Anti-human claudin 18.2 antibody and use thereof

Abstract

Provided are: a novel antibody capable of binding to an extracellular domain of claudin 18.2; a conjugate of the antibody; and others. Also provided is a method for treating cancer with a pharmaceutical composition that contains an anti-human claudin 18.2 antibody. The present inventors have obtained three types of anti-claudin 18.2 monoclonal antibodies which specifically bind to human claudin 18.2 and have binding activities superior to those of existing anti-human claudin 18.2 antibodies. It has been demonstrated that these antibodies have capability of inducing antibody-dependent cytotoxic activity (ADCC) against cells expressing human claudin 18.2 and are promising for the treatment of cancer.

Description

Anti-human claudin 18.2 antibody and uses thereof

[0001] The present disclosure relates to antibodies that specifically bind to human claudin 18.2 and uses thereof.

[0002] In vivo, epithelial cells strongly adhere to each other on their sides via tight junctions (TJs) to form cell sheets with intercellular barrier function. Such epithelial cell sheets compartmentalize various large and small biological sites inside and outside the body and function as an epithelial barrier to maintain homeostasis in the body. The mechanism by which the TJ intercellular barrier restricts and controls the permeability of substances between cells is closely related to the function of the epithelial barrier and is therefore recognized as one of the most fundamental and important issues for life activities.

[0003] Claudin (CLDN) is the only molecule capable of adhering cells to form an intercellular barrier at TJs, and to date, the claudin family consisting of 27 members has been identified in humans and mice. Each of these 27 claudin family members has been shown to be expressed specifically in a particular body site, and it is widely recognized that they form an optimal epithelial barrier specifically in that body site, thereby establishing the foundation for various biological functions (Non-Patent Document 1).

[0004] Claudin 18.2 (CLDN18 isoform 2: NP_001002026.1) is a four-transmembrane protein with a molecular weight of 27.72 kDa that belongs to the claudin family. It is expressed only in the gastric mucosa in normal tissues, but its expression has also been observed in cancer tissues such as gastric cancer and pancreatic cancer (Non-Patent Document 2). To date, various anti-claudin 18.2 monoclonal antibodies that selectively bind to claudin 18.2 have been obtained for the purpose of cancer treatment, and new application methods using these antibodies, such as bispecific antibodies, antibody-drug conjugates, and chimeric antigen receptor-introduced T cell therapy, are being actively developed (Non-Patent Document 2).

[0005] On the other hand, although zolbetuximab, an antibody against claudin 18.2, has been approved as a therapeutic agent for claudin 18.2-positive, HER2-negative unresectable locally advanced or metastatic gastric adenocarcinoma and gastroesophageal junction adenocarcinoma, the development of antibodies with strong binding and selectivity to claudin 18.2 is desired from the viewpoints of reducing side effects and manufacturing costs.

[0006] WO2007059997

[0007] Trends Biochem Sci. , 2019, Vol. 44, p. 141-152 Cancers, 2023, Vol. 15,5742

[0008] The present disclosure aims to provide new antibodies and fusions thereof that bind to the extracellular domain of claudin 18.2. The present disclosure also provides a method for treating cancer using a pharmaceutical composition comprising an anti-human claudin 18.2 antibody. The present disclosure also aims to provide a method for confirming the cross-reactivity of an anti-claudin 18.2 antibody with claudin family members. The present disclosure further aims to provide a kit that can be used in the method for confirming the cross-reactivity of an anti-claudin 18.2 antibody with claudin family members.

[0009] The present inventors conducted extensive research and obtained 16 types of anti-claudin 18.2 monoclonal antibodies that specifically bind to human claudin 18.2 (Example 2). The monoclonal antibodies provided by the present disclosure are highly selective, specifically binding to human claudin 18.2 but barely binding to many other human claudin family members (Example 3). Furthermore, the anti-claudin 18.2 monoclonal antibodies of the present disclosure were shown to have the ability to induce antibody-dependent cellular cytotoxicity (ADCC) targeting cells expressing human claudin 18.2 (Example 4).

[0010] That is, the present invention relates to the following [1] to

[15] . [1] An anti-human claudin 18.2 (hCLDN18.2) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region according to any of the following (a) to (f): (a) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 41; (c) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 45, CDR2 consisting of the amino acid sequence of SEQ ID NO: 46, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 48, CDR2 consisting of the amino acid sequence of SEQ ID NO: 49, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 59;or (f) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 63, CDR2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 65. [2] The anti-hCLDN18.2 antibody or antigen-binding fragment thereof according to [1], comprising a heavy chain variable region and a light chain variable region according to any of the following (a) to (c): (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 67; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 69; or (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 70 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 71. [3] The monoclonal antibody according to [1] or [2], which is a humanized antibody. [4] An anti-hCLDN18.2 antibody according to any one of [1] to [3], comprising a heavy chain and a light chain according to any one of the following (a) to (c): (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 72 and a light chain consisting of the amino acid sequence of SEQ ID NO: 73; (b) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 74 and a light chain consisting of the amino acid sequence of SEQ ID NO: 75;or (c) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 76 and a light chain consisting of the amino acid sequence of SEQ ID NO: 77. [5] A post-translationally modified anti-hCLDN18.2 antibody or antigen-binding fragment thereof according to any of [1] to [4]. [6] A fusion or complex of the anti-hCLDN18.2 antibody or antigen-binding fragment thereof according to any of [1] to [5], or a cell expressing the anti-hCLDN18.2 antibody of the present invention or its antigen-binding fragment on its cell surface. [7] A pharmaceutical composition for treating a cancer expressing claudin 18.2, comprising the monoclonal antibody or antigen-binding fragment thereof according to any of [1] to [6]. [8] The pharmaceutical composition according to [7], wherein the cancer expressing claudin 18.2 is gastric cancer, pancreatic cancer, lung cancer, or esophageal cancer. [9] A method for treating a cancer expressing claudin 18.2, comprising administering to a subject the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

[10] The pharmaceutical composition according to [9], wherein the cancer expressing claudin 18.2 is gastric cancer, pancreatic cancer, lung cancer, or esophageal cancer.

[11] A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5].

[12] An expression vector comprising the polynucleotide of claim 11.

[13] A diagnostic agent for cancer expressing claudin 18.2, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5].

[14] A kit for confirming the cross-reactivity of anti-human claudin antibodies, comprising in combination an epithelial cell line in which all claudins have been knocked out, and one or more epithelial cell lines that express 1 to 26 types of human claudins and do not express other claudins.

