Anti-CLDN18.2 monoclonal antibody and its use

Abstract

The present invention provides an anti-CLDN18.2 monoclonal antibody and its preparation. Specifically, the present invention provides a novel anti-CLDN18.2 humanized antibody. The antibody of the present invention binds to antigen with high specificity, has high affinity and high biological activity, specifically binds to human CLDN18.2 antigen molecule, and can effectively inhibit tumors both in vitro and in vivo.

Description

[Technical Field]

[0001] This invention relates to the field of pharmaceuticals, and more specifically to an anti-CLDN18.2 humanized monoclonal antibody and its formulation. [Background technology]

[0002] Tight junctions (TJs) are multimolecular complexes that regulate permeability between epithelial tissue cells. In tight junction complexes, claudins form tight channels between adjacent cells, regulating the flow of substances such as ions and solutes.

[0003] CLDN18 is a major component of tight junctions in the epithelial cells of the lung and stomach. CLDN18 has two subtypes, CLDN18.1 and CLDN18.2, with CLDN18.1 being primarily expressed in the lungs and CLDN18.2 primarily expressed in the stomach.

[0004] Studies have shown that CLDN18.2 is highly expressed in many tumors, including primary and metastatic gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, bile duct cancer, and ovarian cancer (Sahin U et al., Clin Cancer Res, 2008;14(23):7624-34). Due to characteristics such as rapid proliferation and a tendency to invasively metastasize, tumor cells lose their tight junction structure, making the CLDN18.2 molecule on their surface easily exposed, thus making CLDN18.2 an ideal target for tumor therapy (Hewitt KJ et al., BMC Cancer, 2006; 6:186).

[0005] IMAB362, a chimeric antibody against CLDN18.2 developed by Ganymed Pharmaceuticals AG, is already in Phase III clinical trials. In Phase II clinical trials for terminal or recurrent gastroesophageal cancer, IMAB362 significantly extended patients' PFS and OS more effectively as a monotherapy than in combination with the first-line chemotherapy drug EOX (epirubicin, oxaliplatin, capecitabine), making it a potential drug for the treatment of gastric cancer, esophageal cancer, and other diseases (Lordick F et al., Gastric Cancer, 2021; 24(3): 721-730).

[0006] Due to the potential of CLDN18.2 in tumor therapy, there is a significant clinical demand for the development of highly active antibody drugs.

[0007] The high degree of similarity between human CLDN18.1 and CLDN18.2 presents a challenge in developing CLDN18.2-specific antibodies. Furthermore, mouse CLDN18.2 has a sequence that is a perfect match to the extracellular loop 1 of human CLDN18.2, meaning that screening for specific anti-human CLDN18.2 antibodies in mice must overcome the mouse immune resistance. Therefore, developing highly active drugs that specifically recognize human CLDN18.2 antibodies requires overcoming many technical challenges.

[0008] Therefore, in this field, the development of CLDN18.2 antibodies with high affinity and high biological activity, and their applications, is still eagerly awaited. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a CLDN18.2 antibody with high affinity and high biological activity, and its use. [Means for solving the problem]

[0010] In a first aspect of the present invention, the present invention provides a heavy chain variable region of an antibody, comprising the following three complementarity-determining regions (CDRs): (1) Complementary determining region CDR1 having an amino acid sequence shown by SEQ ID NO: 3; (2) Complementary determining region CDR2 having an amino acid sequence shown by SEQ ID NO: 4 or 16; and (3) Complementary determining region CDR3 having an amino acid sequence shown by SEQ ID NO: 5 or 17.

[0011] In another preferred example, the sequences of the three complementary determining regions CDRs of the heavy chain variable region are SEQ ID NOs: 3, 4 and 5, or 3, 16 and 5, or 3, 4 and 17.

[0012] In another preferred example, the heavy chain variable region contains the following four framework regions FR, where the four framework regions FR are derived from the four framework regions FR corresponding to SEQ ID NOs: 1, 11, 12 and 13. In another preferred example, the heavy chain variable region has an amino acid sequence shown by SEQ ID NO: 1, 11, 12 or 13.

[0013] In the second aspect of the present invention, a heavy chain of an antibody having the heavy chain variable region of the antibody described in the first aspect of the present invention is provided. In another preferred example, the constant region of the heavy chain is of human origin. In another preferred example, the constant region of the heavy chain is a heavy chain constant region such as human IgG1, IgG2.

[0014] In the third aspect of the present invention, a light chain variable region of an antibody is provided, which includes the following three complementary determining regions CDR': (1) Complementary determining region CDR1' having an amino acid sequence shown by SEQ ID NO: 8; (2) Complementary determining region CDR2' having an amino acid sequence' shown by SEQ ID NO: 9; and (3) Complementary determining region CDR3' having an amino acid sequence' shown by SEQ ID NO: 10.

[0015] In another preferred example, the light chain variable region includes the following four framework regions FR, where the four framework regions FR are derived from the four framework regions FR corresponding to SEQ ID NOs: 6, 14, and 15. In another preferred example, the light chain variable region has an amino acid sequence represented by SEQ ID NO: 6, 14, or 15.

[0016] In the fourth aspect of the present invention, an antibody light chain having the light chain variable region of the antibody described in the third aspect of the present invention is provided. In another preferred example, the constant region of the light chain is of human origin. In another preferred example, the constant region of the light chain is a light chain constant region such as human κ or λ.

[0017] In the fifth aspect of the present invention, (1) the heavy chain variable region described in the first aspect of the present invention, and / or (2) the light chain variable region described in the third aspect of the present invention An antibody having the same or an antibody having the heavy chain described in the second aspect of the present invention and / or the light chain described in the fourth aspect of the present invention is provided.

[0018] In another preferred example, the antibody further has a heavy chain constant region and a light chain constant region. In another preferred example, the antibody has a heavy chain variable region represented by SEQ ID NO: 1, 11, 12, or 13, and / or a light chain variable region represented by SEQ ID NO: 6, 14, and 15. In another preferred example, the antibody has a heavy chain variable region represented by SEQ ID NO: 1, 11, 12, or 13, and / or a light chain variable region represented by SEQ ID NO: 15.

[0019] In another preferred example, the antibody is selected from the following group: (Z1) An antibody having a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 6; (Z2) An antibody having a heavy chain variable region as indicated by SEQ ID NO: 11 and a light chain variable region as indicated by SEQ ID NO: 14; (Z3) An antibody having a heavy chain variable region as indicated by SEQ ID NO: 11 and a light chain variable region as indicated by SEQ ID NO: 15; (Z4) An antibody having a heavy chain variable region as indicated by SEQ ID NO: 12 and a light chain variable region as indicated by SEQ ID NO: 14; (Z5) An antibody having a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 15; (Z6) An antibody having a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 14; (Z7) An antibody having a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 15.

[0020] In another preferred example, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NOs: 18, 20, 21, or 22. In another preferred example, the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 19. In another preferred example, the antibody is a humanized antibody.

[0021] In another preferred example, the antibody specifically binds to CLDN18.2. In another preferred example, the antibody is a double-chain antibody or a single-chain antibody. In another preferred example, the antibody is a monoclonal antibody.

[0022] In another preferred example, the antibody is a bispecific antibody. In another preferred example, the antibody is in the form of a drug conjugate. Furthermore, the present invention provides an antibody corresponding to 624, having a heavy chain variable region indicated by SEQ ID NO: 23 and a light chain variable region indicated by SEQ ID NO: 24.

[0023] In a sixth aspect of the present invention, a recombinant protein, (i) a heavy chain variable region described in the first aspect of the present invention, a heavy chain described in the second aspect of the present invention, a light chain variable region described in the third aspect of the present invention, a light chain described in the fourth aspect of the present invention, or an antibody described in the fifth aspect of the present invention, and (ii) Tag sequences to aid in expression and / or purification of any type It provides proteins.

[0024] In another preferred example, the aforementioned tag array includes 6His tags. In another preferred example, the recombinant protein (or polypeptide) includes a fusion protein. In another preferred example, the recombinant protein is a monomer, a dimer, or a polymer.

[0025] A seventh aspect of the present invention is an antibody preparation, (a) The antibody described in the fifth aspect of the present invention, and (b) A vehicle containing buffering agent, sterile water, and optionally a surfactant. The present invention provides a formulation containing the above.

[0026] In another preferred example, the concentration of the antibody in the formulation is 5-100 mg / mL, preferably 10-70 mg / mL, and more preferably 20-60 mg / mL. In another preferred example, the buffer is selected from the group consisting of a citrate buffer system, a histidine buffer system, or a combination thereof.

[0027] In another preferred example, the pH range of the formulation is 5.0-7.5, preferably 5.5-7. In another preferred example, the preparation is an injectable preparation.

[0028] In the eighth aspect of the present invention, a kit is provided which includes the antibody preparation described in the seventh aspect of the present invention, and a container containing the antibody preparation.

[0029] In a ninth aspect of the present invention, the present invention provides a CAR construct wherein the scFv fragment of the antigen-binding region of the CAR construct is a binding region that specifically binds to CLDN18.2, and the scFv has a heavy chain variable region as described in the first aspect of the present invention and a light chain variable region as described in the third aspect of the present invention.

[0030] In the tenth aspect of the present invention, recombinant immune cells are provided which express an exogenous CAR construct described in the ninth aspect of the present invention. In another preferred example, the immune cells are selected from a group consisting of NK cells and T cells. In another preferred example, the immune cells are derived from humans or non-human mammals (e.g., mice).

[0031] In an eleventh aspect of the present invention, an antibody-drug conjugate, (a) an antibody portion selected from the group consisting of the heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, the light chain described in the fourth aspect of the present invention, or the antibody described in the fifth aspect of the present invention, or a combination thereof, (b) A complex portion which is combined with the antibody portion, selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof. A complex containing the above is provided. In another preferred example, the antibody portion and the composite portion are compounded via a chemical bond or a linker.

[0032] In the twelfth aspect of the present invention, the use of an active ingredient, wherein the active ingredient is selected from the group consisting of the heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, the light chain described in the fourth aspect of the present invention, or the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, the immune cell described in the tenth aspect of the present invention, the antibody-drug conjugate described in the eleventh aspect of the present invention, or a combination thereof, and the active ingredient is (a) Manufacturing of detection reagents or kits, (b) manufacture of drugs or formulations that prevent and / or treat CLDN18.2 related diseases, and / or (c) manufacture of drugs or formulations that prevent and / or treat cancer or tumors It provides a use that is intended for that purpose.

[0033] In another preferred example, the tumor is selected from the group consisting of hematological malignancies, solid tumors, or combinations thereof. In another preferred example, the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, or a combination thereof.

[0034] In another preferred example, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer with peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), gliocytoma, endometrial cancer, or the like. In another preferred example, the tumor is a tumor that highly expresses CLDN18.2.

