Anti-CLDN18_2 antibody and in vitro diagnostic kit

Abstract

This disclosure relates to a kit comprising an anti-claudin 18.2 antibody useful for evaluating the expression of claudin 18.2 in human biological samples, for example, from human patients with cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to China Patent Application No. 202310675165.8, filed on 7 June 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated in its entirety herein by reference. A copy of the sequence listing was created on 4 June 2024, named 025471.WO017.xml, and is 10,134 bytes in size. [Background technology]

[0003] Gastric cancer is one of the most common cancers worldwide. In 2020 alone, there were 479,000 new cases of gastric cancer and 374,000 deaths in China. While the five-year survival rate for gastric cancer in China rose from 27.4% in 2003 to 35.1% in 2015, it remains significantly lower than in other countries / regions (Chinese Guidelines for Screening, Early Diagnosis and Treatment of Gastric Cancer (2022), National Cancer Center of China). These statistics highlight the urgent need for improved diagnosis and treatment of gastric cancer.

[0004] Antibody drugs have been widely used to treat cancer. Antibody-based targeted therapies offer higher specificity and fewer side effects compared to traditional cancer treatments such as chemotherapy. Claudin 18.2 (CLDN18.2 or CLDN18_2) has been found to be a promising antibody target for the treatment of gastric and esophageal cancer (Singh et al., J Hematol Oncol. (2017) 10(1):105). It is also being studied as an antibody target for pancreatic cancer. Currently, several anti-CLDN18.2 monoclonal antibodies, including zolbetuximab (a chimeric antibody) and ASKB589 (a humanized antibody), are in clinical development.

[0005] CLDN18.2 belongs to the family of claudins, proteins that constitute tight junctions between cells. Tight junctions determine the permeability of epithelial cells and inhibit the diffusion of cell surface proteins and lipids. Claudin 18, i.e., members of the family, are encoded by the claudin 18 gene. The human claudin 18 gene has two distinct exon 1s, which undergo alternative splicing during transcription, ultimately resulting in two isoforms with different N-terminal sequences: claudin 18.1 and claudin 18.2. The expression of claudin 18.1 and claudin 18.2 in humans is tissue-specific. Claudin 18.1 is mainly expressed in lung tissue but not in gastric tissue or gastric cancer. Claudin 18.2 is a tight junction molecule mainly found in normal gastric epithelium, allowing it to approach the surface of cancer cells that become targets for antibodies during malignant transformation. Due to its expression in various epithelial cancers and its highly restricted expression pattern in normal tissues, claudin 18.2 is a target for the development of treatments for epithelial cancers.

[0006] To improve the efficacy of cancer drugs targeting claudin 18.2, it is necessary to evaluate claudin 18.2 expression in cancer patients in order to appropriately guide the use of these drugs. [Overview of the project]

[0007] In a first aspect, the present disclosure provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. The anti-CLDN18.2 antibody comprises (1) an antibody heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and (6) an antibody light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0008] In some embodiments, the anti-CLDN18.2 antibody includes an antibody heavy chain variable region having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 7, and an antibody light chain variable region having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 8.

[0009] In some embodiments, the anti-CLDN18.2 antibody is referred to as the DS-3 antibody in this disclosure.

[0010] In some embodiments, the anti-CLDN18.2 antibody comprises an antibody heavy chain having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 9, and an antibody light chain having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 10.

[0011] In a second aspect, the Disclosure provides an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in the first aspect of the Disclosure.

[0012] In a third aspect, the Disclosure provides a recombinant expression vector containing the nucleic acid described in the second aspect of the Disclosure.

[0013] In a fourth aspect, the disclosure provides an in vitro diagnostic kit containing an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. The anti-CLDN18.2 antibody comprises (1) an antibody heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and (6) an antibody light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0014] In some embodiments, the anti-CLDN18.2 antibody includes an antibody heavy chain variable region having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 7, and an antibody light chain variable region having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 8.

[0015] In some embodiments, the anti-CLDN18.2 antibody comprises an antibody heavy chain having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 9, and an antibody light chain having 90% or more similarity (e.g., 90% or more, or 95% or more, or 98% or more, or 99% or more) to the amino acid sequence shown in SEQ ID NO: 10.

[0016] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment is bound to at least one detectable marker.

[0017] In some embodiments, the anti-CLDN18.2 antibody preparation of the antibody diagnostic kit includes the anti-CLDN18.2 antibody, a phosphate buffer solution, sodium chloride, a surfactant, bovine serum albumin, and a preservative, and has a pH between 7 and 8.

[0018] In some embodiments, the anti-CLDN18.2 antibody preparation of the antibody diagnostic kit 0.05 - 0.2 μg / mL of the anti-CLDN18.2 antibody which is the antibody 0.05 - 0.2 M phosphate buffer solution, 0.05 - 0.2 M sodium chloride, a surfactant, 0.5 - 2% (w / v) bovine serum albumin, and a preservative, and has a pH between 7 and 8.

[0019] In some embodiments, the anti-CLDN18.2 antibody preparation of the antibody diagnostic kit includes the anti-CLDN18 antibody: 0.1 μg / mL of the anti-CLDN18.2 antibody which is the antibody 0.1 M phosphate buffer solution, 0.05 - 0.06 M sodium chloride, a surfactant which is 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol at a concentration of approximately 0.5% (v / v), 1.0% (w / v) bovine serum albumin, and a preservative, and has a pH between 7.2 and 7.5.

[0020] In some embodiments, the anti-CLDN18.2 antibody preparation of the antibody diagnostic kit includes the anti-CLDN18 antibody: 0.1 μg / mL of the anti-CLDN18.2 antibody which is the antibody 0.1 M phosphate buffer solution, 0.05 - 0.06 M sodium chloride, a surfactant which is Triton X-100 at a concentration of approximately 0.5% (v / v), 1.0% (w / v) bovine serum albumin, and, ProClin at a concentration of approximately 0.05% (v / v) 商標 It has a pH of 300, contains preservatives, and has a pH between 7.2 and 7.5.