[15] The kit according to

[14] , comprising an epithelial cell line in which all claudins have been knocked out, an epithelial cell line that expresses only one type of human claudin and does not express other claudins, and an epithelial cell line that expresses one or more types of human claudins other than the one type of human claudin and does not express other claudins.

[0011] The anti-claudin 18.2 antibody of the present invention exerts an anti-tumor effect against cancers that express claudin 18.2, either alone or in combination with other modalities (e.g., antibodies, cells, drugs). Thus, pharmaceutical compositions containing the anti-claudin 18.2 antibody of the present invention can be used for the treatment of cancer.

[0012] Figure 1 shows the cross-reactivity of mouse anti-hCLDN18.2 antibody with human claudin family members. The numbers indicate the binding ability of the antibody to cells on a scale of 0 to 5. - indicates that the test was not performed. Figure 2A shows the ADCC activity of mouse anti-hCLDN18.2 antibody or human anti-hCLDN18.2 antibody in cells expressing only claudin 18.2 (CLDN18.2-expressing EpH4KO cells) or cells in which all claudins have been knocked out (EpH4KO cells). Figure 2B shows the ADCC activity of mouse anti-hCLDN18.2 antibody or human anti-hCLDN18.2 antibody at 15 μg / ml in CLDN18.2-expressing EpH4KO cells or EpH4KO cells. Zolbetuximab was used as a control. The horizontal axis shows antibody concentration, and the vertical axis shows relative light units (RLU). Figure 3 shows the binding ability of anti-hCLDN18.2 antibody to purified human claudin 18.2 in a fluorescent gel filtration test. The horizontal axis shows elution time, and the vertical axis shows fluorescence intensity. Figure 4 shows the binding ability of zolbetuximab to purified human claudin 18.2 in a fluorescent gel filtration test. The horizontal axis shows elution time, and the vertical axis shows fluorescence intensity. Figure 5 shows a sensorgram measuring the binding ability of anti-hCLDN18.2 antibody and zolbetuximab to human claudin 18.2 by surface plasmon resonance.

[0013] <Definition> As used herein, an "antibody" is an immunoglobulin molecule that binds to an antigen. An immunoglobulin molecule is composed of one or more units comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.

[0014] There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. Each class shares the same basic structure of antibody molecules, with two heavy chains and two light chains bound by disulfide bonds and non-covalent bonds to form a Y-shaped, four-chain antibody molecule. Heavy chains typically consist of polypeptide chains containing approximately 440 amino acids and have a characteristic structure for each class, referred to as Igγ, Igμ, Igα, Igδ, and Igε, corresponding to IgG, IgM, IgA, IgD, and IgE antibodies, respectively. IgG subclasses include IgG1, IgG2, IgG3, and IgG4, with the corresponding heavy chains referred to as Igγ1, Igγ2, Igγ3, and Igγ4. Light chains typically consist of two types of polypeptide chains, lambda and kappa, containing approximately 220 amino acids, referred to as Igλ and Igκ, respectively. The two types of light chains can pair with either type of heavy chain.

[0015] The amino acid sequence of the C-terminal domain downstream of the variable region is nearly constant for each class or subclass and is called the constant region. The heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH) from the N-terminus to the C-terminus. The CH is further divided into three domains from the N-terminus: the CH1 domain, the CH2 domain, and the CH3 domain. The light chain has a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus.

[0016] The light chain variable region and heavy chain variable region are composed of three CDRs and four framework regions (FRs), arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDRs in the heavy chain variable region are also referred to as "CDR-H1, CDR-H2, and CDR-H3," and the three CDRs in the light chain variable region are also referred to as "CDR-L1, CDR-L2, and CDR-L3." CDRs contain most of the residues that form specific interactions with antigens, and their sequences vary significantly from antibody to antibody. On the other hand, the sequences of framework regions show relatively little variation.

[0017] As used herein, a "framework region" or "framework sequence" refers to any one of framework regions 1-4. Engineered human antibodies and antigen-binding fragments thereof encompassed by the present disclosure include molecules in which any one or more of framework regions 1-4 are substantially or fully human (i.e., any possible combination of individual substantially or fully human framework regions 1-4 is present). For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc., are substantially or fully human. A substantially human framework is one that has at least about 80% sequence identity to known human germline framework sequences. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) or from Academic Press, 2001, ISBN 012441351.

[0018] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody that recognizes only a single epitope. The mAb of the present disclosure refers to, for example, an antibody derived from a single cell clone, and preferably exists in a homogeneous or substantially homogeneous population. Monoclonal antibodies can be produced by methods commonly performed by those skilled in the art (e.g., hybridoma technology, recombinant technology, phage display technology, synthetic technology), or by a combination of other techniques known in the art.

[0019] As used herein, the term "humanized antibody" refers to an antibody that has a combination of CDRs provided in the present disclosure and that has been generated and / or engineered such that the framework regions surrounding the CDRs have framework sequences that are substantially similar or identical to those of human variants. Antibody humanization is well known in the art. The humanized antibodies provided in the present disclosure may be intentionally modified compared to the native sequence, e.g., in the constant region, to alter effector or biofunctional properties, or biophysical properties (such as stability, developability, and / or solubility, among others).

[0020] When an antibody is treated with the protease papain, three antibody fragments are obtained. The two fragments on the N-terminal side are called Fab (Fragment, antigen binding) regions. The Fab region refers to a region consisting of the VH, CH1 domain, and part of the hinge region of the heavy chain, and the light chain (VL and CL), and binds to the antigen via the VH and VL (antigen binding sites) formed by the Fab region. The fragment on the C-terminal side is called Fc (Fragment, crystallizable) region.

[0021] As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment that retains the antigen-binding region of the antibody and has the ability to bind to the same epitope as the antibody. Examples of such fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain Fv fragments (scFv), diabodies, triabodies, and minibodies.

[0022] CDR sequences can be defined by various methods, including the Kabat definition (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), as used herein, as well as the IMGT definition (Lefranc et al., 2003, Dev Comparat Immunol Vol. 27:55-77) and Chothia definition (Al-Lazikani et al., 1997, J. Mol. Biol. Vol. 273:927-948). CDR sequences identified by these different definitions each specify the same antibody sequence and may be used interchangeably.

[0023] As used herein, the term "post-translational modification" refers to post-translational modification of an antibody when the antibody is expressed in a cell. Examples of post-translational modifications include pyroglutamylation, glycosylation, oxidation, deamidation, glycation, and other modifications of glutamine or glutamic acid at the N-terminus of the heavy chain, and lysine deletion due to cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008, Vol. 97, pp. 2426-2447).