[0035] In another preferred example, the drug or formulation is for the manufacture of a drug or formulation for the prevention and / or treatment of a disease associated with CLDN18.2 (expression positive). In another preferred example, the antibody is in the form of a drug conjugate (ADC).

[0036] In another preferred example, the detection reagent or kit is for the diagnosis of CLDN18.2-related diseases. In another preferred example, the detection reagent or kit is for the detection of the CLDN18.2 protein in a sample. In another preferred example, the detection reagent is a detection sheet.

[0037] In a thirteenth aspect of the present invention, a drug composition is provided, (i) an active component selected from the group consisting of a heavy chain variable region described in the first aspect of the present invention, a heavy chain described in the second aspect of the present invention, a light chain variable region described in the third aspect of the present invention, a light chain described in the fourth aspect of the present invention, or an antibody described in the fifth aspect of the present invention, a recombinant protein described in the sixth aspect of the present invention, an immune cell described in the tenth aspect of the present invention, an antibody-drug conjugate described in the eleventh aspect of the present invention, or a combination thereof, (ii) A pharmaceutically acceptable carrier and The present invention provides a composition containing the following:

[0038] In another preferred example, the drug composition further comprises a secondary antitumor active ingredient. In another preferred example, the second active ingredient is selected from the group consisting of cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or combinations thereof. In another preferred example, the second active ingredient comprises an antibody targeting EGFR and an antibody targeting HER2.

[0039] In another preferred example, the drug composition is a liquid formulation. In another preferred example, the drug composition is an injectable preparation. In another preferred example, the drug composition is used to treat a tumor. In another preferred example, the tumor is a tumor that highly expresses CLDN18.2.

[0040] In a fourteenth aspect of the present invention, we provide polynucleotides encoding polypeptides selected from the following group: (1) A heavy chain variable region described in the first aspect of the present invention, a heavy chain described in the second aspect of the present invention, a light chain variable region described in the third aspect of the present invention, a light chain described in the fourth aspect of the present invention, or an antibody described in the fifth aspect of the present invention, (2) Recombinant proteins according to the sixth aspect of the present invention, and / or (3) The ninth aspect of the present invention of a CAR structure.

[0041] In a fifteenth aspect of the present invention, a vector comprising the polynucleotide described in the fourteenth aspect of the present invention is provided. In another preferred example, the vector includes a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, such as an adenovirus, a retrovirus, or other vector.

[0042] In a sixteenth aspect of the present invention, a genetically modified host cell is provided, which contains the vector described in the fifteenth aspect of the present invention, or a host cell in which the polynucleotide described in the fourteenth aspect of the present invention is incorporated into its genome.

[0043] In a seventeenth aspect of the present invention, a method for detecting (including diagnostic and non-diagnostic) the CLDN18.2 protein in a sample in vitro is provided, comprising the following steps: (1) In vitro, the sample is brought into contact with the antibody described in the fifth aspect of the present invention; (2) Detect whether an antigen-antibody complex has been formed, where the formation of a complex means that the CLDN18.2 protein is present in the sample.

[0044] In the eighteenth aspect of the present invention, a detection plate is provided which includes a substrate (support plate) and a test strip containing the antibody described in the fifth aspect of the present invention or the antibody-drug conjugate described in the eleventh aspect of the present invention.

[0045] In nineteen aspects of the present invention, the kit is, (1) A first container containing the antibody described in the fifth aspect of the present invention, and / or (2) A second container containing a secondary antibody against the antibody described in the fifth aspect of the present invention, Alternatively, the present invention provides a kit containing the detection plate described in the eighteenth aspect of the present invention.

[0046] In a twentieth aspect of the present invention, a method for producing recombinant polypeptides is provided, comprising the following steps: (a) Culture the host cells described in the fourteenth aspect of the present invention under conditions suitable for expression; (b) From the culture, a recombinant polypeptide is isolated, which is the antibody described in the fifth aspect of the present invention or the recombinant protein described in the sixth aspect of the present invention.

[0047] In the twenty-first aspect of the present invention, a method for treating a CLDN18.2-related disease is provided, comprising the step of administering to a subject in need thereof an antibody described in the fifth aspect of the present invention, an antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof.

[0048] Of course, within the scope of the present invention, it is understood that the above-mentioned technical features of the present invention and the technical features specifically described below (for example, in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, a detailed explanation will not be provided here. [Brief explanation of the drawing]

[0049] [Figure 1] Figure 1 shows the detection results of ELISA binding to hybridoma supernatant cells. [Figure 2A] Figure 2 shows the detection results of flow cytometry. Figure 2A shows the detection results for chimeric antibody ch429, Figure 2B shows the detection results for chimeric antibody ch623, Figure 2C shows the detection results for chimeric antibody ch624, Figure 2D shows the detection results for chimeric antibody ch782, and Figure 2E shows the detection results for reference antibody Ref.ch175. [Figure 2B]Figure 2 shows the detection results of flow cytometry. Figure 2A shows the detection results for chimeric antibody ch429, Figure 2B shows the detection results for chimeric antibody ch623, Figure 2C shows the detection results for chimeric antibody ch624, Figure 2D shows the detection results for chimeric antibody ch782, and Figure 2E shows the detection results for reference antibody Ref.ch175. [Figure 2C] Figure 2 shows the detection results of flow cytometry. Figure 2A shows the detection results for chimeric antibody ch429, Figure 2B shows the detection results for chimeric antibody ch623, Figure 2C shows the detection results for chimeric antibody ch624, Figure 2D shows the detection results for chimeric antibody ch782, and Figure 2E shows the detection results for reference antibody Ref.ch175. [Figure 2D] Figure 2 shows the detection results of flow cytometry. Figure 2A shows the detection results for chimeric antibody ch429, Figure 2B shows the detection results for chimeric antibody ch623, Figure 2C shows the detection results for chimeric antibody ch624, Figure 2D shows the detection results for chimeric antibody ch782, and Figure 2E shows the detection results for reference antibody Ref.ch175. [Figure 2E] Figure 2 shows the detection results of flow cytometry. Figure 2A shows the detection results for chimeric antibody ch429, Figure 2B shows the detection results for chimeric antibody ch623, Figure 2C shows the detection results for chimeric antibody ch624, Figure 2D shows the detection results for chimeric antibody ch782, and Figure 2E shows the detection results for reference antibody Ref.ch175. [Figure 3A] Figure 3 shows the results of detecting the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to recombinant cells. Figure 3A shows the results of the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to HEK293-hCLDN18.1 cells, and Figure 3B shows the results of the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to HEK293-hCLDN18.2 cells. [Figure 3B] Figure 3 shows the results of detecting the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to recombinant cells. Figure 3A shows the results of the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to HEK293-hCLDN18.1 cells, and Figure 3B shows the results of the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to HEK293-hCLDN18.2 cells. [Figure 4]Figure 4 shows the results of detecting the binding activity of the anti-CLDN18.2 human mouse chimeric antibody to NUGC4 cells. [Figure 5] Figure 5 shows the results of flow cytometry detection of the cell binding activity of the anti-CLDN18.2 human mouse chimeric antibody with recombinant cells. [Figure 6] Figure 6 shows the results of ADCC activity of the anti-CLDN18.2 human mouse chimeric antibody against HEK293-hCLDN18.2 cells. [Figure 7A] Figure 7 shows the binding activity of the humanized antibody to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2, and NUGC4 cells. [Figure 7B] Figure 7 shows the binding activity of the humanized antibody to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2, and NUGC4 cells. [Figure 7C] Figure 7 shows the binding activity of the humanized antibody to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2, and NUGC4 cells. [Figure 7D] Figure 7 shows the binding activity of the humanized antibody to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2, and NUGC4 cells. [Figure 7E] Figure 7 shows the binding activity of the humanized antibody to human CLDN18.1&CLDN18.2, mouse CLDN18.1&CLDN18.2, and NUGC4 cells. [Figure 8A] Figure 8 shows the ADCC activity of the humanized antibody. [Figure 8B] Figure 8 shows the ADCC activity of the humanized antibody. [Figure 8C] Figure 8 shows the ADCC activity of the humanized antibody. [Figure 9] Figure 9 shows the detection results for the CDC activity of humanized antibodies. [Figure 10] Figure 10 shows the results of the humanized antibody's HEK293-hCLDN18.2 proliferation inhibitory activity. [Figure 11A]Figure 11 shows a comparison of the major binding sites of the antibody hu782 of the present invention and the reference antibody. Here, Figure 11A shows the major binding site of Ref.ch175, and Figure 11B shows the major binding site of the antibody hu782 of the present invention. [Figure 11B] Figure 11 shows a comparison of the major binding sites of the antibody hu782 of the present invention and the reference antibody. Here, Figure 11A shows the major binding site of Ref.ch175, and Figure 11B shows the major binding site of the antibody hu782 of the present invention. [Figure 12] Figure 12 shows the in vivo antitumor activity of the antibody of the present invention. [Modes for carrying out the invention]

[0050] Through extensive and in-depth research, the inventors unexpectedly obtained an anti-human CLDN18.2 antibody with high affinity and high antitumor activity. Specifically, after conducting a large-scale screening, the inventors obtained a mouse-derived antibody with high affinity for multiple strains of human CLDN18.2 for the first time. In this invention, based on the mouse-derived antibody, chimeric antibodies and humanized antibodies were further produced, and the obtained humanized antibodies were screened for mutations. The antibody of this invention, in particular the humanized antibody after mutation, has affinity close to that of the chimeric antibody and has the potential to be further developed into a humanized monoclonal antibody drug for targeted therapy. The antibody of this invention effectively binds to human CLDN18.2, suppresses the proliferation of many tumor cells, inhibits tumor growth in nude mice, and exhibits excellent antitumor activity. Based on this, the present invention was completed.

[0051] Specifically, the inventors constructed multiple forms of hCLDN18.2 DNA, alternately immunized mice with these as antigens, and then performed enhanced immunization with recombinant cell lines expressing hCLDN18.2. Through hybridoma fusion technology, they obtained mouse monoclonal antibodies mab429, mab623, mab624, and mab782 that specifically recognize hCLDN18.2.

[0052] The chimeric antibody ch782 specifically binds to hCLDN18.2 and can recognize the CLDN18.2 molecule on the cell surface of tumor cell NUGC4, exhibiting higher affinity for CLDN18.2 than the reference antibody Ref.ch175. ch782 possesses ADCC, CDC, and proliferation inhibitory activity against hCLDN18.2 cells.

[0053] The chimeric antibody ch782 was humanized to obtain four combinations of IgG1 type humanized antibodies: hu782H1.1L1, hu782H1.2L1, hu782H1.1L2, and hu782H1.2L2. All four humanized antibodies specifically recognize hCLDN18.2 and have corresponding affinity for ch782.