[0021] In a fifth aspect, the Disclosure provides a method for detecting a sample using an in vitro diagnostic kit described in a fifth aspect of the Disclosure.

[0022] In some embodiments, the method detects claudin 18.2 (CLDN18.2) in biopsy samples obtained from humans (e.g., human cancer patients); the method comprises contacting the biopsy sample with an anti-CLDN18.2 antibody or antigen-binding fragment as defined herein, and detecting the binding of the antibody or antigen-binding fragment to the biopsy sample, the presence of which indicates the presence of CLDN18.2 in the biopsy sample.

[0023] In some embodiments, the method diagnoses cancer in humans; the method comprises contacting a biopsy sample derived from a human with the anti-CLDN18.2 antibody or antigen-binding fragment described herein, and detecting the binding of the antibody or antigen-binding fragment to the biopsy sample, the presence of which indicates the presence of cancer in the human.

[0024] In some embodiments, a method for detecting a sample includes: (i) immobilizing the sample on a solid base; (ii) contacting the sample with an anti-CLDN18.2 antibody or its antigen-binding fragment contained in an in vitro diagnostic kit according to a fifth aspect of the present disclosure; (iii) contacting the anti-CLDN18.2 antibody or its antigen-binding fragment with a detectable marker; and (iv) determining the binding level of the anti-CLDN18.2 antibody or its antigen-binding fragment to the sample. The detectable marker is used to label the anti-CLDN18.2 antibody or its antigen-binding fragment.

[0025] In some embodiments, a method is provided for detecting a sample, which includes: (i) immobilizing the sample on a solid base; (ii) contacting the sample with an anti-CLDN18.2 antibody or its antigen-binding fragment contained in an in vitro diagnostic kit according to a fifth aspect of the present disclosure; and (iii) determining the binding level of the antibody or its antigen-binding fragment to the sample via a detectable marker. The anti-CLDN18.2 antibody or its antigen-binding fragment binds to at least one detectable marker.

[0026] In some embodiments, the detectable marker is horseradish peroxidase.

[0027] In some embodiments, the sample is a tissue section.

[0028] In some embodiments, the tissue is selected from the pancreas, esophagus, ovaries, lungs, stomach, bronchi, mammary glands, or ears, nose, and throat.

[0029] In some embodiments, the tissue is selected from tumors of gastric cancer, pancreatic cancer, biliary tract cancer, or esophageal cancer.

[0030] In some embodiments, the tissue is selected from tumors located in the pancreas, esophagus, ovaries, lungs, stomach, bronchi, mammary glands, large intestine, biliary tract, or throat.

[0031] In some embodiments, the solid base is a glass slide.

[0032] In a sixth aspect, the Disclosure provides a method for treating cancer in a person in need; the method comprises detecting the presence of CLDN18.2 in a biopsy sample obtained from a person by the detection method, and, if CLDN18.2 is found to be present in the biopsy sample, treating the patient with an anti-CLDN18.2 antibody drug.

[0033] Those skilled in the art may readily determine the CDR sequence based on the sequence of the heavy chain or light chain variable region. To define the CDR on the antibody variable region, an antibody numbering scheme and a definition scheme are required. Antibody numbering schemes include, but are not limited to, IMGT, Chothia, Kabat, and Martin (an extension of Chothia), and definition schemes include, but are not limited to, Chothia, Kabat, IMGT, and Contact.

[0034] This disclosure provides an anti-CLDN18.2 antibody or its antigen-binding fragment, and an in vitro diagnostic kit containing the anti-CLDN18.2 antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment has high binding activity to the CLDN18.2 protein. The in vitro diagnostic kit containing the antibody or its antigen-binding fragment has simplicity and convenience of operation, high detection sensitivity, and storage stability.

[0035] Other features, purposes, and advantages of the present invention are revealed in the detailed description below. However, the detailed description illustrates embodiments and aspects of the present invention, but should be understood to be for illustrative purposes only and not limiting. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0036] [Figure 1-1] Figures 1A and 1B show a comparison of the performance of antibody DS-3 and another antibody (mouse antibody) in staining of gastric cancer tissue sections. In the tissue sections shown, the DS-3 antibody showed more pronounced claudin 18.2 staining compared to the mouse anti-CLDN18.2 antibody (lot number PW03-33) (Figure 1B).

[0037] [Figure 1-2] Figures 1C and 1D show a comparison between the DS-3 antibody and the rabbit antibody.

[0038] In human gastric cancer tissue sections, the DS-3 antibody showed more pronounced claudin 18.2 coloration compared to the rabbit-derived anti-CLDN18.2 antibody (AbCam, clone number EPR19202-244) (Figure 1D) (Figure 1C). [Modes for carrying out the invention]

[0039] Detailed explanation This disclosure provides a method for detecting the presence of claudin 18.2 expression in human patients (e.g., cancer patients) and a companion diagnostic kit for treating cancer patients with anti-claudin 18.2 antibody drugs. The companion diagnostic may be used to select or exclude patients to be treated with a particular treatment based on biological characteristics that determine whether they are likely to be responders or non-responders to the treatment. The detection method and kit may assist in guiding anti-CLDN18.2 cancer treatment and improve the effectiveness of such treatment.

[0040] The CLDN18.2 detection antibodies or antigen-binding fragments provided herein are significantly more sensitive and specific in detecting CLDN18.2 in tissue samples. These antibodies and fragments may be used, for example, in immunohistochemical staining of tissue sections obtained from patients (e.g., paraffin sections of biopsy samples from patients with various cancers such as gastric cancer, gastroesophageal junction cancer, and other epithelial cancers).

[0041] In this disclosure, the terms “homology” or “identity” in relation to sequence alignment refer to the proportion (e.g., percentage) of similar or identical amino acids between the query sequence and the reference sequence over the entire length of the reference sequence. Similarity in amino acid sequence alignment includes two residues at corresponding positions having similar properties, such as size, charge, and hydrophilicity of side chain groups, as well as identical residues. Methods for sequence alignment are common technical knowledge in the art, such as BLAST. (登録商標)It may be executed by a program.