[0024] As used herein, the term "active ingredient" refers to an ingredient having a pharmacological action required for preventing or curing a disease or alleviating the symptoms of a disease.

[0025] As used herein, "effective amount" refers to the amount of active ingredient required to prevent or cure a disease or to ameliorate the symptoms of a disease. The amount of active ingredient used in practicing the present invention to prevent or treat a disease will vary depending on the mode of administration, the age, weight, and general health of the subject.

[0026] As used herein, "subject" refers to an animal to which an agent is administered, for example, a mammal such as a human or a cow, horse, dog, sheep, or cat. In one embodiment, the subject to which the agent is administered is a human. In one embodiment, the pharmaceutical compositions provided in the present disclosure are particularly suitable for use in treating human subjects.

[0027] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disease and / or its associated symptoms.

[0028] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates, and, as known to those skilled in the art, includes any aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride and Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, infusion and nutritional supplements, and combinations of such materials. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0029] <First Aspect: Anti-Human Claudin 18.2 Monoclonal Antibodies> The first aspect of the present disclosure is novel anti-human claudin 18.2 monoclonal antibodies, h#3, h#6, and h#15 antibodies, and #3, #6, and #15 antibodies (also referred to herein as "anti-hCLDN18.2 antibodies"). The heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3) of each antibody are shown below. The antibody regions used herein are defined according to the Kabat numbering system.

[0030] The anti-hCLDN18.2 antibodies provided by the present disclosure have the CDR sets set forth in Tables 1 to 6. These CDR sets can be used in any form of mouse antibody, chimeric antibody, or humanized antibody. In one embodiment, the anti-hCLDN18.2 antibodies provided by the present disclosure are mouse antibodies or humanized antibodies. In one embodiment, the anti-hCLDN18.2 antibodies provided by the present disclosure are humanized antibodies.

[0031] The CDR sequences identified by the Kabat method are listed below.

[0032] In one embodiment, the anti-hCLDN18.2 antibody provided by the present disclosure may be an anti-human claudin18.2 antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region described in any of the following (a) to (f): (a) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 41; (c) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 45, CDR2 consisting of the amino acid sequence of SEQ ID NO: 46, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 48, CDR2 consisting of the amino acid sequence of SEQ ID NO: 49, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 59;or (f) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 63, CDR2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 65;

[0033] In one embodiment, the antibody of the present disclosure may be a chimerized or humanized antibody by conventional methods. In one embodiment, the antibody of the present disclosure is a humanized antibody.

[0034] In one embodiment, the anti-hCLDN18.2 antibody provided by the present disclosure may be an anti-hCLDN18.2 antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region described in any of the following (a) to (c): (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 67; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 69; or (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 70 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 71.

[0035] During humanization of the anti-hCLDN18.2 antibody of the present invention, the heavy chain constant region can be selected from Igγ, Igμ, Igα, Igδ, or Igε. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. In one embodiment, the constant region of the humanized anti-hCLDN18.2 antibody is derived from the human Igγ1 constant region.

[0036] In one embodiment, the anti-hCLDN18.2 antibody provided by the present disclosure may be an anti-hCLDN18.2 antibody comprising a heavy chain and a light chain described in any of the following (a) to (c): (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 72 and a light chain consisting of the amino acid sequence of SEQ ID NO: 73; (b) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 74 and a light chain consisting of the amino acid sequence of SEQ ID NO: 75; or (c) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 76 and a light chain consisting of the amino acid sequence of SEQ ID NO: 77.

[0037] Furthermore, the present disclosure may further provide an antibody that competes with #3, #6, or #15 obtained in the Examples of the present application, or h#3, h#6, or h#15, for binding to claudin 18.2.

[0038] When the anti-hCLDN18.2 antibody of the present invention comprises an Fc region, the Fc region of the bispecific antibody may contain mutations that enhance or reduce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In one embodiment, the anti-hCLDN18.2 antibody of the present invention contains a mutation that enhances ADCC activity.

[0039] In one embodiment, the anti-hCLDN18.2 antibody of the present invention may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of the C-terminal lysine of the heavy chain. It is known in the art that post-translational modification by pyroglutamylation of the N-terminus or deletion of the C-terminal lysine does not affect the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0040] The present disclosure also provides polynucleotides encoding the anti-hCLDN18.2 antibodies or antigen-binding fragments thereof provided by the present disclosure.

[0041] In one embodiment, the polynucleotide encoding the anti-hCLDN18.2 antibody or antigen-binding fragment thereof provided by the present disclosure is a polynucleotide comprising a base sequence encoding the heavy chain variable region and light chain variable region of an anti-human claudin18.2 (hCLDN18.2) antibody selected from the group consisting of the following (a) to (f): (a) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 41; (c) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 45, CDR2 consisting of the amino acid sequence of SEQ ID NO: 46, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 48, CDR2 consisting of the amino acid sequence of SEQ ID NO: 49, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 59;or (f) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 63, CDR2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 65;

[0042] The present disclosure also provides an expression vector comprising a polynucleotide encoding an anti-hCLDN18.2 antibody or antigen-binding fragment thereof provided by the present disclosure. Each of these polynucleotides may be contained in a separate vector, or multiple polynucleotides may be contained in a single vector.

[0043] The present disclosure also provides a host cell (hereinafter "host cell of the present invention") containing a polynucleotide encoding an anti-hCLDN18.2 antibody or antigen-binding fragment thereof provided by the present disclosure. The polynucleotide may be contained in a vector, or may be delivered directly to the host cell without being contained in a vector. The host cell may be cultured in vitro to produce the antibody, or may be a cell that directly expresses the antibody in vivo.

[0044] In one embodiment, the host cell of the present invention comprises a polynucleotide comprising a base sequence encoding the heavy chain variable region and light chain variable region, or the heavy chain and light chain, of an anti-human claudin 18.2 (hCLDN18.2) antibody, selected from the group consisting of (a) to (l) below: (a) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 41; (c) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 45, CDR2 consisting of the amino acid sequence of SEQ ID NO: 46, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 48, CDR2 consisting of the amino acid sequence of SEQ ID NO: 49, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 59;(f) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 63, CDR2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 65; (g) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 67; (h) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 69; (i) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 70 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 71; (j) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 72 and a light chain consisting of the amino acid sequence of SEQ ID NO: 73; (k) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 74 and a light chain consisting of the amino acid sequence of SEQ ID NO: 75; or (l) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 76 and a light chain consisting of the amino acid sequence of SEQ ID NO: 77.