[0054] By introducing a point mutation at the S60A site in the heavy chain hu782H1.1 of the humanized antibody, a potential glucosylation site was removed, and simultaneously, the expression level of the V101A mutation was increased, resulting in the humanized antibody heavy chain hu782 H1.2-V101A. The S60A and V101A sites are defined according to the Kabat numbering rules.

[0055] The humanized antibodies hu782 (H1.2L2 combination) and hu782-HV (H1.2-V101A L2 combination) exhibit significantly better ADCC activity against tumor cells than the reference antibody Ref.ch175.

[0056] The humanized antibody hu782-DE was obtained by performing the S239D / I332E mutation at amino acids 239 and 332 of the heavy chain constant region of the humanized antibody hu782. The above S239D and I332E are defined by the EU numbering system.

[0057] The humanized antibody hu782-DE exhibits significantly better ADCC against tumor cell NUGC4 than the reference antibody Ref.ch175, and its EC50 value is 24 times lower than that of Ref.ch175.

[0058] The humanized antibodies hu782 and hu782-HV exhibit CDC-killing activity against hCLDN18.2 target cells. The humanized antibodies hu782 and hu782-HV exhibit growth inhibitory activity against hCLDN18.2 target cells, and this inhibitory activity is dose-dependent and significantly better than that of the reference antibody Ref.ch175.

[0059] Epitope analysis revealed that the main recognition site of the humanized antibody hu782 includes the E56 and G48 sites of the hCLDN18.2 molecule. However, the main recognition site of the reference antibody Ref.ch175 includes the A42S, N45A, E56A, and G48 sites, demonstrating some difference in the binding epitopes of the two antibodies.

[0060] In a nude mouse tumor transplantation model, all three antibodies, hu782, hu782-HV, and hu782-DE, were able to clearly suppress tumor growth, and more effectively than the reference antibody Ref.ch175. In particular, the tumor growth inhibitory effect of hu782 and hu782-DE was significantly better than that of the reference antibody Ref.ch175. In all treatment groups using hu782 and hu782-DE, the mouse tumors completely regressed, with tumor suppression rates of 34.68%, 62.88%, and 65.05% for hu782-HV, hu782, and hu782-DE, and 15.5% for the reference antibody Ref.ch175.

[0061] term To facilitate understanding of this disclosure, we first define some terms. Unless otherwise explicitly defined herein, the following terms have the meanings set forth below, as used in this application. Other definitions are provided throughout the application.

[0062] The term "about" means a value or composition that falls within the permissible margin of error of a particular value or composition as determined by those skilled in the art, thereby partially determining how the value or composition is measured or measured. For example, as used herein, the designation "about 100" includes 99, 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0063] As used herein, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include “basically composed of…” or “composed of…”.

[0064] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) and by the number of positions where similar residues appear. This is usually expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method familiar to those skilled in the art.

[0065] As used herein, the terms “heavy chain variable region” and “V H The characters can be used interchangeably. As used herein, the terms “light chain variable region” and “V L The characters can be used interchangeably.

[0066] As used herein, the terms “variable region” and “complementarity determining region (CDR)” are interchangeable.

[0067] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are interchangeable and all refer to antibodies that specifically bind to CLDN18.2, for example, proteins or polypeptides having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID NOs. 1, 11, 12, or 13) and / or a light chain variable region (e.g., the amino acid sequence of SEQ ID NOs. 6, 14, or 15). These may or may not contain initiating methionine.

[0068] Another preferred antibody of the present invention is an antibody that specifically binds to CLDN18.2 corresponding to 624, for example, a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID NO: 23) and / or a light chain variable region (e.g., the amino acid sequence of SEQ ID NO: 24).

[0069] CLDN18.2 Human CLDN18 is a 4-transmembrane protein containing 261 amino acids, with four transmembrane hydrophobic regions. The extracellular portion consists of two loop-like structures: Loop 1 is formed by the circular arrangement of transmembrane regions 1 and 2, and Loop 2 is formed by the circular arrangement of transmembrane regions 3 and 4. Human CLDN18.1 and CLDN18.2 differ in the amino acid composition of their N-terminus, transmembrane region 1, and extracellular loop 1, but the rest of both are completely identical, with a sequence similarity of 92%.

[0070] antibody As used herein, the term “antibody” refers to an immunoglobulin, which has a four-peptide chain structure consisting of two similar heavy chains and two similar light chains linked together by disulfide bonds.

[0071] Existing antibody numbering schemes include the following: 1. The Kabat scheme (Kabat et al., 1991) is based on the location of high-level sequence variant regions between sequences of the same domain type, resulting in different numbers for the heavy chain (VH) and light chain (Vλ and Vκ) variable domains of the antibody. 2. The Chothia scheme (Al-Lazikani, 1997) is similar to the Kabat scheme, but the position of the annotations inserted around the first VH complementarity determination region (CDR) is calibrated to correspond to the structural loop. Similarly, the enhanced Chothia scheme (Abhinandan and Martin, 2008) further structurally modifies the insertion positions. 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Pluckthun, 2001) are both unique schemes that define variable domains of antibodies and T cell receptors (TCRs) (Vα and Vβ). Therefore, the locations of equivalent effects can be easily compared between domain types. IMGT and AHo differ in the number of annotation positions (128 and 149, respectively) and the locations where indels are said to occur.

[0072] The antigenicity of immunoglobulins differs depending on the amino acid composition and sequence order of the heavy chain constant region. This divides immunoglobulins into five types, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains being μ, δ, γ, α, and ε, respectively. The same Ig is further divided into different subtypes based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds; for example, IgG is divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ or λ chains depending on the constant region. Any of the five types of Ig may possess either a κ or λ chain. The subunit structures and three-dimensional structures of each class of immunoglobulin are well known to engineers in this field.

[0073] The light chain of the antibody described in the present invention may further include a light chain constant region, the light chain constant region comprising a human or mouse-derived κ chain, λ chain or a variant thereof.

[0074] In the present invention, the heavy chain of the antibody described herein may further include a heavy chain constant region, the heavy chain constant region comprising human or mouse-derived IgG1, IgG2, IgG3, IgG4 or its variants. The sequences of approximately 110 amino acids near the N-terminus of the antibody's heavy and light chains are highly different and constitute the variable region (Fv region), while the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved skeletal regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In Example 13 of the present invention, the Kabat and Chothia schemes are combined to split the six CDRs of the ch782 antibody.

[0075] In the present invention, the term "mouse-derived antibody" refers to an anti-CLDN18.2 monoclonal antibody manufactured by the knowledge and skills of the art. During manufacturing, a test subject is injected with the CLDN18.2 antigen, and then hybridomas expressing antibodies having the required sequence or functional characteristics are isolated. In one preferred embodiment of the present invention, the mouse-derived CLDN18.2 antibody or its antigen-binding fragment may further include the light chain constant region of a mouse-derived κ chain, λ chain or a variant thereof, or the heavy chain constant region of a mouse-derived IgG1, IgG2, IgG3 or a variant thereof.

[0076] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody, and it can reduce the immune response caused by mouse-derived antibodies.

[0077] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody created by transplanting a mouse CDR sequence into the framework of a human antibody variable region, i.e., a different species of human antibody skeleton sequence. Humanized antibodies overcome the heterogeneity that can be induced by chimeric antibodies due to their large amount of mouse protein components. Such skeleton sequences can be obtained from public DNA databases containing gene sequences of antibodies or from publicly available reference literature. To prevent a decrease in immunogenicity and the resulting decrease in activity, activity can be maintained by introducing minimal retrograde or reverse mutations into the framework sequence of the variable region of the human antibody.

[0078] The term "antibody antigen-binding fragment" (or "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen (e.g., CLDN18.2). It has been shown that full-length antibody fragments can achieve the antigen-binding function of an antibody. Examples of binding fragments included in the term "antibody antigen-binding fragment" include the following: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) A divalent fragment, F(ab')2 fragment, which contains two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) An Fv fragment consisting of the VH and VL domains of one arm of the antibody.

[0079] Fv antibodies are the smallest antibody fragments that contain the heavy chain variable region and light chain variable region of the antibody, but lack a constant region and possess all antigen-binding sites. Generally, Fv antibodies further contain a polypeptide linker between the VH and VL domains and can form the structure necessary for antigen binding.

[0080] The term "CDR" refers to one of six hypervariable regions within the variable domain of an antibody that mainly promotes antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91 - 3242).

[0081] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody binds specifically (e.g., a specific site on the CLDN18.2 molecule). An epitope usually has a unique conformation and contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non - consecutive amino acids.

[0082] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Usually, an antibody binds with an affinity (KD) of less than about 10 -7 M, for example about 10 -8 M, 10 -9 M or less than 10 -10 M or lower. [[ID=十七]]

[0083] The term "competitively binds" refers to an antibody that recognizes and binds to an antigen at an epitope (also called an antigenic determinant) similar to that of the monoclonal antibody of the present invention in the extracellular region of CLDN18.2 or a part of a similar epitope. An antibody that binds to an epitope similar to that of the monoclonal antibody of the present invention is an antibody that recognizes and binds to the amino acid sequence of CLDN18.2 recognized by the monoclonal antibody of the present invention.

[0084] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a specific antibody - antigen interaction. Usually, the antibodies of the present invention have a KD of less than about 10 -7 M, for example about 10 -8 M, 10 -9 M or 10 -10It binds to CLDN18.2 with a dissociation equilibrium constant (KD) less than or equal to M.

[0085] As used herein, the term “antigenic determinant” refers to a discontinuous steric spatial region in an antigen that is recognized by the antibody or antigen-binding fragment of the present invention. The present invention includes not only complete antibodies but also fragments of antibodies having immunoactivity or fusion proteins consisting of antibodies and other sequences. Therefore, the present invention further includes such antibody fragments, derivatives, and analogs.

[0086] In the present invention, antibodies include mouse antibodies, chimeric antibodies, humanized antibodies, or whole-human antibodies produced by techniques familiar to those skilled in the art. Recombinant antibodies, such as chimeric monoclonal antibodies and humanized monoclonal antibodies, include human and non-human portions and can be produced by DNA recombination techniques familiar in the art.

[0087] As used herein, the term “monoclonal antibody” refers to an antibody secreted from a clone derived from a single cell. Monoclonal antibodies are highly specific and target a single epitope. The cell may be a clonal cell line of a eukaryote, prokaryote, or phage. In the present invention, the antibody may be monospecific, bispecific, triplicate, or more.

[0088] In the present invention, the antibody of the present invention further includes its conserved variants, and refers to a polypeptide in which 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids are substituted with amino acids having similar or similar properties when compared with the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably produced by performing amino acid substitutions based on the following table. [Table 1]

[0089] Anti-CLDN18.2 humanized antibody The present invention provides a humanized antibody against CLDN18.2 (hereinafter abbreviated as CLDN18.2 antibody). Specifically, the present invention provides a humanized antibody that is highly specific and highly affinity for CLDN18.2, comprising a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of the heavy chain variable region (VH) and the light chain contains the amino acid sequence of the light chain variable region (VL).