[0042] In this disclosure, the terms “Claudin 18” and “CLDN18” are used interchangeably within this specification. The terms “Claudin 18.1,” “CLDN18.1,” and “CLDN18_1” are used interchangeably within this specification, and the terms “Claudin 18.2,” “CLDN18.2,” and “CLDN18_2” are also used interchangeably within this specification.

[0043] In this disclosure, “mass-volume percentage” or “%(w / v)” or “%w / v” is an expression of mass-volume concentration, representing the grams of solute contained in 100 ml of solution. For example, 20%(w / v) means that there are 20 g of solute per 100 ml of solution.

[0044] I. Kit for evaluating claudin 18.2 expression in vitro This disclosure provides kits for in vitro evaluation of claudin 18.2 expression in biological samples obtained from human patients, for example, human patients who have or are suspected of having cancer. These kits, which may hereafter be referred to as in vitro diagnostic kits, comprise an anti-CLDN18.2 antibody or antigen-binding fragment and a detectable substance that is or should be bound to the antibody or fragment. The kits may also comprise a standard and / or control. The kits may include instructions on how to perform in vitro detection of CLDN18.2 in a biological sample, and may further include instructions on how to use CLDN18.2 expression information to guide treatment. The kits may be used in the process of monitoring hyperplasia in human patients, in the process of diagnosing cancer and predicting prognosis, in the process of monitoring disease progression and treatment, and in the process of evaluating disease status. The kits may be used alone or in combination with other instruments and systems.

[0045] A. Anti-CLDN18.2 antibody The CLDN18.2 detection substance is an anti-CLDN18.2 antibody or its antigen-binding fragment. The antibody or fragment may include one, two, three, four, five, or six complementarity-determining regions (CDRs), heavy chain variable regions (VH) and / or light chain variable regions (VL) of the anti-CLDN18.2 DS-3 antibody described herein, or the heavy chain and / or light chain.

[0046] The delimitation of CDR numbers is known in the art and is incorporated herein. Those skilled in the art can readily determine the CDR of a given delimitation based on the sequence of the heavy chain or light chain variable region. The "Kabat" CDR is commonly used based on sequence diversity (Kabat et al., Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "Chothia" CDR refers to the position of the structural loop (Chothia & Lesk, J Mol Biol. (1987) 196:901-917). The "AbM" CDR is intermediate between the Kabat CDR and the Chothia structural loop and is used in Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on the analysis of available complex crystal structures. Referencing a common antibody numbering scheme, each residue of these CDRs is listed in Table 1 below. Unless otherwise specified herein, amino acid numbers in antibodies refer to the Kabat numbering scheme described above by Kabat et al., including cases where the delimitation of the CDR is performed by referring to the Kabat, Chothia, AbM, or Contact scheme. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids in response to shortening or insertion of the CDR in the framework region (FR) or variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after H2 residue 52 (residue 52a by Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c by Kabat). The Kabat numbering of residues may be determined for a given antibody by aligning the antibody sequence with the “standard” Kabat numbering sequence in regions of homology. Table 1. CDR delimitation using various schemes [Table 1]

[0047] In some embodiments, the CDR is an "extended CDR" encompassing regions that begin or end in different schemes. For example, an extended CDR could be: L24-L36, L26-L34, or L26-L36 (VL-CDR1); L46-L52, L46-L56, or L50-L55 (VL-CDR2); L91-L97 (VL-CDR3); H47-H55, H47-H65, H50-H55, H53-H58, or H53-H65 (VH-CDR2); and / or H93-H102 (VH-CDR3).

[0048] In some embodiments, the anti-CLDN18.2 antibody or fragment described herein comprises heavy chain complementarity-determining regions (CDRs) 1-3 described in SEQ ID NOs: 1-3 and / or light chain CDRs 1-3 described in SEQ ID NOs: 4-6, respectively.

[0049] In some embodiments, the anti-CLDN18.2 antibody or fragment herein comprises a VH having an amino acid sequence of SEQ ID NO: 7 or at least 90% identical thereto, and / or a VL having an amino acid sequence of SEQ ID NO: 8 or at least 90% identical thereto. In further embodiments, the antibody or fragment comprises a VH having the amino acid sequence of SEQ ID NO: 7 and a VL having the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antibody may be a mouse IgG1, IgG2, IgG3, or IgG3 isotype.

[0050] In some embodiments, the anti-CLDN18.2 antibody or fragment described herein comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 9 or at least 90% identical thereto, and / or a light chain containing an amino acid sequence of SEQ ID NO: 10 or at least 90% identical thereto. In further embodiments, the antibody is a DS-3 antibody having the heavy chain and light chain sequences of SEQ ID NO: 9 and 10, respectively.

[0051] B. Detectable Markers In some embodiments, the kits of this disclosure provide a detectable marker. For example, the presence of CLDN18.2 in a sample can be detected by an anti-CLDN18.2 antibody or fragment as specified herein, labeled with a fluorescent moiety, metal, or enzyme. In some embodiments, the detection reagent may be contained in or on the tissue collection device (e.g., an antibody or indicator embedded on a test strip or glass slide). In other embodiments, the detection reagent is provided separately from the collection device as part of the kit.

[0052] In some embodiments, the detectable marker is used either by directly binding to an anti-CLDN18.2 antibody or fragment for direct detection, or in conjunction with a second reagent (such as an antibody that recognizes the anti-CLDN18.2 antibody or fragment and is labeled with the detectable marker) for indirect detection. In other words, the anti-CLDN18.2 antibody or fragment may be directly or indirectly bound to the detectable marker.

[0053] In some embodiments, the detectable marker is a chromogenic reagent using a colored enzyme substrate or a fluorescent substance. Non-limiting examples of chromogenic reagents include horseradish peroxidase or alkaline phosphatase, both of which can catalyze a color reaction in the presence of a chromogenic substrate such as diaminobenzidine (DAB). Non-limiting examples of fluorescent reagents include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), aminomethylcoumarin acetate (AMCA), cyanine 5 (Cy5), or Alexa Fluor (登録商標) Fluorescent dyes are included. Chromogenic, fluorescent, and chemiluminescent reagents are widely known in this field and are available (e.g., from companies such as Thermo Fisher Scientific and AbCam).

