 5,959,177; Nos. 6,040,498, 6,420,548, 7,125,978 and 6,417, Issue 429 (Describes PLANTIBODIESTM technology for producing antibodies in genetically modified plants) Refer to the following:

[0062] Vertebrate cells are also available as hosts. For example, mammalian cells adapted for growth in suspension. Animal cell lines are applicable. Other examples of suitable mammalian host cell lines include SV40 transformed monkey kidney CV1 cell line (COS-7); human fetal kidney cell lines (e.g. 293 cells); Baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells); monkey kidney CV1; African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); Human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060 562); TRI cells; MRC 5 cells; Chinese hamster ovary (CHO) cells, For example, DHFR-CHO cells; and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. Some examples include:

[0063] 8) Immune complex The present invention also relates to a method for the preparation of an anti-CLDN18.2 antibody according to the present invention, comprising administering to said patient a therapeutically effective amount of said antibody or antibody combination .... The present invention provides an immunoconjugate having a cytotoxic agent linked thereto, such as a chemotherapeutic agent. or a chemotherapeutic agent, a growth inhibitor, a toxin (e.g. a protein toxin, bacterial, fungal, plant or animal The toxin may be an enzymatically active toxin derived from a steroid or a fragment thereof, or a radioactive isotope.

[0064] In one embodiment, the immunoconjugate is an antibody-drug conjugate in which an antibody is conjugated to one or more drugs. Antibody complexes (ADCs), including maytansine, orlistat, dolastatin, methotrexate, Cert, vindesine, taxane, trichothecene and CC Including, but not limited to, 1065.

[0065] In another embodiment, the immunoconjugate comprises an anti-CLDN18.2 antibody described herein and an enzymatically active and a complex with an enzymatically active toxin or a fragment thereof, the enzymatically active toxin being diphtheria A chain, diphtheria Non-binding active fragments of toxins, including but not limited to exotoxin A chains and trichothecenes. It's not something like that.

[0066] In another embodiment, the immunoconjugate comprises an anti-CLDN18.2 antibody described herein and a radioactive The radioisotopes include radioactive complexes in which the atom is bound to the radioisotope. It is useful. For example, 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu radioisotopes are included. Can be enjoyed.

[0067] Conjugates of antibodies and cytotoxic agents are prepared using multiple bifunctional protein binding agents. The binding agent may be, for example, N-succinimidyl-3-(2-pyridyldithio ) propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl) cyclohexyl Monohexane-1-carboxylate (SMCC), iminothiolane (IT), imide Bifunctional derivatives of esters (e.g., dimethyl adipate hydrochloride), active esters (e.g., disulfide Cinimidyl suberate), aldehydes (e.g. glutaraldehyde), diazide compounds (e.g. bis(p-azidobenzoyl)hexamethylenediamine), bisdiazo derivatives (e.g. bis(p-diazobenzoyl)-ethylenediamine), diisothiocyanates (e.g. Toluene 2,6-diisothiocyanate) and doubly active fluorine compounds (e.g. 1,5-difluoro The compound is fluoro-2,4-dinitrobenzene.

[0068] 9) Drug Preparations The pharmaceutical formulation of the anti-CLDN18.2 antibody of the present invention comprises an antibody having a desired purity and one or more Mix with multiple pharma- ceutically acceptable carriers to form a freeze-dried preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally suitable for use in the dosages and concentrations used. buffers such as phosphates, citrates and other organic acids; Antioxidants such as scorbic acid and methionine; preservatives (octadecyl dimethyl phenyl acetate) Benzalkonium chloride;Biothrium;Benzalkonium chloride;Benzalkonium chloride phenol, butanol or benzyl alcohol; methyl p-hydroxybenzoate, or or alkyl p-hydroxybenzoates such as propyl p-hydroxybenzoate; Catechol; resorcinol; cyclohexanol; 3-pentanol; and metacrezo low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, immunoglobulins Proteins such as ribonucleic acid; hydrophilic polymers such as polyvinylpyrrolidone; glycine, Amino acids such as thiamine, asparagine, histidine, arginine or lysine; glucose Monosaccharides, disaccharides and other carbohydrates, including mannose and dextrins; EDTA Chelating agents such as sucrose, mannitol, fucose, and sorbitol; sugars such as sodium salt-forming counterions such as amines; metal complexes (e.g., zinc-protein complexes) and / or polymers; Non-ionic surfactants, including but not limited to ethylene glycol (PEG) It is not something that can be achieved.

[0069] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006 / 044908. Including.

[0070] The formulations herein contain one or more active ingredients essential for the particular indication being treated. Preferably, active ingredients have complementary activities that do not adversely affect each other. For example, additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or In some cases, the active ingredients may need to be administered in combination with other drugs (anti-hormonal agents). and are suitable to be present in an amount effective for the intended purpose.

[0071] 10) Products In another aspect of the invention, there is provided an article of manufacture containing an antibody or pharmaceutical composition of the invention. The product shall consist of a container and any label or package insert (protocol) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Such containers may be formed from a variety of materials, such as glass or plastic. The container contains the composition of the present invention alone or in combination with another composition, and may include a sterile inlet (e.g., the container may be puncturable by an IV bag or hypodermic needle). (The vial may be provided with a stopper that can be used for the purpose.) At least one activator of the composition is The level or package insert indicates that the composition is used to treat a particular tumor. The article of manufacture further comprises: (a) a first container containing a composition comprising an antibody of the invention; (b) a second container containing another tumor therapeutic agent or a composition comprising another antibody. The product of this embodiment of the invention may further comprise a package insert indicating the tumors that such composition may treat. Optionally, the product may also contain a pharma- ceutically acceptable buffer, e.g. Antibacterial Water for Injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution The device may further include a second (or third) container for containing the composition. The composition may further include other materials that facilitate the synthesis of the composition. Such materials may include other buffers, diluents, filters, etc. , needles and syringes.