[0045] Second Aspect: Method for Confirming Cross-Reactivity with Claudin Family Members In a second aspect of the present disclosure, a method for confirming the cross-reactivity of an anti-claudin antibody that specifically binds to a target claudin with a claudin family member is provided. The method includes the steps of: (1) preparing an epithelial cell line that expresses one human claudin per cell and no other claudins; one or more epithelial cell lines that express 1 to 26 human claudins other than the target per cell and no other claudins; and an epithelial cell line that does not express any claudins; (2) adding a test antibody to a culture of each epithelial cell line; and (3) measuring the binding of the test antibody to each cultured epithelial cell line.

[0046] In a second aspect of the present disclosure, an epithelial cell line that expresses one target human claudin per cell and does not express other claudins, one or more epithelial cell lines that express 1 to 26 non-target human claudins per cell and do not express other claudins, and an epithelial cell line that does not express any claudins are used.

[0047] An epithelial cell line that does not express all claudins can be obtained, for example, by knocking out all claudins expressed in a mouse mammary epithelial cell line. An epithelial cell line that expresses one target human claudin per cell but does not express other claudins, as well as an epithelial cell line that expresses 1 to 26 non-target human claudins per cell but does not express other claudins, can be obtained by transfecting an epithelial cell line that does not express all claudins with the cDNA of the human claudin of interest.

[0048] The epithelial cells are not particularly limited, and any stably available epithelial cell line may be used. Examples include, but are not limited to, the mouse mammary gland-derived epithelial cell line EpH4. First, the claudin mRNA expressed in the original wild-type cell line is confirmed by RT-PCR or the like, and then all of the expressed claudins are knocked out.

[0049] The genomic sequence of each mouse claudin is known. Knockout of each claudin expressed in the mouse mammary gland-derived epithelial cell line EpH4 may be performed by a known method, and is not particularly limited. For example, genome editing technology may be used. Examples of genome editing technology include the CRISPR system (such as CRISPR-Cas9), TALEN, and ZFN. An example of a genome editing method is a method using the CRISPR-Cas system. CRISPR-Cas9 vectors that can simultaneously achieve multiple gene knockouts are commercially available, and such vectors may also be used.

[0050] Next, DNA encoding the human claudin of interest is transfected into the epithelial cell line in which all claudins have been knocked out, according to standard methods. Currently, 26 types of human claudins are known, and the cDNA sequences of various human claudins used for knockout, each with a stop codon at the end, are listed in SEQ ID NOS: 1 to 28.

[0051] The expression of claudins in transfected cells can be confirmed by standard methods using antibodies specific to each transfected claudin. If suitable specific antibodies are not available, a tag can be attached to the transfected protein when transfecting each claudin, and the expression of the target claudin can be confirmed by standard methods using an antibody specific to the tag.

[0052] Cells transfected with DNA encoding each claudin are cultured, and clones in which the target claudin is produced on the cells are picked and used in an assay for cross-reactivity of antibodies to claudin family members.

[0053] To confirm the cross-reactivity of an antibody with claudin family members, first culture an epithelial cell line expressing only the target claudin, an epithelial cell line expressing the claudin for which cross-reactivity is to be confirmed, and a control epithelial cell line expressing no claudins. The test antibody or the culture supernatant of a hybridoma containing the test antibody is added to each culture, and the binding of the test antibody to the claudin expressed in each epithelial cell line is confirmed.

[0054] Binding of the test antibody to the claudin expressed in each epithelial cell line can be confirmed by a standard method, such as a fluorescent antibody technique. This method allows for the determination of reactivity to the target claudin and cross-reactivity with other claudins.

[0055] <Third Aspect: Method for Confirming Cross-Reactivity with Claudin Family Members> In a third aspect of the present disclosure, a kit for confirming the cross-reactivity of the anti-human claudin antibody used in the second aspect is provided. The kit includes a combination of an epithelial cell line in which all claudins have been knocked out and one or more epithelial cell lines that express 1 to 26 human claudins but do not express other claudins.

[0056] The kit provided in a third aspect of the present disclosure includes a combination of an epithelial cell line in which all claudins have been knocked out, and two or more epithelial cell lines that express 1 to 26 types of claudins and do not express any other claudins. The kit of the present disclosure may further include a medium used for cell culture, a fluorescently labeled secondary antibody, and the like. The kit provided in the present disclosure is preferably one that includes a combination of a cell line that expresses only one type of claudin and a cell line that expresses one or more claudins selected from claudins that do not include the one type. Examples of cell lines that express one or more types of claudins include cell lines that express 1 to 5 types or 1 to 3 types, for example, one type of claudin each.

[0057] <Fourth Aspect: Fusion of Anti-hCLDN18.2 Antibody or Antigen-Binding Fragment Thereof> In a fourth aspect of the present disclosure, an anti-hCLDN18.2 antibody or an antigen-binding fragment thereof (also referred to as the "fusion of the present invention") is provided, which is linked to a protein (including an antibody) or polypeptide other than human claudin-18.2. The protein or polypeptide used in the fusion of the present invention is not particularly limited, and may be, for example, various antibodies, cytokines, chemokines, human serum albumin, various tag peptides, artificial helix motif peptides, maltose-binding protein, glutathione S-transferase, or other peptides or proteins that can promote multimerization. In one embodiment, the fusion of the present invention has a structure in which a protein or polypeptide is linked to an anti-hCLDN18.2 antibody or an antigen-binding fragment thereof. In one embodiment, the protein or polypeptide used in the fusion of the present invention may be, for example, an antibody or antigen-binding fragment thereof against a surface antigen of blood cells such as immune cells, dendritic cells, or macrophages, or a polypeptide that activates immune cells, such as various interleukins. In this case, the protein or polypeptide used in the fusion of the present invention may be directly linked to the anti-hCLDN18.2 antibody of the present invention or its antigen-binding fragment, or may be linked via any linker (e.g., a peptide linker). In one embodiment, the fusion of the present invention may be a multispecific antibody such as a bispecific antibody or a trispecific antibody.