[0054] C. Antibody composition In some embodiments, the kit provides the antibody or fragment in a dry powder form (e.g., lyophilized) which can then be reconstituted in a buffered aqueous solution optionally included in the kit to produce an anti-CLDN18.2 antibody or fragment preparation for use. In other embodiments, the kit provides the antibody or fragment in a buffered aqueous solution or preparation.

[0055] The aqueous solution may have a pH of about 7 to 8 and may contain phosphate buffer solution, sodium chloride, surfactant, albumin (e.g., bovine serum albumin), and a preservative. In some embodiments, the aqueous solution contains 0.01–0.5 μg / mL of anti-CLDN18.2 antibody or fragment.

[0056] In some embodiments, a 0.01–0.5 M phosphate buffer solution is used in aqueous solution. The phosphate buffer solution may be prepared by combining disodium hydrogen phosphate and sodium dihydrogen phosphate; for example, the combination of disodium hydrogen phosphate and sodium dihydrogen phosphate may be used in a ratio of 1:1, 2:1, or 1:2. The buffer solution may further contain sodium chloride at a concentration of, for example, 0.01–0.5 M.

[0057] In some embodiments, the surfactant is used in an aqueous solution. Non-limiting examples of surfactants include Triton 商標 X-100 (Sigma Aldrich; CAS No.: 9036-19-5; 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol) is included.

[0058] In some embodiments, 0.1-5% (w / v) bovine serum albumin is used in an aqueous solution. In some embodiments, the aqueous solution has a pH of about 6-9. In certain embodiments, the anti-CLDN18.2 antibody is present at a pH of about 7-8, or in more specific embodiments, the anti-CLDN18.2 antibody is present at a pH of about 7-7.5, such as 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0059] In some embodiments, the aqueous solution contains a preservative or bacteriostatic substance such as ProClin 商標 300 at a concentration of, for example, about 0.05% (v / v). ProClin 商標 300 (Sigma Aldrich) contains 3% 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT) in a glycol free of salts containing alkyl carboxylic acid stabilizing substances. It has a recommended working pH range of 2.5 - 8.5.

[0060] As an example, the aqueous solution in which the antibody or fragment is provided or reconstituted contains 0.05 - 0.2 μg / mL of anti-CLDN18.2 antibody, 0.05 - 0.2 M phosphate buffer solution, 0.05 - 0.2 M sodium chloride, a surfactant, 0.5 - 2% (w / v) bovine serum albumin, and a preservative, and the aqueous solution has a pH of about 7 - 8. In some embodiments, the aqueous solution contains 0.1 μg / mL of anti-CLDN18.2 antibody, 0.1 M phosphate buffer solution, 0.05 - 0.06 M sodium chloride, Triton 商標 X-100 at a concentration of approximately 0.5% (v / v) as a surfactant, 1.0% (w / v) bovine serum albumin, and a preservative, and has a pH of about 7 - 7.5, preferably 7.2 - 7.5. In further embodiments, the aqueous solution contains 0.1 μg / mL of anti-CLDN18.2 antibody, 0.1 M phosphate buffer solution, 0.05 - 0.06 M sodium chloride, Triton 商標 X-100 at a concentration of approximately 0.5% (v / v) as a surfactant, 1.0% (w / v) bovine serum albumin, and ProClin 商標 300 at a concentration of approximately 0.05% (v / v), and the aqueous solution has a pH of about 7 - 7.5, preferably 7.2 - 7.5.

[0061] D. Other Kit Components In some embodiments, the kit provides means or devices for collecting biological samples, such as one or more test tubes, syringes, glass slides, test strips, and containers for storing biological samples. It may also include any other devices known in the art for collecting and processing biological samples, such as biopsy samples.

[0062] In some embodiments, the kit may further include a device for quantifying or semi-quantifying CLDN18.2 expression in a biological sample. Alternatively, such quantification may be performed by means outside the kit. Non-limiting examples of such means include microscopes, colorimeters, spectrometers, and electromagnetic frequency spectrometers (e.g., UV-VIS, IR, or NMR).

[0063] The kit may include instructions for obtaining, processing, and staining biological samples obtained from human patients. In some embodiments, the kit further includes instructions for comparing the level of CLDN18.2 expression in the biological sample to a reference level.

[0064] II. How to use This kit and method may be used to detect CLDN18.2 (e.g., its presence and / or level) in biological samples (e.g., tissue biopsy samples) taken from subjects (e.g., human patients) who are known to have or suspected to have cancer (e.g., epithelial carcinoma). In some embodiments, the subject has gastric cancer or gastroesophageal junction cancer. In some other embodiments, the cancer is of the ear, nose, throat, esophagus, stomach, pancreas, biliary tract, liver, colorectal, ovarian, mammary gland, lung, bronchus, or skin.

[0065] Biological samples may be taken from tissue containing or suspected of containing cancer cells. For example, a biological sample may be a biopsy sample taken from peripheral or internal tissue. Biological samples may be taken from, for example, the ear, nose, throat, esophagus, stomach, pancreas, biliary tract, liver, colon, ovary, mammary gland, lung, bronchi, or skin. A biological sample may be a liquid biopsy sample, which may contain cancer cells (e.g., circulating tumor cells). Examples include plasma, whole blood (e.g., dried blood spots), and serum.

[0066] As described above, claudin 18.2 is a tightly bound molecule primarily found in non-malignant gastric epithelium, which becomes accessible to the tumor cell surface during malignant transformation. Therefore, subjects screened or diagnosed with cancer according to this method may express detectable claudin 18.2 on the surface of cancer cells, in contrast to normal cells in the same tissue. In some embodiments, cancer cells express 2-5%, 5-10%, 5-20%, 5-30%, 5-40%, or at least 50% more CLDN18.2 than normal cells in humans (e.g., adult, adolescent, and pediatric patients).