[0090] In 1986, Jones et al. were the first to transplant the heavy chain CDR of a mouse monoclonal antibody into the heavy chain framework region of a human antibody, and then assembled it with the light chain of the mouse monoclonal antibody to form a complete antibody, maintaining affinity similar to the original mouse monoclonal antibody, thus providing a new concept for the development of antibody humanization technology. In 1989, Queen et al. succeeded in constructing an anti-CD25 humanized antibody using the CDR transplantation method, which uses human antibody framework region humanization, maintaining affinity by leaving amino acids from the mouse-derived antibody in a part of the framework region. In 1992, Presta et al. reported a method in which they succeeded in constructing humanized antibodies by transplanting CDRs using the consensus sequence of a human antibody subgroup as a template. In 1994, Pedersen et al. reported the humanization of antibodies by the resurfacing method. In 1994, Hsiao et al. reported a humanization method using CDR transplantation in the framework region of human antibody germline sequences. Also in 1994, Jespers et al. succeeded in constructing a humanization method using a phage library (shuffling library) method.

[0091] In antibody humanization, there are typically two types of human framework regions to choose from: one is a known mature antibody, and the other is a human germline sequence. Framework regions of known mature antibodies usually contain somatic mutation sites, which can lead to potential immunogenicity. Compared to mature antibodies, human germline framework regions are theoretically less immunogenic, more structurally mobile, more adaptable, and more readily accept different CDR regions. Human antibody germline genes tend to have a certain frequency of use in the human body, and antibodies humanized using germline framework regions with high frequency of use have advantages such as lower immunogenicity, higher expression levels, and structural stability.

[0092] In one preferred embodiment of the present invention, instead of selecting the germline sequence with the highest similarity to mouse-derived antibodies during humanization, a framework region of the optimized sequence is selected through extensive experimental screening that balances similarity with frequency of use in humans. CDR transplantation is performed using the framework region of the human antibody germline, and the humanized antibody constructed in this way has a more stable structure, higher expression levels, lower immunogenicity, and greater drug-like properties.

[0093] In another preferred example, the heavy chain steady region and / or light chain steady region may be a humanized heavy chain steady region or light chain steady region. More preferably, the humanized heavy chain steady region or light chain steady region is a heavy chain steady region such as human IgG1, IgG2, or a human κ,λ light chain steady region.

[0094] In another preferred example, the sequence obtained through the addition, deletion, modification, and / or substitution of at least one amino acid is preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology. The antibody of the present invention may be a double-chain or single-chain antibody, and preferably a whole-humanized antibody.

[0095] The antibody derivatives according to the present invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or derivatives of other known antibodies in the art, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies. The antibody of the present invention may be a humanized antibody targeting CLDN18.2, or an antibody grafted and / or modified with CDR.

[0096] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the original amino acid sequence, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20%.

[0097] Antibody production Any method suitable for the production of monoclonal antibodies can be used to produce the CLDN18.2 antibody of the present invention. For example, animals can be immunized with concatenated or naturally occurring CLDN18.2 protein or fragments thereof. Appropriate immunization methods, including adjuvants, immunostimulants, and double immunization, may be used, or one or more of these methods may be used.

[0098] Any suitable form of CLDN18.2 can be used as an immunogen (antigen) to generate non-human antibodies specific to CLDN18.2, and the biological activity of these antibodies can be selected for use. The immunogen may be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen may be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed in a suitable genetic vector, which includes, but is not limited to, adenovirus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0099] Humanized antibodies may be selected from any type of immunoglobulin, but include IgM, IgD, IgG, IgA, and IgE. Similarly, any light chain can be used in the compounds and methods herein. Specifically, κ chains, λ chains, or their variants are useful in the compounds and methods of the present invention.

[0100] The DNA molecule sequences of the antibody or fragment of the present invention can be obtained by conventional techniques, such as PCR amplification or genome library screening. Alternatively, the light chain and heavy chain coding sequences may be fused together to form a single-chain antibody.

[0101] Once a related sequence is obtained, a large quantity of it can be acquired using recombination. In this case, the sequence is usually cloned into a vector, introduced into cells, and then the related sequence is isolated from the host cells that have been grown using conventional methods. Furthermore, especially when the fragment length is short, the relevant sequence may be synthesized by artificial synthesis methods. Typically, a long fragment can be obtained by first synthesizing a large number of small fragments and then concatenating them. This DNA sequence may then be introduced into various known DNA molecules (or vectors, etc.) or cells, as is well known in this field.

[0102] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but double-stranded DNA is preferred. When nucleic acids are functionally related to another nucleic acid sequence, they are "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

[0103] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA ring to which another DNA fragment can be linked. Furthermore, the present invention relates to vectors comprising the above-mentioned suitable DNA sequence and a suitable promoter or regulatory sequence. These vectors can be used to transform suitable host cells to express proteins.

[0104] The term "host cell" refers to a cell into which an expression vector has already been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

[0105] The process of transforming host cells by DNA recombination as described in this invention may be carried out using techniques well known in the art. The resulting transformants can be cultured by conventional methods and express the polypeptide encoded by the gene of this invention. Depending on the host cells used, they may be cultured in a conventional medium under appropriate conditions.

[0106] Typically, host cells obtained by transformation are cultured under conditions suitable for the expression of the antibody of the present invention. Then, the antibody of the present invention is obtained by purifying it using conventional globulin purification methods familiar to technicians in this field, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and affinity chromatography.

[0107] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of monoclonal antibodies can be measured by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0108] Antibody preparations Antibodies exhibit different stabilities in different formulation buffers, manifesting as changes in charge heterogeneity, degradation of antibody molecules, and polymerization. Since these changes in mass properties are related to the physical and chemical properties of the antibody itself, it is necessary to screen formulation buffers suitable for different antibodies during the antibody drug development process. Currently, commonly used buffer systems for antibody production include phosphate buffers, citrate buffers, and histidine buffers. At the same time, antibody stability may be maintained by adding different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as twin, depending on the properties of the antibody.

[0109] Drug composition Furthermore, the present invention provides compositions. In a preferred example, the composition is a drug composition containing the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier, and the pH value is usually around 5 to 8, preferably around 6 to 8, depending on the properties of the substances formulated and the disease to be treated. The formulated drug composition can be administered via a normal route, including, but not limited to, intratumoral, intraperitoneal, intravenous, or local administration.

[0110] The antibody according to the present invention may be expressed in cells according to its nucleotide sequence and used for cell therapy. For example, the antibody can be used in chimeric antigen receptor T cell immunotherapy (CAR-T). The drug composition of the present invention can be used for direct binding to the CLDN18.2 protein molecule, and is therefore useful for the prevention and treatment of CLDN18.2-related diseases. It may also be used in combination with other therapeutic agents.

[0111] The drug composition of the present invention comprises a safe effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the monoclonal antibody (or its complex) of the present invention and a pharmaceutically acceptable carrier or excipient. Such vehicles include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The formulation of the drug corresponds to the dosage form. The drug composition of the present invention may also be an injectable preparation and can be prepared by conventional methods, for example, in physiological saline or an aqueous solution containing glucose and other excipients. The drug composition, in the case of an injectable preparation or solution, is prepared under sterile conditions. The dose of the active ingredient is a therapeutic effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight daily. Furthermore, the polypeptide of the present invention can be used in combination with other therapeutic agents.

[0112] When using a drug composition, a safe and effective dose is administered to a mammal. This safe and effective dose is usually at least about 10 μg / kg body weight, and in many cases less than about 50 mg / kg body weight, preferably between about 10 μg / kg body weight and about 20 mg / kg body weight. Of course, the specific dose should also be determined considering factors such as the mode of administration and the patient's health condition, all of which are within the skill range of a qualified physician.

[0113] Detection applications and kits The antibodies of the present invention are useful for detection and can provide diagnostic information, for example, by detecting a sample. In the present invention, the specimens used include cell specimens, tissue specimens, and biopsy specimens. The term "biopsy" as used in the present invention includes all types of biopsies known to those skilled in the art. Therefore, the biopsies used in the present invention include, for example, tissue specimens prepared by endoscopic methods or by organ puncture or needle stick.

[0114] The specimens used in this invention include fixed or preserved cell or tissue specimens. Furthermore, the present invention provides a kit containing the antibody (or a fragment thereof) of the present invention, in one preferred example of the present invention, the kit further includes a container, instructions for use, buffer solution, etc. In a preferred example, the antibody of the present invention may be immobilized on a detection plate.

[0115] The main advantages of this invention are as follows: 1. The antibody of the present invention has a well-structured embedding of typical tryptophan and tyrosine, a stable template structure for the screened human VH and VL framework regions, good adaptability to the CDR region of mouse monoclonal antibodies, suitable match between light and heavy chain variable regions, high affinity, and structural stability. 2. Compared to chimeric antibodies, the humanized antibodies of the present invention possess superior biological activity and specificity, maintaining affinity equivalent to CLDN18.2 while exhibiting lower immunogenicity and higher expression levels. 3. The humanized antibody of the present invention has remarkable in vitro biological activity and can efficiently suppress the activity of tumor cell proliferation in in vitro culture, and its activity is better than that of the control antibody. 4. The humanized antibody of the present invention has remarkable biological activity in the body, exhibiting excellent inhibitory activity against tumor cells such as human gastric cancer cells, and its activity is better than that of the control antibody.

[0116] The present invention will be further described below with reference to specific examples. These examples are used solely for the purpose of illustrating the present invention and are not intended to limit the scope of the invention. Experimental methods in the following examples where specific conditions are not given will generally follow standard conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight. Unless otherwise specified, all materials or reagents used in the examples of this invention are commercially available.

[0117] Materials and methods 1. Reference antibody The reference antibody used in the experiments of this invention has a sequence derived from the antibody sequence of the 175D10 clone in patent US8168427. The 175D10 antibody is also known as IMAB362. The variable region sequence of the 175D10 antibody was synthesized as a whole gene, constructed in a eukaryotic expression vector containing the constant region of the IgG1 chimeric antibody, expressed, and purified. In this invention, the manufactured reference antibody is abbreviated as Ref.ch175.

[0118] The reference antibody Ref.ch175 (or IMAB362) specifically recognizes the extracellular loop 1 portion of CLDN18.2, does not bind to CLDN18.1, and kills tumor cells primarily through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0119] Example 1: Construction of a stably expressing cell line of CLDN18.1 or CLDN18.2 In this example, cell lines that stably express human CLDN18.1, human CLDN18.2, mouse CLDN18.1, and mouse CLDN18.2 were constructed. The method is as follows.