[0072] 11) Detection reagents and kits The present invention also relates to a method for detecting cancer comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention. The company provides reagents for cancer detection. The cancer detection reagents are used to test tumor or blood tissue from actual patients. The test may be performed, for example, by PCR, microarray or chip technology. Upon detection of the presence of cancer detection agents, these agents are selected to guide or direct treatment of the patient. It is used for guidance.

[0073] In one embodiment, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is chemically labeled. Specifically, enzymes such as alkaline phosphatase and glucose oxidase Labels: fluorescent labels such as fluorescein and rhodamine; iodine (125I, 131I), charcoal C (14C), S (35S), Tritium (3H), Indium (121In) and Thermo isotope labels such as 99mTc; or chemicals such as luminol or luciferase It may also be a luminescent label.

[0074] The present invention also relates to a method for detecting cancer comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention. The kit includes a labeled anti-CLDN18.2 antibody or an antigen-binding Specifically, the cancer detection kit is useful for detecting gastric cancer, pancreatic cancer, or esophageal cancer. It is useful.

[0075] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings. Although specific examples of the present invention are shown, the present invention is not limited to these examples. It will be understood that the present invention may be embodied in various forms without departing from the spirit and scope of the present invention. The following examples are provided to enable a more complete understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. is provided to. Working Example

[0076] Example 1 Preparation of immunization and screening materials (1) Human hCLDN18.2-TCE (T-cell epitope peptide) retroviral Construction of virus expression plasmid The hCLDN18.2 cDNA (SEQ ID NO:1) was cloned and expressed as C- A T-cell epitope peptide was fused to the end to obtain hCLDN18.2-TCE. The antigen overcomes immune tolerance by using cellular epitope peptides, and induces the production of antibodies (Percival-Alwyn J. et al, mAbs, 2015, 7(1), 129-137). The retrovirus vector was cloned to generate the hCLDN18.2-TCE retrovirus. The hCLDN18.2-TCE retroviral expression plasmid was obtained. HEK293-T cells were transiently transfected and incubated for 72 hours after transfection. Afterwards, the expression of hCLDN18.2 on the surface of HEK293-T cells was examined by flow cytometry. The primary antibody (1st Ab) was a self-made positive control (Benchmark). ker) chimeric antibody 163E12. First, cells were incubated with the primary antibody (1 μg / mL). The plates were incubated at 4°C for 45 min, washed with PBS, and then incubated with Alexa Flu or 488-labeled goat anti-human Fc secondary antibody (Thermofisher, part number A-110 13)) (2nd Ab, diluted 1:200) and incubated at 4°C for 45 minutes. After washing twice with PBS, flow cytometry (FACS) analysis was performed. The control cells were incubated with the secondary antibody alone. No hCLDN18.2 expression was detected on the surface of 293-T cells. HEK293 transiently transfected with -TCE retroviral expression plasmid -T cells (HEK293T CLDN18.2 TCE) showed 80.9% positive cells. The vector plasmid expressed hCLDN18.2. Used in epidemic schemes. (2) Mouse tumor cells expressing high levels of hCLDN18.2-TCE hCLDN18.2-TCE retroviral expression plasmid and lentiviral package The resulting mixture was mixed with the genomic plasmid and transfected into 293-T cells to express hCLDN1. hCLDN18.2-TCE lentivirus particles were produced. The particles were transfected into mouse tumor cell lines and analyzed by flow cytometry 72 hours later. We performed gene detection to select a mouse tumor cell pool that highly expressed hCLDN18.2-TCE. The primary antibody (1st Ab) was a self-made positive control (Benchmarker). First, the cells were incubated with the primary antibody (1 μg / mL) at 4°C. After incubation at 4 °C for 45 min and washing with PBS, Alexa Fluor 488 With labeled goat anti-human Fc secondary antibody (2nd Ab, 1:200 dilution) at 4°C After washing twice with PBS, the cells were analyzed by flow cytometry (FA As shown in Figure 