[0067] As an example, the present disclosure provides a method for detecting CLDN18.2 expression in a biological sample obtained from a human patient with cancer, which includes: immobilizing the biological sample on a solid substrate; contacting the biological sample with an aqueous solution of a kit disclosed herein comprising an anti-CLDN18.2 antibody; contacting the biological sample with a signal-generating reagent that enables the detection of the anti-CLDN18.2 antibody bound to the solid substrate; and determining the presence or level of CLDN18.2 binding by the antibody.

[0068] In some embodiments, a biopsy sample (e.g., a cancer biopsy sample) is embedded in a paraffin section for IHC staining with Claudine 18.2. In some embodiments, the biopsy sample is sectioned and fixed onto a solid base (e.g., a glass slide) for staining. After staining is complete, the slide may be dehydrated and mounted using a microtome apparatus available in the art to produce high-quality sections. Non-limiting examples of such apparatus include the Dakewe fully automated staining and mounting machine. IHC apparatuses are well known in the art; non-limiting examples include the Leica Bond 商標 III is a fully automated IHC (Intravascular Cooperative Hydraulics) system.

[0069] If cancer is determined to be CLDN18.2 positive, the patient may be treated with anti-CLDN18.2 immunotherapy. One such example is immunotherapy using zolbetuximab. Another example is immunotherapy using AKSB589.

[0070] III. Exemplary Embodiments To better understand the present invention, exemplary embodiments are described below. These embodiments are illustrative and should be construed in any way as limiting the scope of the invention. 1. An in vitro diagnostic kit containing an anti-CLDN18.2 antibody or its antigen-binding fragment, wherein the anti-CLDN18.2 antibody comprises the following: (1) Antibody heavy chain CDR1 having the amino acid sequence shown in Sequence ID No. 1, (2) Antibody heavy chain CDR2 having the amino acid sequence shown in Sequence ID No. 2, (3) Antibody heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (4) Antibody light chain CDR1 having the amino acid sequence shown in Sequence ID No. 4, (5) an antibody light chain CDR2 having the amino acid sequence shown in Sequence ID No. 5, and (6) An antibody light chain CDR3 having the amino acid sequence shown in Sequence ID No. 6. 2. An in vitro diagnostic kit according to Embodiment 1, comprising the anti-CLDN18.2 antibody as follows: A variable region of the antibody heavy chain having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 90% or more similarity to the amino acid sequence shown in SEQ ID NO: 7, and A variable region of an antibody light chain having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 90% or more similarity to the amino acid sequence shown in SEQ ID NO: 8. 3. An in vitro diagnostic kit according to Embodiment 1 or 2, wherein an anti-CLDN18.2 antibody or its antigen-binding fragment is bound to at least one detectable marker. 4. An in vitro diagnostic kit according to Embodiment 1 or 2, wherein the anti-CLDN18.2 antibody preparation comprises the anti-CLDN18.2 antibody, phosphate buffer solution, sodium chloride, surfactant, bovine serum albumin, and preservative, and has a pH between 7 and 8. 5. Anti-CLDN18.2 antibody preparations, The aforementioned antibody, anti-CLDN18.2 antibody, is present in a concentration of 0.05-0.2 μg / mL. 0.05-0.2 M phosphate buffer solution, 0.05-0.2M sodium chloride, surfactant, 0.5-2% (w / v) bovine serum albumin, and preservative An in vitro diagnostic kit according to Embodiment 1 or 2, comprising and having a pH between 7 and 8. 6. Anti-CLDN18.2 antibody preparations The aforementioned antibody, anti-CLDN18.2 antibody, is present in a concentration of 0.1 μg / mL. 0.1M phosphate buffer solution, 0.05-0.06 M sodium chloride, The surfactant is 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol at a concentration of approximately 0.5% (v / v). 1.0% (w / v) bovine serum albumin, and preservative An in vitro diagnostic kit according to Embodiment 1 or 2, comprising and having a pH between 7.2 and 7.5. 7. Anti-CLDN18.2 antibody preparations, The aforementioned antibody, anti-CLDN18.2 antibody, is present in a concentration of 0.1 μg / mL. 0.1M phosphate buffer solution, 0.05-0.06 M sodium chloride, Trixton X-100 is a surfactant at a concentration of approximately 0.5% (v / v). 1.0% (w / v) bovine serum albumin, and A preservative containing Proclin 300 at a concentration of approximately 0.05% (v / v). An in vitro diagnostic kit according to Embodiment 1 or 2, comprising and having a pH between 7.2 and 7.5. 8. A method for detecting a sample, (i) Fixing the sample onto a solid base; (ii) Contacting a sample with an anti-CLDN18.2 antibody or its antigen-binding fragment contained in the in vitro diagnostic kit according to claim 1 or 2; (iii) Contacting an anti-CLDN18.2 antibody or its antigen-binding fragment with a detectable marker; and (iv) Determine the binding level of the anti-CLDN18.2 antibody or its antigen-binding fragment to the sample; A method comprising a detectable marker used to label an anti-CLDN18.2 antibody or its antigen-binding fragment. 9. A method for detecting a sample, (i) Fixing the sample onto a solid base; (ii) Contacting a sample with the anti-CLDN18.2 antibody or its antigen-binding fragment contained in the in vitro diagnostic kit described in claim 7; and (iii) Determining the binding level of the antibody or its antigen-binding fragment to the sample via a detectable marker. Methods that include... 10. The method according to Embodiment 8 or 9, wherein the detectable marker is horseradish peroxidase. 11. The method according to Embodiment 8 or 9, wherein the sample is a tissue section. 12. The method according to Embodiment 11, wherein the tissue is selected from the pancreas, esophagus, ovaries, lungs, stomach, bronchi, mammary glands, or ears, nose, and throat. 13. The method according to Embodiment 11, wherein the tissue is selected from tumors located in the pancreas, esophagus, ovaries, lungs, stomach, bronchi, mammary glands, biliary tract, large intestine, or ears, nose, and throat. 14. The method according to Embodiment 11, wherein the tissue is selected from tumors of gastric cancer, pancreatic cancer, biliary tract cancer, or esophageal cancer. 15. The method according to embodiment 8 or 9, wherein the solid base is a glass slide. 16. A method for detecting claudin 18.2 (CLDN18.2) in a biopsy sample obtained from a human, Contacting the biopsy sample with an anti-CLDN18.2 antibody or its antigen-binding fragment containing heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6, respectively; and A method comprising detecting the binding of the antibody or antigen-binding fragment to a biopsy sample, wherein the presence of binding indicates the presence of CLDN18.2 in the biopsy sample. 17. A method for diagnosing cancer in humans: Contacting a human-derived biopsy sample with an anti-CLDN18.2 antibody or its antigen-binding fragment containing heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6, respectively; and A method comprising detecting the binding of the antibody or antigen-binding fragment to a biopsy sample, wherein the presence of binding indicates the presence of cancer in a human. 18. The method of Embodiment 17, wherein the biopsy sample is taken from tissue that does not normally express detectable claudin 18.2, and optionally the tissue is tissue other than gastric epithelium. 19. The antibody or antigen-binding fragment A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and Light chain variable region containing an amino acid sequence of SEQ ID NO: 8 or at least 90% identical thereto A method from any one of embodiments 16 to 19, including the method described above. 20. The antibody or its antigen-binding fragment Heavy chains containing an amino acid sequence identical to or at least 90% of SEQ ID NO: 9, and Light chain containing the amino acid sequence of SEQ ID NO: 10 or at least 90% identical thereto A method from any one of embodiments 16 to 19, including the method described above. 21. Any one of embodiments 16 to 20, wherein an antibody or its antigen-binding fragment is linked to a directly or indirectly detectable marker. 22. The method of Embodiment 21, wherein the detectable marker is horseradish peroxidase. 23. Any one of Embodiments 16 to 22, wherein the biopsy sample is obtained from the ear, nose, throat, esophagus, stomach, pancreas, liver, biliary tract, large intestine, lung, bronchi, breast, or ovary. 24. Any one of the embodiments 16 to 23, wherein the biopsy sample is sectioned and fixed onto a solid base, optionally the solid base being a glass slide, before being brought into contact with the anti-CLDN18.2 antibody or fragment. 25. A kit comprising an anti-CLDN18.2 antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6. 26. The antibody or antigen-binding fragment A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and Light chain variable region containing an amino acid sequence of SEQ ID NO: 8 or at least 90% identical thereto A kit of embodiment 25, including the kit of embodiment 25. 27. The antibody or antigen-binding fragment Heavy chains containing an amino acid sequence identical to or at least 90% of SEQ ID NO: 9, and Light chain containing the amino acid sequence of SEQ ID NO: 10 or at least 90% identical thereto A kit of embodiment 25, including the kit of embodiment 25. 28. Any one of the kits from Embodiments 25 to 27, wherein the antibody or antigen-binding fragment is present in a phosphate-buffered solution comprising sodium chloride, a surfactant, bovine serum albumin, and a preservative, with a pH of 7 to 8. 29. The kit is Lyophilized antibody or antigen-binding fragment and A reconstituted phosphate buffer solution containing sodium chloride, surfactants, bovine serum albumin, and preservatives, with a pH of 7-8. A kit including any one of embodiments 25 to 27. 30. A method for treating cancer in a person who needs it, To detect the presence of CLDN18.2 in a biopsy sample obtained from a human using any one of the methods in Embodiments 15 to 24, and If CLDN18.2 is found to be present in the biopsy sample, the patient should be treated with an anti-CLDN18.2 antibody drug. Methods that include... 31. The method of Embodiment 15, wherein the biopsy sample is taken from tissue that does not normally express detectable claudin 18.2, and optionally the tissue is tissue other than gastric epithelium. 32. The method of Embodiment 30 or 31, wherein the anti-CLDN18.2 antibody drug is zolbetuximab or ASKB589.