[0058] <Fifth Aspect: Conjugate of Anti-hCLDN18.2 Antibody or Antigen-Binding Fragment Thereof> In a fifth aspect of the present disclosure, an anti-hCLDN18.2 antibody of the present invention or an antigen-binding fragment thereof (also referred to as the "conjugate of the present invention") is provided, which is conjugated to a carbohydrate, lipid, metal (including a radioisotope), organic compound (including a toxin, a near-infrared fluorescent dye, a chelating agent), etc. (also referred to as a "modifying agent"). As used herein, the term "modifying agent" refers to a non-peptide substance that is bound to an antibody or its antigen-binding fragment directly or via a linker, etc. The modifying agent used in the conjugate of the present invention is not particularly limited, and examples thereof include polyethylene glycol, sugar chains, phospholipids, radioisotopes (e.g., zirconium-89 (89Zr), yttrium-90 (90Y), indium-111 (111In), astatine-211 (211At), actinium-225 (225Ac)), organic compounds, toxins, near-infrared fluorescent dyes (e.g., IRDye (registered trademark)), chelating agents, and the like. The modifying agent used in the conjugate may be directly bound to the anti-hCLDN18.2 antibody or antigen-binding fragment thereof of the present invention, or may be bound via any linker. In one embodiment, the conjugate of the present invention is a drug conjugate (antibody drug conjugate; ADC) of an anti-hCLDN18.2 antibody or antigen-binding fragment thereof. The drug and linker used in the ADC may be selected from among drugs and linkers commonly used by those skilled in the art. In one embodiment, the conjugate of the present invention is a radioisotope-labeled antibody in which a radioisotope is bound to an anti-hCLDN18.2 antibody or an antigen-binding fragment thereof.

[0059] Sixth Aspect: Cells Expressing an Anti-hCLDN18.2 Antibody or an Antigen-Binding Fragment Thereof on the Cell Surface In a sixth aspect of the present disclosure, cells (e.g., chimeric antigen receptor-T cells; CAR-T cells) are provided in which the anti-hCLDN18.2 antibody of the present invention or an antigen-binding fragment thereof is expressed on the cell surface. Such cells can be produced by those skilled in the art using a polynucleotide encoding the anti-hCLDN18.2 antibody of the present invention or an antigen-binding fragment thereof. Various immune cells (T cells, NK cells, NKT cells, etc.) can be used as cells expressing the anti-hCLDN18.2 antibody of the present invention or an antigen-binding fragment thereof.

[0060] The anti-hCLDN18.2 antibody or antigen-binding fragment thereof of the present invention, the fusion of the present invention, the complex of the present invention, and the cell expressing the anti-hCLDN18.2 antibody or antigen-binding fragment thereof on the cell surface (hereinafter, one or more of the above or collectively referred to as the "active ingredient of the present invention") bind to human claudin 18.2. Binding to human claudin 18.2 can be confirmed using a known binding activity measurement method. Methods for measuring binding activity include, for example, surface plasmon resonance (SPR), ELISA, flow cytometry, etc. In one embodiment, the antibody or antigen-binding fragment portion of the anti-hCLDN18.2 antibody or antigen-binding fragment thereof of the present invention, the fusion of the present invention, the complex of the present invention, and the cell expressing the anti-hCLDN18.2 antibody or antigen-binding fragment thereof on the cell surface may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of a lysine at the C-terminus of the heavy chain.

[0061] The anti-hCLDN18.2 antibody or antigen-binding fragment thereof of the present invention, the fusion of the present invention, and the complex of the present invention, as well as cells expressing the anti-hCLDN18.2 antibody or antigen-binding fragment thereof on the cell surface, can be prepared by a person skilled in the art using methods known in the art based on the VH and VL sequence information of the anti-hCLDN18.2 antibody or antigen-binding fragment thereof disclosed herein, other peptides or proteins (e.g., antibodies) used in the fusion of the present invention, and information on the modifying agent used in the complex of the present invention.

[0062] Seventh Aspect: Pharmaceutical Use of Anti-hCLDN18.2 Antibodies and the Like In a seventh aspect of the present disclosure, a pharmaceutical composition for use in the prevention or treatment of diseases in a subject, including cancers expressing claudin 18.2, or a method for preventing or treating the disease is provided. The pharmaceutical composition used for the treatment, etc., contains a pharmaceutically acceptable excipient, etc. in addition to the active ingredient of the present invention (hereinafter also referred to as the "pharmaceutical composition of the present invention"). The pharmaceutical composition of the present invention can be used to treat cancer and other diseases caused by abnormalities in claudin 18.2. This aspect also provides a method for treating cancer, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of the present invention or an anti-hCLDN18.2 antibody, etc. to a subject, the anti-hCLDN18.2 antibody, etc. for use in cancer treatment, and the use of the anti-hCLDN18.2 antibody, etc. of the present invention in the manufacture of a pharmaceutical composition for cancer treatment. The pharmaceutical use of the pharmaceutical composition, etc. of the present invention can be carried out by methods commonly used by those skilled in the art.

[0063] The pharmaceutical compositions of the present invention include pharmaceutical compositions containing the active ingredient of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers, etc. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered by intravenous administration, subcutaneous administration, intraperitoneal administration, etc. When formulating, excipients, carriers, additives, etc. appropriate for these dosage forms can be used within a pharmaceutically acceptable range. The pharmaceutical compositions of the present invention may contain post-translationally modified forms of the anti-hCLDN18.2 antibody of the present invention. For example, pharmaceutical compositions containing antibodies that have undergone both or either of C-terminal lysine deletion and N-terminal pyroglutamylation are also included in the present invention.

[0064] Furthermore, the anti-hCLDN18.2 antibodies provided herein can be suitably used for the treatment or diagnosis of cancers that express claudin 18.2. In one embodiment, the pharmaceutical composition of the present invention is suitable for the treatment of cancers that express claudin 18.2, particularly cancers that express claudin 18.2 on the surface of cancer cells.

[0065] Cancers that can be treated by the present invention are not particularly limited, but include, for example, various peritoneal disseminated cancers, gastric cancer, lung cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), blood cancers such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, and other cancers. Examples of cancers include solid cancers such as cervical cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma.

[0066] Examples of cancers that express claudin 18.2 include, but are not limited to, gastric cancer, lung cancer, pancreatic cancer, and esophageal cancer. In one embodiment, the cancers treated with the pharmaceutical composition of the present invention are gastric cancer, lung cancer, pancreatic cancer, and esophageal cancer. In one embodiment, the cancers treated with the pharmaceutical composition of the present invention are gastric cancer and pancreatic cancer.

[0067] Pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the present disclosure can be administered to a subject by, for example, intravenous (IV), intramuscular (IM), subcutaneous (SC), parenteral, spinal, or epidermal administration by injection or infusion.