2, negative control cells were incubated with the secondary antibody alone. The results showed that 16.8% of wild mouse tumor cells (UBER parental) The only positive signal was from hCLDN18.2-TCE retrovirus. In transfected mouse tumor cells (UBER CLDN18.2 TCE), the % of the cells were positive. The results of flow cytometry analysis showed that the positive cells were 95.7%. hCLDN18.2-TCE was expressed at high levels in many mouse tumor cells. The mouse tumor cells of hCLDN18.2-TCE were used in accordance with the cell immunization scheme in Example 2(2). Used. (3) Production of hCLDN18.2 extracellular loop 1 virus-like particles The method published by Thorsten K. (Thorsten K., et al, Cancer Res, 2011, 71(2), 515-52 According to the above, the extracellular loop 1 (ECL1) of hCLDN18.2 was ular Loop1) (SEQ ID NO:2) is a hepatitis B virus core antigen (HBV cAg: Hepatitis B virus core antigen) is the main immunological Inserted into the major immunodominant region (MIR) Then, G4SG4 linkers were introduced into the N-terminus and C-terminus of ECL1, respectively, to obtain a full-length fusion protein. A 6xHis tag (SEQ ID NO:3) was added to the C-terminus of the fusion protein. The full-length gene of the fusion protein was synthesized and cloned into the pET24a(+) plasmid. Then, the fusion protein was expressed by transfecting it into the BL21(DE3) E. coli expression system. After purification of the fusion protein, the HBV hCLDN18.2 cells were incubated with renaturation buffer. The hCLDN18.2 extracellular loop 1 virus-like particles were produced. The preparation of the virus-like particles was carried out according to the method described in Example 2 (3) Virus-like particle multi-site repeated immunization-cell immunization scheme. and (4) virus-like particle hock immuno-cellular immunization schemes. (4) Construction of CLDN18 stable transfected cell line hCLDN18.2(SEQ ID NO:4), hCLDN18.1(SEQ ID NO:4) NO:5), mouse mhCLDN18.2 (SEQ ID NO:6), mCLDN1 The full-length gene of 8.1 (SEQ ID NO: 7) was synthesized and inserted into the pcDNA3.4 vector. The plasmids were then transformed into HEK293 cells. Then, neomycin (G418) was added and pressure screening was performed. hCLDN18.2, hCLDN18.1, mhCLDN18.2, and mCLDN18.1 were identified by field dilution. HEK293 stably transfected cell lines expressing CLDN18.1 were As a result of flow cytometry detection, hCLDN18.2 and mCLDN18 HEK293 stably transfected cell line (HEK In HEK293-hCLDN18.2 and HEK293-mCLDN18.2, paraformaldehyde Both aldehyde-fixed and non-fixed cells were treated with a self-made anti-CLDN18.2 positive control antibody. The antibodies were specifically identified by the antibodies 163E12 or 175D10 (Fig. 3A, C). Broad-spectrum anti-CLDN18 antibody 34H14L15 (Abcam part number ab203563) hCLDN18.1 and mCLDN18.1 can be distinguished by the following (Figure 3A, C). Transfected HEK293 stable transfected cell line (HEK293 -hCLDN18.1, HEK293-mCLDN18.1) were treated with paraformaldehyde. Both fixed and non-fixed cells were stained with a self-made anti-CLDN18.2 positive control antibody 16. It could not be specifically identified by 3E12 or 175D10 (Fig. 3B, D), whereas the commercially available It can be identified by the broad spectrum anti-CLDN18 antibody (34H14L15) (Figure 3B, D). The above HEK293 stably transfected cell line was hybridized with the hybridomask of Example 3. Used for leaning. (5) Preparation of positive control antibodies 43A11, 175D10, and 163E12 Heavy chain (SEQ ID NO:8) and light chain of human-mouse chimeric positive control antibody 43A11 (SEQ ID NO:9), the heavy chain of 175D10 (SEQ ID NO:10) and the light chain of The heavy chain of 163E12 (SEQ ID NO:11), and the heavy chain of 163E12 (SEQ ID NO:1 2) and light chain (SEQ ID NO: 13) antibody genes were synthesized in their entirety, A signal peptide was added to each N-terminus, and then the resulting product was inserted into the pcDNA3.4 eukaryotic expression vector. Heavy and light chain expression of 43A11, 175D10 and 163E12 The plasmids were mixed and co-transfected into CHOS cells, and transient transfection was performed. The protein was expressed by transfection, the supernatant was collected, and the resultant was purified using Protein A. Affinity purification was performed and SDS-PAGE detection was performed, and the results are shown in FIG.