[0071] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings generally understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of this disclosure, but exemplary methods and materials are listed below. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, unless the context requires otherwise, singular terms shall include plurals, and plural terms shall include singulars. Throughout this specification and the embodiments, variations of the words “have” and “comprise” or “has,” “having,” “comprises,” or “comprising” shall be understood to mean the inclusion of the integer or group of integers described, but not to exclude any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference as if each individual reference were specifically and individually indicated to be incorporated by reference in whole. Many documents are cited herein, but this citation does not constitute an endorsement that any of these documents form part of the common technical knowledge of the art. Where used herein, the terms “approximately” or “about” apply to one or more numerical values ​​and refer to values ​​similar to the given reference value. In certain embodiments, unless otherwise stated or the context makes otherwise clear, the terms refer to a range of values ​​that fall within either direction (greater than or less than) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than the given reference value.

[0072] According to this disclosure, forward references in dependent claims mean abbreviations for all combinations of claims indicated by each direct and unambiguous disclosure and forward reference. Any compound or kit disclosed herein can be used in any manner disclosed herein. Furthermore, headers herein are provided for ease of organization and are not intended in any case to limit the scope of the claimed inventions.

[0073] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Examples]

[0074] Example 1: Preparation of anti-CLDN18.2 antibody Female Balb / c mice aged 6-8 weeks were immunized with human claudin 18, and blood was collected one week after three immunizations. Serum titer was measured by indirect ELISA, and an OD-blank > 0.5 in 1000-fold diluted serum was used as the qualifying standard for titer. Mouse serum with qualifying titers was detected by immunohistochemistry (IHC). Mice with histochemical advantages were selected and boosted. Three to four days after boosting, the mice's spleens were removed, and hybridoma fusion was performed.

[0075] Hybridoma cell culture supernatants were subjected to an indirect ELISA binding assay to screen for hybridoma-positive cells. Hybridoma-positive clones were cultured, the supernatant was obtained, and subjected to ELISA and IHC assays. Cells that were double-positive in ELISA and IHC were selected and subjected to 1 to 2 limiting dilutions until stable positive cells were obtained. Using stable positive cells, purified anti-CLDN18.2 monoclonal antibody was further prepared. Antibody clone DS-3 was selected for further investigation. The amino acid sequence of DS-3 is shown in Table 2 (SEQ = sequence number). Table 2. Amino acid sequence of DS-3 [Table 2]

[0076] After determining the amino acid sequence of DS-3, an expression construct was created and transfected into suitable mammalian host cells (e.g., CHO cells) to express a DS-3 monoclonal antibody.