[0120] The full-length gene fragments encoding human CLDN18.1 (NP_057453.1) and human CLDN18.2 (NP_001002026.1) were synthesized as whole genes. These full-length fragments were then cleaved using the Xhol+BamHI enzyme and cloned into pEGFP-N2 eukaryotic expression vectors (purchased from Clontech), and named pEGFP-N2-hCLDN18.1 and pEGFP-N2-hCLDN18.2, respectively.

[0121] HEK293 cells (purchased from the Chinese Academy of Sciences Cell Library, GNHu43) 4 × 10 5The cells were inoculated into a 6-well plate at a rate of one cell per well. The following day, pEGFP-N2-hCLDN18.1 and pEGFP-N2-hCLDN18.2 were transfused into HEK293 cells using the lipofection reagent LipoMax. 24 hours after transfusion, the cells were passaged in a 1:10 ratio. Pressure screening was performed by adding G418 (purchased from Gibco) to achieve a final concentration of 300 μg / ml once the cells adhered to the cell wall. Subcloning yielded HEK293 cells that stably expressed human CLDN18.1 and human CLDN18.2 membrane proteins, which were named HEK293-hCLDN18.1 and HEK293-hCLDN18.2, respectively.

[0122] The full-length gene fragments encoding mouse CLDN18.1 (NP_062789.1) and mouse CLDN18.2 (NP_001181850.1) were synthesized as whole genes, cleaved with the KpnI+XbaI enzyme, and cloned into a pcDNA3.1-P2A-EGFP eukaryotic expression vector (purchased from Nanjing Genscript), and named pcDNA3.1-mCLDN18.1 and pcDNA3.1-mCLDN18.1, respectively. These were transfused into HEK293 cells using the same method as above, and cells stably expressing mouse CLDN18.1 and CLDN18.2 membrane proteins were obtained, and named HEK293-mCLDN18.1 and HEK293-mCLDN18.2, respectively.

[0123] >NP_057453.1 Claudin-18 isoform 1 precursor [Human (Homo sapiens)] SEQ ID NO: 25 MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0124] >NP_001002026.1 Claudin-18 isoform 2 [Human (Homo sapiens)] Sequence ID 26 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0125] >NP_062789.1 Claudin-18 isoform A1.1 precursor [Mus musculus] Sequence ID 27 MATTTCQVVGLLLSLLGLAGCIAATGMDMWSTQDLYDNPVTAVFQYEGLWRSCVQQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICA IIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV

[0126] >NP_001181850.1 Claudine-18 isoform A2.1 [Mus musculus] Sequence ID 28 MSVTACQGLGFVVSLIGFAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGVIGILVSIFALKCIRIGSMDDSAKAKMTLTSGILFIISGICA IIGVSVFANMLVTNFWMSTANMYSGMGGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLTPDDSNFKAVSYHASGQNVAYRPGGFKASTGFGSNTRNKKIYDGGARTEDDEQSHPTKYDYV

[0127] Example 2: Preparation of immunogen Because the amino acid sequences of human CLDN18.2 extracellular loop 1 (CLDN18.2 ECL1) and mouse CLDN18.2 ECL1 are identical, it is difficult to obtain antibodies that bind to CLDN18.2 ECL1. At the same time, considering that the conformation of the CLDN18.2 molecule on the surface of tumor cells may differ from that of normal tissue cells, in this example, we designed multiple forms of DNA plasmids as antigens to overcome immune resistance and obtained antibodies that recognize tumor cells with high affinity.

[0128] The nucleotides encoding human CLDN18.2 ECL1 and the full-length amino acids of CLDN18.2 were ligated in series with the tetanospasmin P2 epitopes TT830-844 and P30 epitopes TT947-967, respectively, and cloned into the eukaryotic expression vector PTT5 (purchased from Invitrogen). These were named PTT5-TT-hCLDN18.2 and PTT5-TT-hCLDN18.2-ECL1, respectively. In this invention, CLDN18.2 ECL1 was fused to the Fc-C terminus of human IgG1 and named hFc-CLDN18.2-ECL1.

[0129] TT-hCLDN18.2 nucleotide sequence, SEQ ID NO: 29 CAGTACATCAAGGCCAACAGCAAGTTCATCGGCATCACCGAGCTGAAGAAGCTGGGTGGTTCCAACGACATATTCAACAACTTCACCGTGAGCTTCTGGCTGCGCGTGCCGAAGGTGAGCGCCAGCCACCTGGAGCAGTACGGTGGCGGTAGCGGAATGGCCGTGACTGCCTGTCAGGGCTTGGGGTTCGTGGTTTCACTGATTGGGATTGCGGGCATCATTGCTGCCACCTGCATGGACCAGTGGAGCACCCAAGACTTGTACAACAACCCCGTAACAGCTGTTTTCAACTACCAGGGGCTGTGGCGCTCCTGTGTCCGAGAGAGCTCTGGCTTCACCGAGTGCCGGGGCTACTTCACCCTGCTGGGGCTGCCAGCCATGCTGCAGGCAGTGCGAGCCCTGATGATCGTAGGCATCGTCCTGGGTGCCATTGGCCTCCTGGTATCCATCTTTGCCCTGAAATGCATCCGCATTGGCAGCATGGAGGACTCTGCCAAAGCCAACATGACACTGACCTCCGGGATCATGTTCATTGTCTCAGGTCTTTGTGCAATTGCTGGAGTGTCTGTGTTTGCCAACATGCTGGTGACTAACTTCTGGATGTCCACAGCTAACATGTACACCGGCATGGGTGGGATGGTGCAGACTGTTCAGACCAGGTACACATTTGGTGCGGCTCTGTTCGTGGGCTGGGTCGCTGGAGGCCTCACACTAATTGGGGGTGTGATGATGTGCATCGCCTGCCGGGGCCTGGCACCAGAAGAAACCAACTACAAAGCCGTTTCTTATCATGCCTCAGGCCACAGTGTTGCCTACAAGCCTGGAGGCTTCAAGGCCAGCACTGGCTTTGGGTCCAACACCAAAAACAAGAAGATATACGATGGAGGTGCCCGCACAGAGGACGAGGTACAATCTTATCCTTCCAAGCACGACTATGTGTAA

[0130] TT-hCLDN18.2-ECL1 nucleotide sequence: SEQ ID NO: 30 CAGTACATCAAGGCCAACAGCAAGTTCATCGGCATCACCGAGCTGAAGAAGCTGGGTGGTTCCAACGACATATTCAACAACTTCACCGTGAGCTTCTGGCTGCGCGTGCCGAAGGTGAGCGCCAGCCACCTGGAGCAGTACGGTGGCGGTAGCGGACGCATGG ACCAGTGGAGCACCCAAGACTTGTACAACAAACCCCGTAACAGCTGTTTTCAACTACCAGGGGCTGTGGCGCTCCTGTGTCCGAGAGAGCTCTGGCTTCACCGAGTGCGGGGCTACTTCACCCTGCTGGGGCTGCCAGCCATGCTGCAGGCAGTGCGAGCCTAA

[0131] hFc-CLDN18.2-ECL1 nucleotide sequence: SEQ ID NO: 31 GACAAGACCCACACATGCCCACCTTGTCCAGCTCCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAGGATACACTGATGATCTCTAGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTATCGGGTGGTGTCTGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGAGCAATAAGGCCCTGCCTGCTCCAATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCTCCATCCCGCGAGGAGATGACCAAGAACCAGGTGAGCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCTGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTTCCTGTAGCGTGATGCACGAGGCCCTGCACAATCATTACACACAGAAGTCTCTGTCCCTGAGCCCTGGCAAGGGATCCGGAGGAGGCTCTGGAGGCTCCGACCAGTGGAGCACCCAGGATCTGTACAACAATCCCGTGACAGCCGTGTTCAACTATCAGGGCCTGTGGAGGTCTTGCGTGCGGGAGTCCAGCGGCTTCACCGAGTGTAGAGGCTACTTTACACTGCTGGGACTGCCTGCTATGCTGCAGGCTGTGCGCTGA

[0132] Example 3: Production of anti-human CLDN18.2 hybridoma cell line The Balb / c mice used for the experiment were purchased from the Shanghai SIPPR-BK laboratory. All immunization mice were 4-6 week old females, standardized disease-free, and healthy purebred mice. CLDN18.2 is a four-transmembrane protein. To obtain a specific antibody that recognizes tumor cells with high affinity, mice were immunized alternately with multiple plasmids, and then rush-immunized with cells that stably express human CLDN18.2 membrane protein. The method is as follows.

[0133] Day 1 and Day 14: 50 μg of the eukaryotic expression vector plasmid pEGFP-N2-hCLDN18.2 encoding the full-length human CLDN18.2 gene was injected into the quadriceps muscle of mice (im), with the adjuvant being In vivo-Jet PEI-Man translocation reagent (purchased from Polyplus, 203-10G). On day 28, 50 μg of the eukaryotic expression plasmid hFc-CLDN18.2-ECL1 DNA was injected into the quadriceps muscle of mice (im), on day 42, 50 μg of the eukaryotic expression vector plasmid PTT5-TT-hCLDN18.2 was injected into the quadriceps muscle of mice (im), on day 56, 50 μg of the eukaryotic expression vector plasmid PTT5-TT-CLDN18.2 ECL1 was injected into the quadriceps muscle of mice (im), and on day 70, 50 μg of the eukaryotic expression vector plasmid PTT5-TT-hCLDN18.2 and 50 μg of the eukaryotic expression vector plasmid PTT5-TT-CLDN18.2 ECL1 were injected into the quadriceps muscle of mice (im).

[0134] Fourteen days later, blood was collected from the tail vein of the mouse, and serum antibody titers were measured by cell-based ELISA. Three days before fusion, 2 × 10⁶ antibodies were measured. 7 Two mice with the highest antibody titers were enhanced immunized by intraperitoneal injection with one HEK293-hCLDN18.2 cell per mouse. On the day of fusion, blood was collected from the mouse eyes, the spleen was removed, and a single-cell suspension was prepared. Hybridomas were obtained by electrofusion with SP20 cells and placed on plates. [Table 2]

[0135] Example 4: Screening of hybridoma cells that specifically bind to human CLDN18.2 but not to human CLDN18.1 In this example, hybridomas were screened using a cell-based ELISA method to obtain hybridoma cells that specifically bind to human CLDN18.2 but not to human CLDN18.1. The method is as follows.