[0077] Example 2 Mouse immunization program and hybridoma preparation A total of four immunization schemes (Table 1) were employed, and at least Immunization was performed using two different strains of mice (Table 2). In the same scheme, high levels of serum immune titers were observed in some mice. Mice that showed high levels of immune titers in the immunological scheme were killed and their spleens were removed. After isolating and mixing B lymphocytes, they were electro-fused with a mouse myeloma cell line to produce hybridomas. A total of three batches of hybridomas were produced (Table 2).

[0078] [Table 1-1]

[0079] [Table 1-2]

[0080] [Table 2]

[0081] Example 3 Hybridoma screening Hybridomas from three batches were cloned by limiting dilution, and the clone supernatants were The antibodies were screened in triplicate for binding or reverse binding by flow cytometry, and the antibodies were typed and Antibody dependent cell cytotoxicity (ADCC) -mediated cytoxity was measured and found to bind strongly and specifically to CLDN18.2. It can bind to CLDN18.1, but does not or only slightly binds to CLDN18.1, and has ADCC function. 128 hybridoma clones were screened for the screening step and The hybridoma clones obtained by screening at each stage are summarized in Table 3. .

[0082] [Table 3]

[0083] 1) Primary screening: High-level expression products of each clone and CLDN18.2-TCE were The binding of UBER CLDN18.2 TCE expressed in a mouse tumor cell line was examined using The hybridomas were analyzed by flow cytometry. After inoculation into an 84-well plate and culturing for 10 days, the supernatant was taken and positively analyzed by flow cytometry. The hybridoma clones were screened for UBER CLDN18.2. Co-incubate with TCE cells (20,000 cells / well) for 45 min at 4°C. After that, the plate was washed and Alexa Fluor-488-goat anti-mouse IgG ( ThermoFisher product number A28175) was used as a secondary antibody for flow cytometry. Detection was performed, and clones with a mean fluorescence intensity of 100,000 or more were screened and amplified. The saturated supernatant was collected and subjected to confirmation screening. Three batches of hybridomas were isolated from each batch by limiting dilution to obtain 10 384-cell clones. The hybridoma was cloned into well plates. Three batches of hybridoma were obtained by primary screening. The hybridoma was positive for binding of CLDN18.2-TCE to mouse tumor cell lines. Macron was divided into 341 (MFI>100,000) and 336 (MFI>7 5,000 or MFI>80,000) and 89 (MFI>90,000). 2) Confirmatory screening: Positive clones from the previous step, the primary screening The supernatant expression products of HEK293-hCLDN18.2 cells and HEK293-h Incubated with CLDN18.1 cells and HEK293 wild-type cells (20,000 (cells) / well, 4°C, 45 min), Alexa Fluor-488-goat anti-mouse IgG (ThermoFisher product number A28175) was used as the secondary antibody for HEK29 3-Positive binding to hCLDN18.2 cells but not HEK293-hCLDN18. Clones that were negative for binding to HEK293 cells and wild-type HEK293 cells were analyzed by flow cytometry. The plasmid p53 was screened and amplified by, and the saturated supernatant was collected and subjected to a validation screen. After confirmation screening, the three batches of hybridomas were confirmed to be HEK293-hCL It can be strongly and specifically bound to HEK293-hCLDN18.2 cells, but not to HEK293-hCLDN18.1 cells. We selected 16 clones that bind weakly to the vesicles and do not bind to wild-type HEK293 cells. 5 (HEK293-hCLDN18.2 MFI>200,000, HEK293- hCLDN18.1 MFI<100,000), 120 (HEK293-hCLD N18.2 MFI>50,000, HEK293-hCLDN18.1 MFI<40, 000), and 21 (HEK293-hCLDN18.2 MFI>70,000, HEK293-hCLDN18.1 (MFI < 30,000) was obtained. FACS analysis of the cells is shown in FIG. 3) Verification screening: Confirmation screening of hybridomas derived from three batches The supernatants of 306 (165 + 120 + 21) positive hybridomas were expressed. The samples were divided into HEK293-hCLDN18.2 cells and HEK293-hCLDN18 .1 cells, HEK293-mCLDN18.2 cells, HEK293-mCLDN18.1 The cells were incubated with HEK293 wild-type cells (20,000 cells / well). 45 min at 4°C), Alexa Fluor-488-goat anti-mouse IgG (The The secondary antibody was used for flow cytometry analysis. Flow cytometry was performed on five cell lines of some of the clones shown in Figure 6. The analysis results showed that the positive clones were isolated from HEK293-hCLDN18.2 cells and HEK29 3-mCLDN18.2 cells showed a mean fluorescence intensity (MFI) of 500,000 or more. On the other hand, HEK293-hCLDN18.1 cells and HEK293-mCLDN18. 1 cells and the HEK293 wild-type cell line all had mean fluorescence intensities below 50,000. Most of the clones (16 / 22) were expressed in HEK293-hCLDN18.2 cells and In HEK293-mCLDN18.2 cells, the mean fluorescence intensity (MFI) was Mean fluorescence for hCLDN18.1 and HEK293-mCLDN18.1 cells It was shown that the intensity (MFI) was more than 50 times higher. 4) Antibody typing: IgG molecules have different subtypes that mediate ADCC effects. Therefore, prior to the detection of antibody-dependent cell killing, a mouse antibody subtype detection kit was used. 306 hybridomas obtained by confirmation screening from three batches were For the supernatant antibody expression products of the (165 + 120 + 21) positive hybridomas, Mouse antibody subtype typing was performed. Most of the subtypes of 306 clones were These are mouse IgG2a, IgG2b, or IgG3 with ADCC function, and only a few The amount (26 / 306) was IgG1 subtype (human equivalent antibody) that does not have ADCC function. Some clones are of mixed subtypes, which is why hybridization is important. This is probably because the plasmid clones are not monoclonal. All light chains are mouse. It is the κ subtype. 5) Measurement of ADCC effect The results of the above confirmation screening, validation screening and antibody typing will be taken into consideration comprehensively. Considering this, the saturated supernatants of 128 positive clones were selected for functional testing of antibody-dependent cellular cytotoxicity. Effector cells were collected from two donors (No. 45 and No. 46) on the day before the test. The collected blood was stored at room temperature and then centrifuged using Ficoll density gradient. Freshly isolated human peripheral blood mononuclear cells (PBMCs). The target cells were CLDN The cells were HEK293 cells expressing hCLDN18.2 (HEK293-hCLDN18.2). All hybridoma expression products were diluted 4-fold. Freshly obtained PBMCs and target The cells were co-incubated with each sample at an effector / target cell ratio of 25:1 for 4 h. By measuring lactate dehydrogenase (LDH), which is related to cytotoxicity, This represents the ADCC effect of the antibody. The absorbance detected when the target cells spontaneously lyse. The absorbance value detected when all the target cells were lysed was defined as 0% and the absorbance value detected when all the target cells were lysed was defined as 100%. The ADCC effect of each test sample is expressed as a percentage of activity. As shown in Figure 7, the ADCC killing effect of the clone supernatant was 20% or higher on average. The ADCC effect of some clones (9D-1, 8-O11) was higher than 50%. On the other hand, the ADCC effect was not observed for 6-G11 and 4-O2, which are mouse IgG1 subtypes. It fell below 10%.