[0068] Eighth Aspect: Anti-hCLDN18.2 Antibody or Antigen-Binding Fragment Thereof Used for Diagnosis of Disease The seventh aspect of the present disclosure provides an anti-hCLDN18.2 antibody or antigen-binding fragment thereof, or a method for diagnosing cancer, that can be used in diagnosing cancer. The present invention includes a step of confirming the presence or absence of claudin 18.2 expressed in cells contained in a biological sample obtained from a subject. Potential cancers that can be targeted are cancers that express claudin 18.2, including, but not limited to, gastric cancer, lung cancer, pancreatic cancer, and esophageal cancer. In the above diagnostic application, the antibody or antigen-binding fragment thereof of the present disclosure can be used to determine whether a cell population contained in a biological sample obtained from a subject expresses claudin 18.2, for example, by flow cytometry or immunohistochemical assay. Thus, the present application provides a diagnostic agent for cancers that express claudin 18.2, comprising the antibody or antigen-binding fragment thereof of the present disclosure.

[0069] To provide a further understanding of the present invention, reference is now made to specific examples which are provided for purposes of illustration and not limitation.

[0070] Preparation of cells expressing only one member of the claudin family (1) Preparation of all claudin knockout cells The mouse mammary gland-derived epithelial cell line EpH4 was used. To investigate the types of claudins expressed in EpH4, we first performed mRNA expression analysis. As a result, we confirmed the expression of eight claudins (claudins 3, 4, 7, 8, 9, 12, 23, and 25).

[0071] To construct claudin targeting vectors, guide RNA (gRNA) expression cassettes for knocking out each claudin were designed using CRISPRdirect (https: / / crispr.dbcls.jp) (Bioinformatics, 2015, Vol. 31, pp. 1120-1123). The designed gRNA expression cassettes were assembled using the Multiplex CRISPR-Cas9 Assembly System Kit (Addgene, 1000000055) to construct an all-in-one CRISPR-Cas9 vector.

[0072] The all-in-one CRISPR-Cas9 vector and pGK-puro (Addgene, 11349) were co-transfected into EpH4 cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001). The transfected cells were selected in medium containing 5 μg / mL puromycin (Sigma-Aldrich, P8833), and single-cell derived cell clones were picked and genomic DNA was extracted.

[0073] The resulting genomic DNA was amplified by PCR to include the target site of each claudin. The amplified PCR products were subjected to a T7 endonuclease I assay using T7 Endonuclease I Reaction Mix (NIPPON GENE CO., LTD., 313-08801) to determine the presence or absence of indel mutations and select candidate clones. The candidate clones were single-cell cloned by limiting dilution, and genomic DNA was extracted and sequenced to confirm the presence or absence of frameshift mutations within the sequence of each claudin.

[0074] Sequence analysis revealed that claudin-7 and claudin-12 were not completely knocked out in the selected cells. Claudin-7 and claudin-12 were further knocked out from these semi-knockout cells. A new all-in-one CRISPR-Cas9 vector was constructed using the same method as above, and this vector was co-transfected into the semi-knockout cells with pTA-Hyg (Takara Bio, 631750) using Lipofectamine 3000. The transfected cells were selected with 200 μg / mL hygromycin B (Fujifilm Wako Pure Chemical Corporation, 080-07683), and completely knocked-out candidate clones were selected in the same manner as above. The candidate clones were single-cell cloned by limiting dilution, and genomic DNA was extracted. Frameshift mutations within the sequences of each claudin were confirmed by sequence analysis. Finally, cells in which all claudins were knocked out (hereinafter referred to as "EpH4KO cells") were obtained.

[0075] (2) Transepithelial Electrical Resistance (TER) of Total Claudin Knockout Cells Method: Mouse mammary gland-derived epithelial cells EpH4 cells (WT) and EpH4KO cells (KO) lacking all claudins were seeded in a 24-well cell culture insert with a filter (Greiner Bio-One, 662641) and cultured in DMEM medium. The cell count was determined using a hemocytometer to be 0.3 × 10 ５ The cells were evenly seeded onto the filter at 100 cells / well. From the day after seeding, when the cells had adhered to the filter, transepithelial electrical resistance (TER) was measured every day.

[0076] TER was measured using a volt-ohm meter (Merck, MERS00002) and an electrode (Merck, MERSTX01). Using the resistance measurement mode, the electrode was placed in the well and recording began after the value stabilized. The unit area resistance (Ω cm) was calculated by subtracting the measured value of the filter alone and multiplying it by the culture area. ２) was obtained. Compared to WT cells, EpH4KO cells exhibited a decreased TER and a loss of tight junction barrier function. From the perspective of barrier function, it was also demonstrated that all claudins were deleted in EpH4KO cells. (3) Preparation of cultured epithelial cells expressing single claudin subtypes. The cDNAs of the 27 claudins listed in SEQ ID NOS: 1 to 28 were subcloned into the pLVSIN-CMV Neo vector (Takara Bio, 6181) using the In-Fusion HD Cloning Kit (Takara Bio, 639650) to prepare expression vectors containing a single claudin.

[0077] Lentiviral particles were prepared in Lenti-X 293T cells (Takara Bio, 632180) using single claudin expression vectors and Lentiviral High Titer Packaging Mix (Takara Bio, 6194). The resulting lentiviral particles were added to the medium of EpH4KO cells lacking all claudins and incubated for 24 hours to allow infection. Infected cells were selected in medium containing 500 μg / mL G418 (Nacalai Tesque, 09380-44). Cell clones derived from each single cell were examined for expression by fluorescent staining using claudin-specific antibodies, and single claudin-expressing cells were obtained.

[0078] At present, reliable specific antibodies have not been obtained for claudins 12, 13, 16, 17, 20, and 21 to 27. Therefore, for these claudins, we expressed claudins tagged with a flag tag (registered trademark, Sigma-Aldrich (Merck)) at the N-terminus and obtained single-claudin-expressing cell lines using an anti-flag antibody (Fujifilm Wako Pure Chemical Corporation, 018-22381).

[0079] The secondary antibodies used to confirm human Cldn expression in each cell line were selected appropriately from the following: Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch, 711-545-152), Alexa Fluor 488 donkey anti-mouse IgG (H+L) (Invitrogen, A-21202), and Alexa Fluor 488 donkey anti-goat IgG (H+L) (Invitrogen, A-11055), depending on the type of primary antibody.

[0080] Cell lines expressing each claudin were selected, and protein extraction and Western blotting of the resulting cell lines confirmed that each transfected cell line expressed a single specific claudin. Hereinafter, the 27 types of cultured epithelial cell lines expressing each claudin are referred to as "27 types of cultured epithelial cell lines."

[0081] The obtained cells were cryopreserved using standard methods and thawed and cultured as needed. When clones of the obtained cells were plate-cultured, they grew into a monolayer sheet. The obtained EpH4 cell sheet expressed only a single claudin, and no other claudins were expressed.