[0136] After digesting HEK293-hCLDN18.1 cells and HEK293-hCLDN18.2 cells with trypsin, 3 × 10⁻⁶ cells were processed. 5 Cells were inoculated at 200 μL / well into 96-well plates coated with polylysine (P4707, Sigma) at a cell density of cells / mL, and cultured overnight. When cell confluence reached over 90%, the supernatant was removed and the cells were fixed in 10% neutral formalin solution at room temperature for 15 minutes. After washing three times with PBS, the cells were blocked overnight at 4°C in 5% skimmed milk. To detect hybridomas, the supernatant from the same well was placed in plates coated with 80 μl / well of HEK293-hCLDN18.1 cells and HEK293-hCLDN18.2 cells, respectively. After incubation at 37°C for 1 hour and washing three times with PBST, 100 μl / well of 1:5000 diluted sheep anti-mouse IgG(H+L)-HRP was added, incubated at 37°C for 45 minutes, allowed to develop color for 30 minutes, stopped the development by adding 50 μl / well of 2M H2SO4, and the OD450 was read using a microplate reader.

[0137] After screening over 10,000 clones, we obtained several strains of hCLDN18.2-specific antibodies, but the four optimal strains are shown in the table below. [Table 3]

[0138] Example 5: Detection of subtypes of mouse monoclonal antibodies Subtype detection was performed on clonal strains that specifically bind to CLDN18.2 using a subtype detection kit (SBA Clonotyping System-HRP kit, purchased from southernbiotech, 5300-05).

[0139] The detection results are as follows: [Table 4]

[0140] Example 6: Cloning and identification of variable region coding sequence of monoclonal antibody derived from anti-human CLDN18.2 mouse. Since IgM subtype antibodies were present in the mouse monoclonal antibodies obtained through screening, the variable region sequences of the mouse monoclonal antibodies were obtained by RACE-PCR to further evaluate their activity, and then chimeric antibodies were constructed.

[0141] The total mRNA of the hybridoma obtained in Example 4 was extracted using the TakaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767), and the sequence of the variable region of the mouse-derived antibody was obtained by PCR using the SMARTer RACE 5' / 3' Kit (Clontech, 634858). The results are as follows.

[0142] Amino acid sequence of the variable region of the 624 antibody heavy chain (SEQ ID NO: 23): QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARTSYGNSFAYWGQGTLVTVSA

[0143] Amino acid sequence of the variable region of the 624 antibody light chain (SEQ ID NO: 24): DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK

[0144] Nucleotide sequence of the variable region of the 782 antibody heavy chain (SEQ ID NO: 2): GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAGTGATAACACGAAC TACAGCCCATCTCTCAAAGGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGATCTACTATGGTAACTCGTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0145] Amino acid sequence of the variable region of the 782 antibody heavy chain (SEQ ID NO: 1): DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHYSDNTNYSPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARIYYGNSFVYWGQGTLVTVSA

[0146] Nucleotide sequence of the variable region of the 782 antibody light chain (SEQ ID NO: 7): GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTGTTGATCTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAACAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATTTTTATCCGTACACGTTCGGGGGGGGGACCAAGCTGGAAATAAAA

[0147] Amino acid sequence of the variable region of the 782 antibody light chain (SEQ ID NO: 6): DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDYFYPYTFGGGTKLEIK

[0148] Example 7: Construction and expression of an anti-CLDN18.2 human mouse chimeric antibody Primers were designed according to the primer design principles of homologous recombination. Gene fragments of the variable heavy and light chain regions of mouse-derived antibodies obtained in Example 6 were amplified, ligated to the sequences of the constant heavy and light chain regions of human antibodies, and constructed in the expression vector PTT5 (purchased from Invitrogen). Plasmid extraction was performed using a commercial plasmid maxi kit, and then the plasmids were transfused into 293FV suspension cells by PEI transfusion. Seven days after transfusion, the supernatant was collected, and chimeric antibodies were obtained by protein A affinity chromatography.

[0149] Example 8: Specificity analysis of anti-CLDN18.2 human mouse chimeric antibody After stably transfecting HEK293 cells with human CLDN18.2 and HEK293 cells with human CLDN18.1 with trypsin, 5 × 10⁻⁶ cells were processed. 5 Individual cells were placed in 100 μl of hybridoma supernatant at a rate of 100 μl / 100 μl, incubated on ice for 1 hour, washed twice with FACS buffer, then sheep anti-mouse IgG-PE secondary antibody diluted 1:400 was added, incubated on ice for 45 min, washed twice with FACS buffer, then 300 μl of preparation buffer was added, transferred to a BD flow cytometry tube, and analyzed using BD FACS calibur. Median fluorescence intensity (MFI) was analyzed using Flowjo software.

[0150] The results are shown in Figure 2 and the table below. [Table 5]

[0151] The results showed that ch429, ch623, ch624, and ch782 specifically bind to human CLDN18.2 but do not bind to human CLDN18.1. Among these, the MFI of the chimeric antibody ch782 was higher than that of the reference antibody Ref.ch175.

[0152] Example 9: Detection of the binding activity of anti-CLDN18.2 human mouse chimeric antibody to recombinant cells HEK293-hCLDN18.1 and HEK293-hCLDN18.2 cells were digested with trypsin and inoculated at a constant cell density into 96-well plates coated with polylysine (P4707, Sigma), and cultured overnight. When cell confluence reached over 90%, the supernatant was removed and the cells were fixed in 10% formalin solution at room temperature for 15 minutes. After washing once with PBS, the cells were blocked overnight in 5% skimmed milk at 4°C to prepare for use. Candidate chimeric antibodies and reference antibody Ref.ch175 were diluted 3-fold from 1 μg / ml to a gradient of 10, placed in a 96-well plate coated with cells at 100 μl / well, incubated at 37°C for 1 hour, washed three times with PBST, added mouse anti-human IgG Fc-HRP diluted 1:2000, incubated at 37°C for 1 hour, washed three times with PBST, developed with TMB colorant for 15 minutes, and stopped with 2M H2SO4. OD450-OD630 values ​​were read, fitted to graphs using GraphPad Prism 8 software, and EC50 was calculated. The results, as shown in Figure 3, demonstrate that ch429, ch623, ch624, ch782, and the reference antibody Ref.ch175 did not bind to HEK293-hCLDN18.1 cells but specifically bound to HEK293-HCLDN18.2 cells. Furthermore, ch429 and ch782 showed relative binding activity of 257.5% and 179.9% of that of the reference antibody Ref.ch175, respectively. The results are shown in the table below. [Table 6]

[0153] Example 10: Detection of the binding activity of anti-CLDN18.2 human mouse chimeric antibody to NUGC4 cells NUGC4 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were laid in a 96-well plate using the same method as in Example 9. Candidate chimeric antibodies and reference antibody Ref.ch175 were diluted 3-fold from 100 μg / ml to create a gradient of 10, and 100 μl / well was added to the 96-well plate coated with cells. After incubation at 37°C for 1 hour, the plate was washed three times with PBST, mouse anti-human IgG Fc-HRP diluted 1:2000 was added, incubated at 37°C for 1 hour, washed three times with PBST, stained with TMB colorant for 15 minutes, and stopped with 2M H2SO4. OD450-OD630 values ​​were read, fitted using GraphPad Prism 8 software to create a graph, and EC50 was calculated.

[0154] The results are shown in Figure 4 and the table below. [Table 7]

[0155] The EC50 values ​​for chimeric antibodies ch782 and ch429 were 0.57 μg / ml and 5.25 μg / ml, respectively. At high concentrations, ch782 had a higher reading (OD450=2.3) than ch429 (OD450=1.2). EC50 could not be calculated for ch623, ch624, or the reference antibody Ref.ch175.

[0156] This demonstrated that the ch782 antibody exhibited the best binding activity to NUGC4 cells that endogenously express human CLDN18.2 protein, and was clearly stronger than that of the reference antibody Ref.ch175.

[0157] Example 11: Detection of the binding activity of an anti-CLDN18.2 human mouse chimeric antibody to HEK293-hCLDN18.2 cells. Following the method in Example 8, chimeric antibodies ch429, ch624, ch782, and Ref.ch175 were subjected to a 3-fold gradient dilution starting from a concentration of 10 μg / ml, and flow cytometry experiments were performed. The MFI was used as the y-coordinate to create a graph using GraphPad Prism 8 software, and the EC50 was calculated.

[0158] The results are shown in Figure 5 and the table below. [Table 8]

[0159] The chimeric antibodies ch429, ch782, and reference antibody Ref.ch175 of the present invention can all bind to HEK293-hCLDN18.2 cells, with EC50 values ​​of 0.52 μg / ml, 0.81 μg / ml, and 0.85 μg / ml, respectively, although the binding activity of ch624 is clearly weaker. Furthermore, at high concentrations, ch782 shows a higher MFI (Multiple Filtration Indication) for binding to HEK293-hCLDN18.2 cells than Ref.ch175.

[0160] Example 12: Detection of ADCC activity of anti-CLDN18.2 human mouse chimeric antibody against HEK293-hCLDN18.2 cells using reporter gene method. The antibody-mediated ADCC effect, mediated by the antibody's Fc terminus, is an important indicator for evaluating the antitumor activity of an antibody. ADCC is antibody-dependent cell-mediated cytotoxicity, and its mechanism of action involves the antibody binding to an antigen on the tumor surface. The antibody's Fc region then binds to an immune effector cell receptor, primarily targeting and recognizing the Fc receptor on NK cells, directly lysing and killing tumor cells.

[0161] In this example, the ADCC biological activity of the antibody was detected by reporter gene analysis, and engineered modified Jurkat cells that stably express the FcγRIIIa receptor were used as effector cells. The function of the antibody Fc effector factor was quantitatively detected by detecting the luciferase chemiluminescence signal using the Bio-Glo™ Luciferase Assay System kit and measuring the activation of the NFAT signaling pathway.

[0162] After digesting the HEK293-hCLDN18.2 cells constructed in Example 1, 2.5 × 10⁶ cells were obtained. 5 The solution was diluted to a concentration of / ml, then spread at 100 μl / well in a white, opaque cell culture plate. After 24 hours of static incubation at 37°C, the supernatant was discarded, and 25 μl / well of analytical medium containing 0.1% BSA was added. Jurkat / NFAT-luc+FcγRIIIa cells were placed in 6 × 10⁶ cells. 6 The solution was diluted to 1 / mL, and a 96-well plate was prepared. 25 μl of Jurkat / NFAT-luc+FcγRIIIa cell suspension was placed in each well. Then, 25 μL of antibody diluent was added to each well until the final concentration was 6 μg / mL. From this concentration, a 3-fold series dilution was performed, resulting in a total of 10 dilution gradients. The cell plate was placed in a constant temperature incubator and induced cultured for 6 hours. After that, the 96-well plate was allowed to equilibrate at room temperature for at least 15 minutes. Using a continuous pipette, 75 μL / well of Bio-Glo™ Luciferase Assay Reagent was added to all reaction wells (the staining solution must be kept away from light). The mixture was allowed to react at room temperature for 5 minutes, and the plate was set in a multifunction plate reader to detect the Luminescence light signal. The fluorescence values ​​and antibody concentrations were fitted using GraphPad Prism 8 software to create a graph and calculate EC50.