[0084] Example 4 hCLDN18.2 Immunohistochemistry Positive Staining Hybridomas in PPFE Tissue Sections Screening for lydoma In the companion diagnostics, the patient's tumor tissue is biopsied and then paraffin-embedded (FFP) E), tissue sections, and pathology detection are common processes. or associated antigen) is a antigen that is used when performing FFPE tissue sections. The natural structure is lost, and even after antigen retrieval, some The conformational structure of the peptide can be restored to some extent, or some linear peptide segments can be The supernatants of the 128 preferred positive hybridoma clones mentioned above were , the ability to specifically bind to the native CLDN18.2 conformation on the cell surface and AD Although it has CC function, the companion diagnostic antibody does not show antigen specificity on FFPE tissue sections. IHC screening for heterologous binding must be performed. Immunohistochemistry (IHC) of FE tissue sections The 128 positive hybridoma supernatants were isolated from three genetically engineered cell lines ( HEK293-hCLDN18.2 cells, HEK293-hCLDN18.1 cells, and HEK293 wild-type cell block paraffin embedding (F Binding to FPE) tissue sections was detected. First, we stably expressed CLDN18 in HEK293 cell blocks and FFPE tissue sections. IHC staining was performed on HEK293-hCLDN18.2 cells, HE Culture, cell collection, and centrifugation of K293-hCLDN18.1 cells and HEK293 wild-type cells After heart isolation, cell block formation, dehydration, clearing, and paraffin embedding, FFPE tissue sections were prepared. and IHC staining. HEK293-hCLDN18.2 cell block F The FPE tissue sections showed positive staining, but the HEK293-hCLDN18.1 cells and HE Hybridoma clones staining negatively in K293 wild-type cell block FFPE tissue sections The resulting positive hybridoma supernatants were then screened for human gastric cancer tissue. IHC staining validation was performed on FFPE tissue sections. Finally, the validated IHC positive staining hybrids The lysoma supernatants were then analyzed using normal human stomach and gastric cancer FFPE tissue microarrays (TMA: Tiss IHC staining was performed using a genomic microarray, and the staining intensity and staining ratio were evaluated. The final IHC positive staining clones were determined by the above procedure. Cell block FFPE tissue sections, gastric cancer FFPE tissue sections, and normal gastric and gastric cancer FFPE tissue sections. The IHC method for hybridoma clones on PE tissue microarrays (TMA) is as follows: The steps include: 1) Deparaffinization and baking: 60°C for 30 minutes. 2) Deparaffinization 3) Hydration: Ethylene, immersion in three stages (I, II, III), 5 minutes per stage. 2 gradient immersions in 100%, 1 gradient immersion in 95%, 1 gradient immersion in 75%, 1 gradient immersion in distilled water for 3 min each. 5 minutes. 4) Antigen retrieval: Add 100 ml of antigen retrieval solution (Tris-EDTA, pH 9.0) to the sample. ℃ water bath, place the FFPE tissue sections in antigen retrieval solution and incubate for 30 minutes. Cool at room temperature for 30 minutes and wash three times with PBS buffer for 5 minutes each. 5) Blocking: 3 % H2O2-PBS, incubated at room temperature in the dark for 10 minutes, and washed three times with PBS. Then, the plate was incubated with 5% BSA (PBS-BSA) at room temperature for 60 minutes. Wash three times with BS buffer. 6) Incubation with primary antibody: rabbit anti-CLDN18 1.2 Commercially available positive antibody (EPR19202, Abcam, product number ab222512) 1:1 00 or mouse hybridoma supernatants are incubated overnight at 4°C. ) Incubation with secondary antibody: HRP-conjugated goat anti-rabbit (Abc am, product no. ab6721, diluted 1:1000) and HRP-conjugated goat anti- Mouse secondary antibody (ThermoFisher, part number 62-6520, diluted 1:2000 8) Color development: Develop with diaminobenzidine (DAB) for 3 minutes. 9) Hematoxylin Counterstain for 1 min with 0.5% HCl-75% ethanol for a few seconds, then rinse for 20 min. 10) Dehydration and clearing: 70% ethanol, 80% ethanol, Dehydrate the sections using 95% ethanol, absolute ethanol I, and absolute ethanol II for 2 minutes each. Clarify using xylene I and xylene II for 5 minutes each. 11) Mounting: Mount with a neutral mounting medium. Enclose.

[0085] Figure 8 shows the HEK293 cell block FFPE of the positive control commercial antibody EPR19202. IHC staining of tissue sections is shown. The antibody was used to detect HEK293-hCLDN18.2. Specific staining in cell block FFPE tissue sections of stably expressed cell lines and Positive staining was evident at the membrane (see arrow in Figure 8.A), indicating that CLDN18. This is consistent with the conclusion that 2 is a membrane protein (Figure 8A). 3-HCLDN18.1 stably expressing cell lines in cell block FFPE tissue sections In the case of the wild type HEK293 cell line, no obvious color development was observed (Figure 8.B). This was not observed (Figure 8C). Supernatants from 120 hybridoma clones were collected from three cell lines (HEK293-hCLDN 18.2 cells, HEK293-hCLDN18.1 cells, and HEK293 wild-type cells) IHC staining of FFPE tissue sections from cell blocks identified four hybridoma clones (5-I5, 5-I10, 5-G17 and 10-D3) were obtained. The supernatants of these clones were In both cases, FFPE tissue sections of HEK293-hCLDN18.2 cell blocks showed Positive IHC staining was observed, whereas HEK293-hCLDN18.1 and HEK293 -WT cell block FFPE tissue sections showed no or weak staining. HEK293-hCLDN18.2 cell block screening on FFPE tissue sections The supernatants of the four IHC-positive hybridoma clones were subjected to immunofluorescence assay using FFP derived from human gastric cancer tissue. E. IHC color verification was performed on tissue sections, and among these, 5-I5 hybridoma clone The supernatant was analyzed using IHC staining in FFPE tissue sections from three cases of human gastric cancer, as shown in Figure 9 (A, B). As shown in Fig. 3C, positive staining of the antibody was observed only at the location of glandular epithelial cells (indicated by arrows). In the interstitial cells, there was no obvious positive color reaction or only weak staining (indicated by the triangular arrow). 5-I5 was used to identify the positive staining positions and positive staining structures in FFPE tissue sections of three cases of gastric cancer. The tissue structure was similar to the IHC staining results of the positive control antibody (EPR19202) (Figures 9D, E, and F). Matches.