[0082] Obtaining mouse anti-hCLDN18.2 antibodies (1) Production of claudin 18.2 knockout mice Using a known method (Gastroenterology, 2012, Vol. 142, p. 292-304), claudin 18.2-deficient (KO) mice lacking the first exon of the claudin 18.2 gene were produced.

[0083] First, we constructed a targeting vector capable of replacing the first exon of claudin-18.2 in mouse ES cells (129R strain). To enable screening of homologously recombined ES cells, we used a neomycin resistance gene transcribed under the control of the PGK promoter as the replacement site.

[0084] The vector backbone of the targeting vector was removed by restriction enzyme treatment and electrophoresis, and the linearized 5' arm, the homologous recombination region of the 3' arm, and the neomycin resistance gene region flanked by both arms were introduced into mouse ES cells by electroporation. The mouse ES cells were then seeded on MMC-treated mouse fibroblasts, and screening with G418 began the day after electroporation.

[0085] The selected colonies were cultured in the presence of G418 for 7 to 10 days to grow into pickable colonies. The resulting colonies were picked into 96-well plates under a microscope. For positive clones, DNA was extracted from the cells and screened by PCR to confirm the insertion of the neomycin resistance gene.

[0086] Clones confirmed by PCR to have the neomycin resistance gene were further expanded, and the resulting cells were analyzed by Southern blotting to confirm homologous recombination into the first exon of claudin-18.2 and the absence of random insertion into the genome, yielding ES cells in which the first exon of claudin-18.2 had been knocked out.

[0087] The resulting ES cells in which the first exon of claudin-18.2 had been knocked out were injected into blastocysts of fertilized eggs of ICR mice to produce chimeric mice. Among the resulting chimeric mice, those with a high chimerism rate were selected and crossed with 129 wild-type mice to obtain heterozygous founder mice. Claudin-18.2 knockout mice were then obtained by crossing.

[0088] (2) Obtaining anti-hCLDN18.2 antibodies Claudin 18.2 has high homology between humans and mice, so in order to avoid immune tolerance and increase the rate of antibody acquisition, antigen immunization was performed on the prepared Cldn18.2KO mice. In addition, the extracellular region of human claudin 18.2, a four-transmembrane protein, has a characteristic molecular structure containing a β-sheet. In order to obtain a functional antibody that recognizes its native molecular structure, a plasmid vector pDNAimmu3-HA-hCldn18.2 capable of expressing the full-length human claudin 18.2 was prepared, and DNA immunization was performed using the electroporation method. Two months after DNA immunization of Cldn18.2KO mice, B cells were obtained from the spleen and iliac lymph nodes of mice in which an increase in antibody titer against claudin 18.2 in peripheral blood was confirmed, and hybridomas were produced by conventional methods. The mouse B lymphocyte cell line Sp2 / 0-Ag14 (ATCC CRL-1581) was used as the myeloma for preparing hybridomas.

[0089] The cultured epithelial cell lines expressing 27 types of claudins obtained in Example 1 proliferate in sheet form when plate-cultured. A cell line expressing only claudin 18.2 was cultured to obtain a cultured cell sheet, and hybridoma culture supernatant was applied to the cultured cell sheet. Cell lines that express only claudin 18.2 and produce antibodies that bind to cells that do not express other members of the claudin family were selected and cloned.

[0090] Specifically, after the hybridoma culture supernatant was applied to the cell sheet, the cell sheet was washed with HBS and then fixed with formalin solution (0.5% PFA / HBS). After washing away the fixative, a fluorescently labeled secondary antibody was reacted. After washing the cells, they were embedded in a mounting agent to prepare a sample for fluorescent observation with a fluorescent antibody. Donkey anti-mouse IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-21202) was used as the fluorescently labeled secondary antibody.

[0091] Sixteen hybridomas were obtained, whose culture supernatant monoclonal antibodies were confirmed to bind to cells expressing claudin 18.2 by immunofluorescence assay. The antibodies produced by the hybridomas were either IgG1(κ), IgG2a(κ), IgG2b(κ), or IgG3(κ). The antibodies produced by each hybridoma were purified and named monoclonal antibodies #1 to #16.

[0092] Confirmation of the specificity of monoclonal antibodies to the claudin family The 27 types of cultured epithelial cell lines obtained in Example 1 were each cultured, and the binding ability of the 16 types of monoclonal antibodies obtained in Example 2 to each claudin family member was visualized using the fluorescent antibody technique. The binding ability of the antibody to the cells was evaluated on a scale of 0 to 5 based on the fluorescence intensity under a microscope. - indicates that the test was not performed. The results are shown in Figure 1. As a result, strong reactivity to claudin 18.2 was confirmed for all antibodies #1 to #16. Antibodies #4 and #7 showed reactivity to some other claudins, but the other antibodies showed no reactivity to other claudins at all.

[0093] Measurement of antibody-dependent cellular cytotoxicity (ADCC) activity of anti-claudin 18.2 antibodies ADCC is an immune defense mechanism involving effector cells. When an antibody binds to an antigen on a target cell such as a cancer cell, the Fc portion of the antibody binds to the Fc receptor of effector cells such as natural killer cells, macrophages, neutrophils, and eosinophils, resulting in antibody-dependent cytotoxicity. Zolbetuximab (IgG1(κ) antibody), an anti-claudin 18.2 antibody, was used as a control.

[0094] The Mouse FcγRIV ADCC Bioassay Kit (Promega, M1201) was used to measure ADCC activity. The target cells used were EpH4 cultured epithelial cells expressing only claudin 18.2 obtained in Example 1, and claudin knockout EpH4 cultured epithelial cells. The ADCC activity of 16 types of anti-hCLDN18.2 antibodies, which were test antibodies, was measured according to the kit's instructions. The ADCC activity of #2, 3, 4, 6, and 15, which had high ADCC activity, was measured. Zolbetuximab was used as a control. The results are shown in Figure 2.

[0095] As shown in Figure 2, in a test using human claudin 18.2-expressing cells as target cells, the test antibody showed an antibody concentration-dependent increase in ADCC activity. As shown in Figure 2A, for mouse anti-hCLDN18.2 antibodies, #3, #6, and #15 showed significantly high RLU. In addition, for the humanized antibodies shown in Figure 2B, #3 showed a significantly higher RLU of 9.6 times that of human IgG, and #6 showed a significantly higher RLU of 8.6 times, and compared to zolbetuximab, they also showed high ADCC activity of 2.6 times and 2.2 times, respectively. Note that due to the characteristics of the Mouse FcγRIV ADCC Bioassay Kit kit, the affinity of mouse IgG2a is high, so the RLU of mouse IgG2a is about 2 times higher than the RLU of human IgG1.