[0163] The results are shown in Figure 6 and the table below. [Table 9]

[0164] The results show that after gradient dilution of ch782 and the reference antibody Ref.ch175, and incubation at 37°C for 6 hours with an effector target ratio E:T = 6:1, both ch782 and Ref.ch175 can induce strong ADCC activity.

[0165] Example 13: Humanization of anti-CLDN18.2 antibody ch782 To reduce the immunogenicity of the antibody, the human anti-mouse antibody (HAMA) effect upon administration of the antibody into the body was decreased, and the suitable antibody ch782 was humanized. Specifically, a humanization sequence was designed for the variable region sequence of the mab782 antibody in Example 6. The heavy and light chain amino acid sequences were numbered according to the Kabat numbering system, sequence homology alignment was performed using NCBIIgblast (https: / / www.ncbi.nlm.nih.gov / igblast / ), and the sequence of a highly homologous human-derived antibody was used as a framework. Mouse-derived antibody CDR transplantation was performed, and then a core amino acid in a part of the framework region was subjected to regression mutation.

[0166] We used Blast to search for homologous sequences of the heavy chain and human germline of the 782 antibody. [Table 10]

[0167] We used Blast to search for homologous sequences of the light chain and human germline of the 782 antibody. [Table 11]

[0168] The Kabat-defined sequences of FR and CDR in the heavy chain variable region of antibody 782 are as follows: [Table 12]

[0169] The Kabat-defined sequences of FR and CDR in the light chain variable region of antibody 782 are as follows: [Table 13]

[0170] In this invention, the humanization of the antibody heavy chain was performed by replacing the CDR region using the highly homologous IGHV4-61 as a template. During the transplantation of the heavy chain CDR1, the CDR1 site defined by Chothia was simultaneously included, and three sites, I48M / V67I / V71R, were subjected to regressive mutations to obtain hu782H1.1.

[0171] Post-translational modification analysis revealed the presence of a potential glucosylation site at the S60 site. This site was mutated to S60A using point mutation and named hu782H1.2. The expression level was then optimized by mutating the 101st amino acid, V, to A, and this was named hu782 H1.2-V101A.

[0172] In this invention, the humanization of the antibody light chain was performed by replacing the CDR region using the VκI consensus sequence as a template to obtain hu782L1, and by replacing the CDR region using IGKV4-1 as a template to obtain hu782L2. After codon optimization of the above sequence, the entire gene was synthesized, and then cloned into an expression vector containing the heavy and light chain constant regions for expression and purification.

[0173] hu782H1.1 (Sequence ID 11) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSGYSWHWIRQPPGKGLEW M GYIHYSDNTNYSPSLKSR I TIS R DTSKNQFSLKLSSVTAADTAVYYCARIYYGNSFVYWGQGTLVTVSS

[0174] hu782H1.2 (Sequence ID 12) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSGYSWHWIRQPPGKGLEW M GYIHYSDNTNY A PSLKSR I TIS R DTSKNQFSLKLSSVTAADTAVYYCARIYYGNSFVYWGQGTLVTVSS

[0175] hu782H1.2-V101A (Sequence ID 13) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSGYSWHWIRQPPGKGLEW M GYIHYSDNTNYAPSLKSR I TIS R DTSKNQFSLKLSSVTAADTAVYYCARIYYGNSF A YWGQGTLVTVSS

[0176] hu782L1 (Sequence ID 14) DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNDYFYPYTFGQGTKVEIK

[0177] hu782L2 (Sequence ID 15) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYFYPYTFGGGTKLEIK

[0178] Example 14: Detection of specific binding activity of humanized antibodies to CLDN18.2 Using the method for evaluating the binding activity of chimeric antibodies in the above examples, the binding activity of the humanized antibodies to human CLDN18.1 and CLDN18.2, mouse CLDN18.1 and CLDN18.2, and NUGC4 cells was detected.

[0179] The results are shown in Figure 7 and the table below. [Table 14]

[0180] The results showed that all humanized antibodies were able to specifically bind to human CLDN18.2, and that there was no significant decrease in binding activity compared to the chimeric antibody ch782. Furthermore, surprisingly, the EC50 values ​​of the four types of humanized antibodies for cell binding to tumor cells NUGC4 were significantly better than those of the chimeric antibody ch782.

[0181] Example 15: Detection of ADCC activity of humanized antibodies One of the main mechanisms of antibody activity in the body in this invention is ADCC functional activity. Human IgG1 antibody (hIgG1) primarily binds to FcγR via the hinge region and CH2 region of the antibody, thereby exerting the ADCC effect. To enhance the ADCC activity of humanized antibodies, in this example, the S239D / I332E(DE) mutation was introduced into the Fc region of the humanized antibody heavy chain hu782H1.2, thereby increasing its affinity for FcγR and achieving the objective of enhancing ADCC. After expression and purification, the ADCC of wild-type and DE mutant human-derived antibodies was evaluated using the reporter gene method. [Table 15]

[0182] The method is as follows: Following the specific operating steps in Example 12, 2.5 × 10⁶ HEK293-hCLDN18.2 and NUGC4 cells, which are the target cells, were prepared. 5 The solution was diluted to a concentration of / ml, then spread at 100 μl / well in a white, opaque cell culture plate. After 24 hours of static incubation at 37°C, the supernatant was discarded, and 25 μl / well of analytical medium containing 0.1% BSA was added. Jurkat / NFAT-luc+FcγRIIIa cells were placed in 6 × 10⁶ cells. 6The solution was diluted to 1 / mL, and a 96-well plate was prepared. 25 μL of Jurkat / NFAT-luc+FcγRIIIa cell suspension was placed in each well. Then, 25 μL of antibody diluent was added to each well until the final concentration was 6 μg / mL. From this concentration, a 3-fold series dilution was performed, resulting in a total of 10 dilution gradients. The cell plate was placed in a constant temperature incubator and induced cultured for 6 hours. After that, the 96-well plate was allowed to equilibrate at room temperature for at least 15 minutes. Using a continuous pipette, 75 μL / well of Bio-Glo™ Luciferase Assay Reagent was added to all reaction wells (the staining solution must be kept away from light). The mixture was allowed to react at room temperature for 5 minutes, and the plate was set in a multifunction plate reader to detect the Luminescence light signal. The fluorescence values ​​and antibody concentrations were fitted using GraphPad Prism 8 software to create a graph and calculate EC50. [Table 16]

[0183] The results showed that, in recombinant cells with high human CLDN18.2 expression, the chimeric antibody ch782 and the humanized antibodies hu782 and hu782-HV all effectively induced ADCC activity equivalent to that of the reference antibody (EC50 values ​​of 47.64 ng / ml, 64.03 ng / ml, and 70.94 ng / ml, respectively), and that hu782-DE, with the Fc primer S239D / I332E mutation, significantly improved ADCC activity (Figure 8).

[0184] Similar results were observed in NUGC4 cells endogenously expressing human CLDN18.2, and the ADCC activity of the candidate antibodies ch782 and hu782 obtained in this invention against NUGC4 cells was better than that of the reference antibody Ref.ch175.

[0185] Furthermore, when NUGC4 cells stably transfected with hCLDN18.2 were used as target cells for detection of ADCC activity, it was found that hu782-DE and hu782-HV-DE showed significantly better ADCC activity than the control antibody Ref.ch175, with EC50 values ​​of 6.87 ng / ml and 11.48 ng / ml, respectively (Figure 8C).

[0186] Example 16: Detection of CDC activity of humanized antibodies CDC is complement-dependent cytotoxicity, and its mechanism of action involves an antigen-antibody mixture bound to complement ultimately creating pores in the cell membrane, altering the cell's osmotic pressure, and ultimately leading to cytotoxicity. In this example, HEK293-hCLDN18.2 cells were used as target cells, complement was supplied using infant rabbit complement serum, and the antibody-CDC effect was detected using the CCK-8 cell proliferation-toxicity detection kit.

[0187] In Example 1, HEK293-hCLDN18.2 cells were used as target cells, and after digestion, 2.0 × 10⁶ cells were obtained. 5 The solution was diluted to a concentration of cells / mL and spread in a 96-well light-transmitting black culture plate at a volume of 100 μL / well. The cells were incubated statically for approximately 4 hours until they completely adhered to the wall. All of the growth medium was aspirated, and 50 μL / well of analytical medium was added. Diluted antibody was added to the reaction wells at a rate of 50 μL / well, followed by 50 μL of 1:15 diluted infant rabbit complement, until the final volume of each well reached 150 μL. The sample volume in each well was 1 / 3 of the total volume, the initial concentration was 3 times the target concentration, and the target concentration was 6 μg / mL. The cell plate was placed in a constant-temperature incubator and induced for 12 hours. Afterward, the medium was aspirated, fresh analytical medium was added at a rate of 100 μL / well, and 10 μL / well of CCK-8 solution was added at room temperature. The plate was then placed in a 5% CO2 incubator at 37°C, away from light, for 4 hours. The plate was set in a multifunction plate reader, and the OD value was detected at a wavelength of 450 nm. I used GraphPad Prism 8 software to fit the model, create a graph, and calculate the EC50 value.

[0188] The results are shown in the table below.

Table 17

[0189] The results showed that ch782, hu782, and hu782HV in the present invention were all able to induce a CDC effect equivalent to that of the reference antibody Ref.ch175 against HEK293-hCLDN18.2 cells (the EC50 values were 30.9 ng / ml, 49.46 ng / ml, 47.58 ng / ml, and 40.89 ng / ml, respectively).

[0190] Example 17 Detection of the growth inhibitory effect of a humanized antibody against HEK293-hCLDN18.2 cells HEK293-hCLDN18.2 cells in the logarithmic growth phase were taken. The target cells were digested, counted, and then diluted to 1.0×10 5 cells / mL. They were seeded in a 96-well optical transmission black culture plate at a volume of 100 μL / well and statically cultured for about 4 h until the cells completely adhered to the wall. After diluting the antibody to 100 μL / well in the reaction wells, with a final volume of 200 μL / well in each well, the 96-well plate was placed in a 5% CO2 incubator and induced to culture at 37 °C for 96 h. Then, the medium was aspirated, 100 μL / well of fresh analysis medium was added, and at room temperature, CCK-8 solution was added at 10 μL / well. It was placed in a 5% CO2 incubator and reacted at 37 °C in the dark for 4 h. It was set on a multifunctional plate reader to detect the OD value at a wavelength of 450 nm. The result was calculated by the cell growth inhibition rate = 1 - (OD450 抗体 / OD450 ブランク ).

[0191] From the results, the antibody hu782 of the present invention has a better growth inhibitory effect on target cells at a low concentration (1 μg / ml) than the reference antibody Ref.ch175 at a high concentration (30 μg / ml) (18.3% vs. 7.3%), and shows a dose-dependent tendency that the growth inhibitory effect becomes stronger as the antibody concentration increases. At a concentration of 30 μg / ml, the cell growth inhibition rates of ch782, hu782, and hu782-HV reached 67.8%, 76%, and 63.2% respectively, indicating that they were significantly better than the reference antibody Ref.ch175.