[0086] To accurately identify patients who highly express tumor-associated antigens as companion diagnostic antibodies This has important clinical significance for treatment. This leads to discrepancies in IHC color development results between different diagnostic antibodies against the same tumor-associated antigen. This is often the case. A mixture of 4 IHC positive candidate hybridoma expressing supernatants and 4 supernatants (Cocktail) was prepared using normal human stomach and gastric cancer FFPE tissue microarrays ( IHC color development was performed using a TMA chip (Biomax, ST807). In the study, incubation of a single hybridoma clone supernatant and a positive control antibody was performed. The incubation time for the mixture of four hybridoma supernatants was 30 minutes, and the incubation time for the mixture of four hybridoma supernatants was 120 minutes. Table 4 shows the tissue sites where the cell membrane was positively stained and the tissue sites where the cytoplasm was positively stained. , tissue types and corresponding positive staining clones are indicated. 5-I10, 5-G17, 5-I5, 10-D3 and the mixture of the four clone supernatants were , and the cell membrane positive staining rates were 28.9%, 17.1%, 14.5%, 11.8%, and The cell membrane positivity rate for the positive control antibody (EPR19202) was 23.7%. In addition, the clone supernatant and positive antibody stained almost all normal stomach tissues positively (5 -I10 87.5%, 5-G17 87.5%, 5-I5 100%, 10-D3 8 7.5%, Mix. Of 4 100%, EPR19202 100%), As reported in the literature, CLDN18.2 is specifically expressed in normal gastric mucosal epithelial cells. This is consistent with the findings shown in Sahin, U. et al, Human Cancer Biology, 2008, 14(23), 7624-7634. Compared to the high positive staining rate in normal gastric tissue, each hybridoma supernatant showed only a small positive staining rate in gastric cancer tissue. There was a large difference in color, and in gastric cancer tissue, the cell membrane positive staining rate was 5-I10 23.5 %, 5-G17 10%, 5-I5 4.4%, 10-D3 2.9%, and 4 high The mixture of brinoma supernatants (Mix. Of 4) was 22%, which is a highly heterogeneous mixture of gastric cancer tissues. Among these, the 5-I10 hybridoma clone supernatant The positive staining rate was the highest in gastric cancer tissue cell membranes, reaching 23.5%, and the positive control antibody EPR This is much higher than the 14.7% of 19202, and Zhu G. et al. (Zhu G. et al, Sci Rep., This is roughly consistent with the positivity rate of 16-23% reported by the National Institute of Infectious Diseases (NIID) and the National Institutes of Health (NIH). However, 5-I10 and 5-G17 showed high positive staining rates in gastric cancer FFPE tissues, and The positive rate of both combinations (5-I10 + 5-G17) reached a maximum of 26%, and positive staining was observed in gastric cancer. The FFPE tissue section contains positive sites that appear approximately at 5-I5 and 10-D3. Recombinantly expressed and purified 5-I10 and 5-G17 antibodies were mixed in a given ratio to form an antibody mixture. By using it as a companion diagnostic, we can identify gastric cancer patients with high CLDN18.2 expression as much as possible. This could guide the clinical dosing of drugs targeting CLDN18.2. has important significance for

[0087] [Table 4-1]

[0088] [Table 4-2]

[0089] Example 5. FFPE tissue sections: hCLDN18.2 positive staining of hybridoma clones Sequencing of the clones and antibody molecule sequences In this example, four hybridomas were identified that stained positively with IHC on FFPE tissue sections. The loans (5-I10, 5-G17, 5-I5 and 10-D3) were analyzed by limiting dilution method. The subclones and the variable region sequences of the antibody molecules were sequenced. The hybridoma clones were then subjected to limiting dilution and plated at a density of 0.8 cells / well in 192 wells. The cells were inoculated onto well plates, and monoclones were selected by microscopic examination. After 14 days of culture, The supernatant was collected and analyzed by flow cytometry to subclone the HEK293 cells. -Monoclonal clones were amplified and cultured while detecting specific binding to hCLDN18.2 cells. The flow cytometric assay was performed to compare the expression of the monoclonal supernatant and HEK293-CLDN18. After co-incubation with 20,000 cells / well at 4°C for 45 minutes, The plates were washed and incubated with Alexa Fluor-488 goat anti-mouse IgG (Therm oFisher product number A28175) was used as the secondary antibody for flow cytometry detection. For each hybridoma, 10 (5-I5, 9) monoclones were selected and expanded. The FFPE tissue sections were then subjected to IHC validation by extraction, cryopreservation, and collection of the supernatant. The four hybridomas were cloned into HEK293-hCLDN18.2 cells shown in 0. Flow cytometry analysis of the clone supernatants demonstrated that the monoclonal supernatants were HEK293-hCLD The monoclonal supernatants maintained specific binding to N18.2 cells and were also stained with HEK293- The binding strength to hCLDN18.2 cells was measured using the corresponding hybridoma supernatant (Parenta The binding strength of each hybridoma clone was shown to be equivalent to that of the IgG1111A ... The first five monoclonal antibodies with the highest binding strength to HEK293-hCLDN18.2 cells were Select the clone (shown in blue box in Figure 9) supernatant for IHC validation on FFPE tissue sections. As a result, positive IHC staining was observed in all the monoclonal supernatants. For the study, we selected two monoclones (5-I) that showed the highest IHC intensity in FFPE tissue sections. 5-31 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) were selected to obtain the heavy and light chains. Gene clone sequencing of the variant region was performed. Using the RACE (rapid amplification of cDNA ends) method, hybridoma monoclones were isolated. The DNA sequences of VH and VL were amplified. RNA was extracted from the 5'-terminal region of the cDNA, reverse transcribed, and the 5'-universal fragment was isolated by 5'-RACE reaction. A combination of a monkey primer and a degenerate primer for the 3'-H, L(κ) or L(λ) FR1 region Using the combination of the heavy and light chain V regions, PCR was performed and the PCR products were cloned using the TOPO clone method. The CR amplified fragment was ligated into a sequencing vector and sequenced. As seen, 5-I5-31 and 5-I5-25 have the same VH but different VL sequences. This indicates that 5-I5-31 and 5-I5-25 are different monoclones. In addition to 5-I5, 10-D3, 5-G17, and 5-I10 were The two monoclones sequenced had identical VH and VL. The DNA sequences of VH and VL of 5-I10 and 5-G17 are shown in Table 5. The amino acid sequences are shown in Table 6, where the CDR regions of the light and heavy chains are numbered according to the Chothia numbering system. It is divided according to:

[0090] [Table 5]

[0091] [Table 6]

[0092] Example 6 Expression of Recombinant Mouse Monoclonal Antibodies The heavy and light chain V regions of 5-I10 and 5-G17 were compared with mouse IgG2a heavy chain V regions, respectively. The constant region of the IGHC2A chain (Table 7, Mouse IGHC2A, SEQ ID NO:30) and Mouse kappa light chain constant region (Table 7, Mouse IGKC, SEQ ID NO:31) and a signal peptide was added to the N-terminus of each of the heavy and light chain sequences. The full-length mouse heavy chain genes of I10 and 5-G17 (SEQ ID NO: 32, 33) The full-length light chain gene (SEQ ID NO: 34, 35) was synthesized and cloned into an expression vector. The candidate molecules were screened and verified by sequencing (Kin Weizhi Biotechnology Co., Ltd., Suzhou). The expression plasmid constructed in the above was amplified and extracted, and the results were analyzed by agarose gel electrophoresis. The resulting plasmid was then verified by electrophoresis and used as a transfection material. HEK293 cells were seeded in shake flasks and cultured in a chemically defined serum-free medium. Suspension seed culture was performed. The day before transfection, amplified HEK293 cells were A new shake flask was inoculated and the medium was changed to the new medium. The heavy and light chain expression plasmids were co-transfected into HEK293 cells. The cells were cultured in shake flasks until cell activity decreased, and the supernatant was collected for product purification. Preferably, the supernatant is centrifuged and filtered to remove most of the cell debris and insoluble microorganisms. After removing the particles, the sample liquid is injected into a Protein A column, and the antibody molecules are adsorbed onto the packing material. After the equilibration step, most of the impurities are removed. The antibody protein was eluted with a low pH elution solution and fractions were collected. The solution was replaced with the formulation buffer through filtration. Finally, the OD280 was measured to calculate the protein concentration. As shown in FIG. 11, the reduced SDS of the 5-I10 and 5-G17 monoclonal antibodies The band positions of the heavy and light chains were the same as those of the positive control IgG. The positions of the ribozyme and light chains were shown to match, the molecular weights were as expected, and there were no obvious impurity bands. There was no evidence of any cleavage and the protein purity was greater than 95%.

[0093] [Table 7]

[0094] Example 7 Recombinant Mouse Monoclonal Antibody Binding to HEK293-hCLDN18.2 Activity Flow cytometry revealed that 5-I10 and 5-G17 purified monoclonal antibodies Binding to HEK293-hCLDN18.2 was verified, as was binding to HEK293-hCLDN18. 2. Place in a 96-well U-bottom plate (1.5 x 10 5 100 cells / well) and containing 1% FBS The plate was washed once with PBS buffer and the 5-I10 and 5-G17 purified monoclonal antibodies were added. Each antibody was diluted in PBS (1% FBS) and diluted three-fold starting from 10 μg / mL. Dilute the solution into a 96-well U-bottom plate at 100 μL / well to obtain a total of eight concentrations. The plate was then incubated at 4°C for 1 hour. The plate was washed once with PBS (1% FBS) and diluted 1:1 000 diluted Alexa488-conjugated goat anti-mouse secondary antibody (eBioscience, A 11001) was added at 100 μL / well and incubated at 4° C. for 1 hour. After washing twice with 1% FBS, the cells were resuspended in 200 μL PBS (1% FBS) and then subjected to FACS. FIG. 12 shows the HE staining of purified monoclonal antibodies 5-I10 and 5-G17. The binding curves of K293-hCLDN18.2 were shown, and both were dose-dependently expressed in HEK293 - It was able to specifically bind to hCLDN18.2 and had the same binding ability.

[0095] Example 8 Positive staining of recombinant mouse monoclonal antibodies in gastric cancer FFPE tissue sections C-reactivity of purified monoclonal antibodies 5-I10 and 5-G17 in FFPE tissue sections from six cases of gastric cancer LDN18.2 IHC positive staining was verified, and EPR19202 was used as a positive control. The IHC staining steps were the same as those described in Example 4, except that the primary antibody was incubated In the injection step, the working concentration of the positive control antibody EPR19202 was 1:100 dilution, 5 The working concentrations of purified antibodies 5-I10 and 5-G17 were 5 μg / mL. As a result, the recombinant purified mouse IgG2a type 5-I10 and 5-G17 monoclonal antibodies In several cases of gastric cancer FFPE tissue sections, positive staining was observed. The positive staining rates of 0 reached 83.3% (5 / 6) and 66.7% (4 / 6), respectively. This was significantly higher than the 50% (3 / 6) achieved by the positive control antibody EPR19202. In 37358-2A, S08-19346 and S10-36009-3, gastric cancer cells The clear gastric glandular epithelial structure was formed, and 5-I10, 5-G17 and the positive control antibody EPR192 In both cases, strong specific staining of glandular epithelial cells was observed, while staining of interstitial cells was not observed. In the S11-4725-1 tissue, gastric cancer cells formed glandular structures, but as previously described The positive control SPR19202 showed no clear IHC staining, which was clearly different from the glandular structure in the control. On the other hand, 5-I10 and 5-G17 showed diffuse strong positive staining, resembling glandular epithelial cells. Staining was observed in both cytoplasmic and interstitial cells. The tumor cells in the 7939-C section did not form obvious glandular structures. In 9-15785-2FS, the positive controls SPR19202, 5-I10 and 5-G17 None of them showed positive IHC staining. In S07-7939-C, only 5-G17 showed positive IHC staining. HC showed weak positive staining.