[0077] Example 2: Determination of DS-3 antibody binding activity The antigen-binding activity of DS-3 was evaluated by ELISA using a substrate coated with CLDN18.2 capture protein (5 μg / ml). The maximum detection concentration of the DS-3 antibody was set to 1.2 μg / ml, and the DS-3 antibody was diluted in a 3-fold gradient. Goat anti-mouse IgG was used as the secondary antibody at a dilution ratio of 1:1500. The data represent the EC of binding between DS-3 and CLDN18.2. 50 However, this indicates that the level was 3.59 ng / ml.

[0078] Example 3: Use of DS-3 as an in vitro diagnostic reagent Claudin 18.2 staining experiment using Leica Bond 商標 III. The procedure was performed using a fully automated immunohistochemistry apparatus, and the experimental steps were as follows: (i) Sample preparation: Place sections and quality control slides of the samples to be evaluated in an oven at 65±5°C and bake for 30-60 minutes; then remove the sections and allow them to cool to room temperature; (ii) Staining: The cooled sections were stained according to the following protocol: [Table 3] (iii) Washing and mounting: After the staining process was completed, the sections were removed from the staining solution and dehydrated and mounted using a Dakewe fully automated staining and mounting machine.

[0079] Bond 商標 The dewaxing solution is a commercially available product, and its medical device registration number is National Medical Device Registration No. 20140293.

[0080] Antigen recovery buffer solution (Bond 商標 Epitope Retrieval Solution 2) is a commercially available product with medical device registration number National Medical Device Preparation 20150327.

[0081] Bond 商標The cleaning solution is a commercially available product, and its medical device registration number is National Medical Device Registration No. 20150492.

[0082] Immunochromogenic reagent (Bond 商標 Polymer Refine Detection is a commercially available product with medical device registration number National Medical Device Preparation No. 20150558; it contains peroxide blocking solution, primary antibody post-reagent (containing rabbit anti-mouse IgG), polymer (containing anti-rabbit polyHRP-IgG), DAB reagent 1 (DAB Part 1), DAB reagent B (DAB Part B), and hematoxylin.

[0083] The negative control reagent used was mouse IgG2a monoclonal antibody (clone number E5Y6Q, supplier CST) dissolved in antibody diluent.

[0084] The DS-3 antibody in vitro diagnostic reagent has a pH of 7.2-7.5 and contains 0.1 μg / mL DS-3 antibody, 0.1 M phosphate buffer solution (combination of disodium hydrogen phosphate and sodium dihydrogen phosphate), 0.05-0.06 M sodium chloride, surfactant (specifically, an additional 0.5% (v / v) amount of Triton X-100), 1% (w / v) bovine serum albumin, and a bacteriostatic agent (specifically, an additional 0.05% (v / v) amount of ProClin). 商標 Includes 300).

[0085] Example 4: Determination of staining results using DS-3 detection reagent Cell staining using the CLDN18.2 antibody reagent was localized to the cell membrane and / or cytoplasm. In stained tumor tissue samples, only the staining results of the cell membrane of tumor cells were determined, and cytoplasmic staining was not considered. The entire sample was evaluated: all effective tumor cells on the section should be evaluated, and it was necessary to detect more than 100 tumor cells. Criteria for determining staining with the CLDN18.2 antibody reagent in tumor tissue: only the staining results of the cell membrane of tumor cells were determined, and the staining ratio (TC) of tumor cells was calculated separately for each staining intensity of the cell membrane.

[0086] The calculation formula is as follows:

number

[0087] Stain intensity is scored on a scale of 0, 1+, 2+, and 3+, and the stain intensity score is determined as follows: 0: The target was not stained in tumor cells; 1+: Tumor cells showing weak or incomplete cell membrane staining; 2+: Tumor cells showing moderate-intensity cell membrane staining; and 3+: Tumor cells showing strong cell membrane staining.

[0088] Fifty-one different samples were obtained, and the presence of CLDN18.2 was determined in them using the DS-3 detection reagent and the control antibody R detection reagent (antibody 43-14A, AbCam) as described in Example 3. The antibody R detection reagent is a commercially available CLDN18.2 diagnostic reagent and was used according to the manufacturer's instructions.

[0089] When the cutoff was defined as TC(2+ and 3+) = 40%, i.e., TC(2+ and 3+) ≥ 40%, the result was determined to be positive, and a comparison of the detection results is shown in Table 3. In Table 3, two samples (Samples B1786-1 and 221208-18; Group B) were determined to be positive when using DS-3 and negative when using the antibody R detection reagent. The staining results for the two samples are shown in Table 4. Table 3. Comparison of DS-3 detection reagent and antibody R detection reagent * [Table 4] Table 4. Staining results at 40% cutoff. [Table 5]

[0090] In sample B1786-1, TC(2+ and 3+) was 45% when using the DS-3 detection reagent and 35% when using the antibody R detection reagent. In sample 221208-18, TC(2+ and 3+) was 50% when using the DS-3 detection reagent and 30% when using the antibody R detection reagent. DS-3 and antibody R show similar staining patterns, but staining with DS-3 is stronger in some tumor cells, which is likely due to different epitopes recognized by the two clones or due to the higher sensitivity of DS-3.

[0091] When the cutoff was defined as TC(2+ and 3+) = 75%, i.e., TC(2+ and 3+) ≥ 75%, the result was determined to be positive, and a comparison of the detection results is shown in Table 5. Table 6 shows the specific results for three samples where differences were observed when using the DS-3 detection reagent and the antibody R detection reagent. Table 5. Comparison of DS-3 detection reagent and antibody R detection reagent * [Table 6] Table 6: Staining results at 75% cutoff [Table 7]

[0092] These three samples showed relatively small staining differences, and DS-3 demonstrated higher sensitivity.