[0192] Example 18: Analysis of the binding site of the humanized antibody to hCLDN18.2 protein In order to explore the important amino acid sites that bind to the hCLDN18.2 protein but not to the hCLDN18.1 protein of the antibody in the present invention, in this example, the amino acid difference sites between hCLDN18.2 ECL1 and hCLDN18.1 ECL1 and the surrounding amino acids in the pEFGP-N2-hCLDN18.2 plasmid constructed in Example 1 were mutated to alanine by means of point mutation. Here, A42 was mutated to the corresponding amino acid S in hCLDN18.1 ECL1 (Q29A, S31A, N37A, A42S, N45A, Q47A, G48A, S52A, E56A, G65A, L69A, G71A). The mutated plasmid and the wild-type plasmid were respectively transfected into 293Fv cells by the method in Example 1, and 48 hours later, the cells were taken for flow cytometry detection.

[0193] hu782, Ref.ch175, and isotype control (IsotypecontrolhIgG1) were diluted to 10 μg / ml and incubated with transfected cells of at least 10 μg / ml on ice for 1 hour. After washing twice with FACS buffer, sheep anti-mouse IgG-PE secondary antibody diluted 1:400 was added and incubated on ice for 45 minutes. After washing twice with FACS buffer, 300 μl of the preparation buffer was added, and the cells were transferred to a BD flow cytometry tube and analyzed using BD FACS calibur. The median fluorescence intensity (MFI) results were analyzed using Flowjo software. In the evaluation of the results, if the MFI of transfected cells after mutation decreased by more than 50% compared to the wild type, that site was considered an important amino acid site affecting antibody binding.

[0194] The results, as shown in Figure 11, showed that the A42S, N45A, and E56A mutations of the present invention had a significant effect on the binding of the reference antibody Ref.ch175. However, the A42S mutation had no effect on the binding of hu782, the N45A mutation had a small effect on the binding of hu782, and the E56A mutation had a significant effect on the binding of hu782. At the same time, surprisingly, the present invention found that when the same amino acid site G48A in hCLDN18.2 ECL1 and hCLDN18.1 ECL1 was mutated, neither the reference antibody Ref.ch175 nor hu782 could bind.

[0195] Example 19: Evaluation of the in vivo activity of humanized antibodies In this example, a CDX animal model was constructed by subcutaneous transplantation of tumor cells into BALB / c nude mice, and the in vivo efficacy of humanized antibodies was evaluated.

[0196] Expanded culture was performed using the NUGC4-hCLDN18.2 stable translocation cell line constructed by the method of Example 1. Female SPF-grade BALB / c nude mice aged 4-6 weeks were selected, and after 3-4 days of adaptive rearing, tumor inoculation was prepared. Recombinant NUGC4-hCLDN18.2 cells in the logarithmic growth stage were digested, washed twice with serum-free 1640 medium, and then cultured in 2.5 × 10⁶ cells.7 Dilute to the desired concentration of / ml and administer 0.2ml subcutaneously under the right armpit of each mouse, for a total of 5 × 10 6 Individual cells / animals were inoculated. One week after inoculation, the tumors grew to 70-100 mm. 3 Once the mice grew, they were randomly divided into groups of 6 per group. The blank model group served as an IgG1 isotype control. In the experimental group, the drug was administered at a dose of 10 mg / kg twice a week, alternately by intraperitoneal and intravenous injection, and tumor volume and mouse body weight data were recorded before each administration.

[0197] The tumor volume is V(mm 3 )=(W 2 It is calculated as x (L) / 2, where W is the minor axis near the midline of the tumor and L is the major axis near the midline of the tumor. Tumor volume is 2000 mm 3 Mice were euthanized by cervical dislocation if they exceeded the limit, experienced a weight loss of more than 20%, or showed interference with important physiological functions or tumor ulceration / necrosis.

[0198] The formula for calculating the tumor suppression rate (TGI) is as follows:

number

[0199] In a NUGC4-hCLDN18.2 mouse subcutaneous tumor model, the antitumor growth activity of the humanized antibody and its mutants of the present invention was observed, and both showed some degree of efficacy. The tumor volumes of each group at the end of the study are shown in the table below. [Table 18]

[0200] In the humanized antibody hu782 and its mutants hu782-HV and hu782-DE, the tumor suppression rate was significantly higher than in the Ref.ch175 group. In particular, the tumor suppression rates reached 62.88% and 65.05% in the hu782 and its ADCC-enhancing mutant antibody hu782-DE groups, respectively. Furthermore, complete tumor regression was observed in one out of six mice in the hu782 group, and in two out of six mice in the hu782-DE group, but this phenomenon was not observed in either the Ref.ch175 group or the hu782-HV group.

[0201] The results above demonstrate that the antibody of the present invention (especially the humanized antibody) exhibits significantly better in vivo efficacy than the reference antibody Ref.ch175.

[0202] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the above, those skilled in the art will understand that various variations and modifications of the present invention may be made, and that equivalent forms thereof are included within the scope of the claims of the present invention.

Claims

1. An anti-human CLDN18.2 antibody comprising a heavy chain variable region containing the following three complementarity-determining regions (CDRs): (1) Complementarity-determining region CDR1, whose amino acid sequence is shown in SEQ ID NO: 3; (2) Complementarity-determining region CDR2, whose amino acid sequence is shown in SEQ ID NO: 4 or 16; and (3) Having a complementarity-determining region CDR3 whose amino acid sequence is shown in SEQ ID NO: 5 or 17; and a light chain variable region containing the following three complementarity-determining regions (CDR'): (1) Complementarity-determining region CDR1', whose amino acid sequence is shown in SEQ ID NO: 8; (2) Complementarity-determining region CDR2', whose amino acid sequence is shown in SEQ ID NO: 9; and (3) Complementarity-determining region CDR3', whose amino acid sequence is shown in SEQ ID NO: 10 The anti-human CLDN18.2 antibody, characterized by having the following characteristics.

2. The heavy chain variable region consists of the following three CDRs: (1) CDR1 indicated by Sequence ID 3, CDR2 indicated by Sequence ID 4, and CDR3 indicated by Sequence ID 5; or (2) CDR1 shown in Sequence ID No. 3, CDR2 shown in Sequence ID No. 16, and CDR3 shown in Sequence ID No. 5; or (3) The antibody according to claim 1, characterized in that it comprises CDR1 represented by SEQ ID NO: 3, CDR2 represented by SEQ ID NO: 4, and CDR3 represented by SEQ ID NO:

17.

3. The antibody according to claim 1, characterized in that the antibody has a heavy chain variable region indicated by SEQ ID NO: 1, 11, 12, or 13; and / or a light chain variable region indicated by SEQ ID NO: 6, 14, or 15.

4. (Z1) Heavy chain variable region indicated by Sequence ID 1 and light chain variable region indicated by Sequence ID 6; (Z2) Heavy chain variable region shown in Sequence ID 11 and light chain variable region shown in Sequence ID 14; (Z3) Heavy chain variable region shown in Sequence ID 11 and light chain variable region shown in Sequence ID 15; (Z4) Heavy chain variable region shown in Sequence ID No. 12 and light chain variable region shown in Sequence ID No. 14; (Z5) Heavy chain variable region shown in Sequence ID 12 and light chain variable region shown in Sequence ID 15; (Z6) Heavy chain variable region shown in Sequence ID 13 and light chain variable region shown in Sequence ID 14; or (Z7) The antibody according to claim 1, having a heavy chain variable region indicated by SEQ ID NO: 13 and a light chain variable region indicated by SEQ ID NO:

15.

5. The antibody according to claim 1, having a heavy chain indicated by SEQ ID NO: 18, 20, 21, or 22.

6. The antibody according to claim 1, having the light chain indicated by Sequence ID No.

19.

7. Recombinant protein, (i) an anti-human CLDN18.2 antibody according to any one of claims 1 to 6, and (ii) Any tag sequence to aid in expression and / or purification The protein characterized by having the following properties.

8. It is an antibody preparation, (a) an anti-human CLDN18.2 antibody according to any one of claims 1 to 6, and (b) A vehicle containing a buffer, sterile water, and any surfactant The antibody preparation characterized by containing the above.

9. A CAR construct, wherein the scFv fragment of the antigen-binding region of the CAR construct is a binding region that specifically binds to CLDN18.2, and the scFv has a heavy chain variable region and a light chain variable region of the anti-human CLDN18.2 antibody described in claim 1.

10. Recombinant immune cells characterized by expressing the CAR construct described in claim 9 as an external agent.

11. An antibody-drug conjugate, (a) an antibody portion comprising the anti-human CLDN18.2 antibody described in any one of claims 1 to 6, (b) A complex portion which is combined with the antibody portion, selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof. The antibody-drug conjugate characterized by containing the following:

12. The use of an active ingredient, wherein the active ingredient is an anti-human CLDN18.2 antibody according to any one of claims 1 to 6, and the active ingredient is (a) Manufacturing of detection reagents or kits, (b) manufacture of drugs or formulations for the prevention and / or treatment of CLDN18.2-related diseases, and / or (c) manufacture of drugs or formulations that prevent and / or treat cancer or tumors The use described above is characterized by being used for the purpose of.

13. A drug composition, (i) an active ingredient which is an anti-human CLDN18.2 antibody according to any one of claims 1 to 6, (ii) A pharmaceutically acceptable carrier and The composition characterized by containing the following.

14. A polynucleotide characterized by encoding an anti-human CLDN18.2 antibody according to any one of claims 1 to 6.

15. A vector characterized by comprising the polynucleotide described in claim 14.

16. A genetically modified host cell, wherein the host cell contains the vector described in claim 15.

17. A method for detecting the CLDN18.2 protein in a sample in vitro, comprising the following steps: (1) Contacting the sample with the anti-human CLDN18.2 antibody described in any one of claims 1 to 6 in vitro; (2) Detect whether an antigen-antibody complex has been formed. Here, the formation of a complex means that the CLDN18.2 protein is present in the sample. The method, characterized by including the following:

18. A detection plate comprising a substrate (support plate) and a test strip, wherein the test strip contains the antibody described in claim 1.

19. A kit for detecting CLDN18.2 protein in a sample in vitro, (1) A first container containing the anti-human CLDN18.2 antibody according to any one of claims 1 to 6, and (2) A second container containing a secondary antibody against the anti-human CLDN18.2 antibody. The kit, characterized by containing the following.

20. A composition for use in a method for preventing and / or treating CLDN18.2-related disease, characterized by comprising an anti-human CLDN18.2 antibody according to any one of claims 1 to 6, an antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof.

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