Claims

1. A CLDN18.2 antibody comprising heavy chain CDRs and light chain CDRs, The heavy chain CDRs CDR1 as shown in SEQ ID NO:18, CDR2 as shown in SEQ ID NO:19 CDR2, CDR3 as set forth in SEQ ID NO:20, or CDR1 as shown in SEQ ID NO:21, CDR2 as shown in SEQ ID NO:22 CDR2, CDR3 as shown in SEQ ID NO: 23, The light chain CDRs include CDR1 as shown in SEQ ID NO:24, CDR2 as shown in SEQ ID NO:25 CDR2, CDR3 as set forth in SEQ ID NO: 26, or CDR1 as shown in SEQ ID NO:27, CDR2 as shown in SEQ ID NO:28 CLDN18.2 antibody comprising CDR2, CDR3 as shown in SEQ ID NO: 29 。

2. A heavy chain variable region shown in SEQ ID NO: 36 or SEQ ID NO: 37 is included. The antibody of claim 1.

3. A light chain variable region shown in SEQ ID NO: 38 or SEQ ID NO: 39 is included. The antibody of claim 1.

4. The heavy chain variable region shown in SEQ ID NO: 36 and the The antibody according to any one of claims 1 to 3, comprising a light chain variable region which is

5. The heavy chain variable region shown in SEQ ID NO: 37 and the The antibody according to any one of claims 1 to 3, comprising a light chain variable region which is

6. The antibody according to any one of claims 1 to 5, which is a chimeric antibody, a humanized antibody or a fully human antibody. antibodies.

7. The antibody according to any one of claims 1 to 6, which is a full-length antibody.

8. The antibody of claim 7 which is an IgG antibody.

9. The antibody according to any one of claims 1 to 6, which is an antibody fragment that binds to CLDN18.

2. body.

10. Fab, Fab'-SH, Fv, scFv or (Fab') ２ 10. The method of claim 9, antibodies.

11. The antibody according to any one of claims 1 to 8, which is a multispecific or bispecific antibody. 。

12. The multispecific or bispecific antibody comprises a binding domain that binds to a second biological molecule. The antibody of claim 11 , wherein the second biological molecule is a cell surface antigen.

13. The antibody of claim 12, wherein the cell surface antigen is a tumor antigen.

14. The tumor antigen is CD3, CD20, FcRH5, HER2, LYPD1, LY6G6 D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B , EDAR, GFRA1, MRP4, RET, Steap1, and TenB2. The antibody of claim 13.

15. A therapeutic agent linked to the antibody according to any one of claims 1 to 10, an interferon gene The present invention relates to a method for treating inflammatory bowel disease, comprising administering to the patient a therapeutically effective amount of a substance that is capable of stimulating the immune system (STING) receptor agonist, cytokine, radionuclide or enzyme. Hmm, immune complexes.

16. The immunoconjugate of claim 15 , wherein the therapeutic agent is a chemotherapeutic agent.

17. The immunoconjugate of claim 15 , wherein the therapeutic agent is a cytotoxic agent.

18. A fusion protein or a fusion protein comprising the antibody or antigen-binding fragment according to any one of claims 1 to 10. is a polypeptide.

19. The antibody according to any one of claims 1 to 14, the antibody according to any one of claims 15 to 17 20. A pharmaceutical composition comprising an immunoconjugate of, or a fusion protein or polypeptide of claim 18.

20. SEQ ID NOs: A polynucleotide encoding one of the amino acid sequences 18 to 29. An isolated nucleic acid comprising a nucleic acid sequence.

21. A polynucleotide encoding one of the amino acid sequences of SEQ ID NOs: 36 to 39.

21. The nucleic acid of claim 20, comprising a nucleic acid sequence.

22. A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1 to 10.

22. The nucleic acid of claim 21, comprising an octide sequence.

23. A vector comprising the polynucleotide sequence according to any one of claims 20 to 22.

24. The polynucleotide sequence according to any one of claims 20 to 22 or claim 23 A host cell comprising the vector.

25. 25. A method for producing an antibody comprising culturing the host cell of claim 24 and isolating said antibody from the culture. A method for producing the antibody according to any one of claims 1 to 10.

26. The antibody according to any one of claims 1 to 14, the antibody according to any one of claims 15 to 17 19. An immunoconjugate of claim 18, or a pharmaceutical composition comprising the fusion protein or polypeptide of claim 18. , for use in the manufacture of a reagent for diagnosing cancer.

27. The antibody according to any one of claims 1 to 14, the antibody according to any one of claims 15 to 17 19. An immunoconjugate of claim 18, or a pharmaceutical composition comprising the fusion protein or polypeptide of claim 18. , for use in the manufacture of cancer therapeutics.

28. 28. The use according to claim 26 or 27, wherein the cancer is gastric cancer, pancreatic cancer or esophageal cancer.

29. A cancer detection reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

30. 30. The antibody or antigen-binding fragment thereof of claim 29, wherein the antibody or antigen-binding fragment thereof is chemically labeled. Cancer detection reagents.

31. 31. The method of claim 30, wherein the label is an enzyme label, a fluorescent label, an isotopic label, or a chemiluminescent label. The cancer detection reagent described herein.

32. A cancer diagnostic kit comprising the cancer detection reagent according to any one of claims 29 to 31.

33. 33. The kit of claim 32, wherein the cancer is gastric cancer, pancreatic cancer, or esophageal cancer.

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