[0093] Example 5: Stability of DS-3 detection reagent Three batches of DS-3 IHC companion diagnostic kits (prepared as described in Example 3) were tested and qualified. Each batch was then divided into appropriate volumes, packaged, and stored in a 37°C incubator, with samples removed on days 0, 3, 7, and 14, respectively. Each serial section of the tissue chip was tested once, and the staining results were compared. The stained sections were evaluated by a specialist pathologist, and the results were analyzed.

[0094] The results showed no significant changes in the localization and intensity of positive signals obtained from the three batches of kits over four tests spanning 14 days, and no targeted positive signals were observed with the negative control reagent. There were no significant differences in the results for the same tissue and the same reagent across the three batches of kits over four tests spanning 14 days, with ΔTC(≧2+)≦10%. In each test spanning 14 days, there was little difference in the results for the same tissue when using the ready-to-use CLDN18.2 mouse monoclonal antibody reagent derived from the three batches of kits, with ΔTC(≧2+)≦5%. These results demonstrate the reagent's stability over two weeks.

[0095] Example 6: Phase I / II clinical trial on safety, tolerability, pharmacokinetics, and antitumor activity of ASKB589 In a Phase I / II clinical trial of the safety, tolerability, pharmacokinetics, and antitumor activity of ASKB589 injection in patients with locally advanced or metastatic solid tumors, the DS-3 companion diagnostic reagent was used to screen patients according to the method described in Example 3. The diagnostic test was performed at an institution appointed by the applicant and used as a selection criterion for patients to enroll in the clinical trial. Of the 306 patients screened, 51 had negative CLDN18.2 expression, 116 had low CLDN18.2 expression, 33 had intermediate CLDN18.2 expression, and 106 had high CLDN18.2 expression.

[0096] For in vitro diagnostic purposes, we compared the DS-3 antibody with other CLDN18.2 antibodies. A mouse-derived anti-CLDN18.2 antibody (lot number PW03-33) was obtained by mouse immunization. A rabbit-derived anti-CLDN18.2 antibody (AbCam, clone number EPR19202-244) was also obtained. These two antibodies, along with DS-3, were compared by immunohistochemical staining.

[0097] In human gastric cancer tissue sections, DS-3 showed more pronounced claudin 18.2 coloration compared to mouse anti-CLDN18.2 antibody (PW03-33) (Figure 1B) (Figure 1A). DS-3 also showed more pronounced claudin 18.2 coloration compared to rabbit-derived anti-CLDN18.2 antibody (Figure 1D) (Figure 1C).

Claims

1. A method for detecting claudin 18.2 (CLDN18.2) in a biopsy sample obtained from a human: Contacting the biopsy sample with an anti-CLDN18.2 antibody or its antigen-binding fragment containing heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6, respectively; and A method comprising detecting the binding of the antibody or antigen-binding fragment to a biopsy sample, wherein the presence of binding indicates the presence of CLDN18.2 in the biopsy sample.

2. A method for diagnosing cancer in humans: Contacting a human-derived biopsy sample with an anti-CLDN18.2 antibody or its antigen-binding fragment containing heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6, respectively; and A method comprising detecting the binding of the antibody or antigen-binding fragment to a biopsy sample, wherein the presence of binding indicates the presence of cancer in a human.

3. The method according to claim 2, wherein the biopsy sample is taken from tissue that does not normally express detectable claudin 18.

2.

4. Antibodies or antigen-binding fragments A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and Light chain variable region containing an amino acid sequence of SEQ ID NO: 8 or at least 90% identical thereto The method according to any one of claims 1 to 3, including the method described in any one of claims 1 to 3.

5. Antibodies or antigen-binding fragments A heavy chain containing an amino acid sequence identical to or at least 90% identical to SEQ ID NO: 9, and Light chain containing the amino acid sequence of SEQ ID NO: 10 or at least 90% identical thereto The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein an antibody or antigen-binding fragment is linked to a directly or indirectly detectable marker.

7. The method according to claim 6, wherein the detectable marker is horseradish peroxidase.

8. The method according to any one of claims 1 to 7, wherein the biopsy sample is obtained from ear, nose, throat, esophagus, stomach, pancreas, liver, biliary tract, large intestine, lung, bronchus, breast, or ovarian tissue.

9. The method according to any one of claims 1 to 8, wherein the biopsy sample is sectioned and fixed on a solid base, optionally the solid base being a glass slide, before being brought into contact with an antibody or antigen-binding fragment.

10. A kit comprising an anti-CLDN18.2 antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises heavy chain CDR1-3 containing SEQ ID NOs. 1-3 and light chain CDR1-3 containing SEQ ID NOs. 4-6.

11. Antibodies or antigen-binding fragments A heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and Light chain variable region containing an amino acid sequence of SEQ ID NO: 8 or at least 90% identical thereto The kit according to claim 10, comprising:

12. Antibodies or antigen-binding fragments A heavy chain containing an amino acid sequence identical to or at least 90% identical to SEQ ID NO: 9, and Light chain containing the amino acid sequence of SEQ ID NO: 10 or at least 90% identical thereto The kit according to claim 10, comprising:

13. The kit according to any one of claims 10 to 12, wherein the antibody or antigen-binding fragment is present in a phosphate buffer solution comprising sodium chloride, a surfactant, bovine serum albumin, and a preservative, and having a pH of 7 to 8.

14. The kit is Lyophilized antibody or antigen-binding fragment and A reconstituted phosphate buffer solution containing sodium chloride, surfactants, bovine serum albumin, and preservatives, with a pH of 7-8. A kit according to any one of claims 10 to 12, comprising:

15. A method for treating cancer in a person who needs it, To detect the presence of CLDN18.2 in a biopsy sample obtained from a human by the method of any one of claims 1 to 9, and If CLDN18.2 is found to be present in the biopsy sample, the patient should be treated with an anti-CLDN18.2 antibody drug. Methods that include...

16. The method according to claim 15, wherein the biopsy sample is taken from tissue that does not normally express detectable claudin 18.2.

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