[0096] Binding of monoclonal antibody #3 to claudin 18.2 Cells expressing only claudin 18.2 (CLDN18.2-expressing EpH4KO) and cells in which all claudins were knocked out (EpH4KO cells) obtained in Example 1 were cultured to obtain a cultured epithelial cell sheet (a cell sheet with cells on one side). The obtained sheet was treated with antibody #3, fixed, and visualized with a fluorescently labeled secondary antibody. When antibody #3 was treated with this cell sheet, fluorescence was observed only in CLDN18.2-expressing EpH4KO, confirming that antibody #3 bound to claudin 18.2.

[0097] As a control, zolbetuximab was applied to the same cell sheet as above, fixed in the same manner, and visualized with a fluorescently labeled secondary antibody. The fluorescently labeled secondary antibody used to visualize zolbetuximab binding was Goat anti-Human IgG (H+L) Cross-absorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-11013). Zolbetuximab bound only to a portion of the cells. It is believed that it bound only to cells with particularly high levels of claudin-18.2 expression.

[0098] Sequence analysis of monoclonal antibodies The sequence of the obtained antibody #3 was analyzed by a conventional method. The obtained CDR sequences (Kabat method) are shown in Table 1 above and SEQ ID NOs: 30 to 35. The sequence of the monoclonal antibody #6 was analyzed by a conventional method. The obtained CDR sequences (Kabat method) are shown in Table 2 above and SEQ ID NOs: 36 to 41. The sequence of the monoclonal antibody #15 was analyzed by a conventional method. The obtained CDR sequences (Kabat method) are shown in Table 3 above and SEQ ID NOs: 42 to 47.

[0099] Confirmation of the binding properties of anti-hCLDN18.2 antibody to purified claudin 18.2 protein by fluorescence gel filtration (FSEC) method An EGFP tag was fused to human claudin 18.2 protein. Purified EGFP tag-fused claudin 18.2 was added to a buffer solution (20 mM Hepes-NaOH, pH 7.0, 150 mM NaCl, 1 mM MgCl2, 5 mM CaCl2, 0.05% DDM) containing 10 ng / μL, and antibody #3 or zolbetuximab was added at 50 ng / μL, 250 ng / μL, or 500 ng / μL at 4 ° C. for 60 minutes.

[0100] The protein mixture was then subjected to measurement of EGFP fluorescence using a liquid chromatography system equipped with a fluorescence detector. If the antibody binds to the EGFP fusion protein, the size of the protein changes compared to the EGFP fusion protein alone, confirming the binding of the antibody to the antigen. The results are shown in Figures 3 and 4.

[0101] As shown in Figure 3, a peak shift in EGFP fluorescence was observed in the mixture of antibody #3 and EGFP-fused claudin 18.2. When 50 ng of antibody was added, the amount of antibody relative to the antigen was relatively small, and two peaks were observed: one representing the antigen bound to the antibody and the other representing the antibody-free antigen. On the other hand, when 250 ng / μL and 500 ng / μL of antibody were added, the antibody bound to all of the antigens, resulting in overlapping peaks. On the other hand, as shown in Figure 4, zolbetuximab showed almost no binding to EGFP-fused purified claudin 18.2.

[0102] Humanization of Antibodies #3, #6, and #15 The CDR sequences (cover sequences) determined in Example 6 were extracted, and humanized antibodies were designed while maintaining the cover sequences. The design was performed using methods commonly used by those skilled in the art. For the humanized antibody based on #3, the humanization rate of the heavy chain variable region was 76.1%, and the humanization rate of the light chain variable region was 92.9%. For the humanized antibody based on #6, the humanization rate of the heavy chain variable region was 83.8%, and the humanization rate of the light chain variable region was 92.9%. For the humanized antibody based on #15, the humanization rate of the heavy chain variable region was 83.2%, and the humanization rate of the light chain variable region was 92.0%.

[0103] Humanized antibodies were produced based on the amino acid sequences of these humanized heavy and light chain variable regions. CHO cells were codon-optimized using GeneArt GeneOptimizer, and an artificial gene was synthesized at Eurofins. This artificial gene was seamlessly inserted into the pCEC4.3 vector (originally developed by the Cell Engineering Institute), an antibody expression vector encoding the constant region, to produce a nucleic acid construct. Humanized antibodies were produced using this nucleic acid construct. It was confirmed that the desired humanized antibodies were obtained. The humanized antibodies produced using the CDR sequences of #3, #6, and #15 are referred to as h#3, h#6, and h#15 antibodies, respectively.

[0104] The Kabat CDR sequences of h#3 are shown in Table 4 above and in SEQ ID NOS: 48 to 53. The Kabat CDR sequences of h#6 are shown in Table 5 above and in SEQ ID NOS: 54 to 59. The Kabat CDR sequences of h#15 are shown in Table 6 above and in SEQ ID NOS: 60 to 65.

[0105] Confirmation of the binding properties of humanized anti-claudin 18.2 monoclonal antibodies to human claudin 18.2 protein As shown in Figure 5, the binding of three types of antibodies, h # 3, h # 15, and zolbetuximab, to human claudin 18.2 protein was confirmed by surface plasmon resonance (SPR) using a Cytiva Biacore S200 (29136649). The measurement data was recorded using the Kiretics culture using capture program of Cytiva's Biacore S200 Control Software (29136649). The sensor chip used was a Cytiva Series S Sensor Chip CM5 (BR100530), and anti-human IgG from the Cytiva Human Antibody Capture Kit (BR100839) was immobilized on the chip's gold membrane surface by amine coupling using the Cytiva Amine Coupling Kit (BR100050). The running buffer used was HBS (Cytiva, BR100670), 0.02% n-Dodecyl-β-D-maltoside (anatrace, 69227-93-6) solution. Next, h#3 antibody adjusted to 1 μg / mL was passed through the column at a flow rate of 10 μL / min for 25 seconds, Running Buffer was passed through the column at a flow rate of 30 μL / min for 720 seconds, and Regeneration solution included in Cytiva's Human Antibody Capture Kit (BR100839) was passed through the column at a flow rate of 20 μL / min for 30 seconds. This series of three steps was repeated three times. Then, h#3 antibody prepared at 1 μg / mL was run at a flow rate of 10 μL / min for 25 seconds, purified human claudin 18.2 protein of the desired concentration was run at a flow rate of 30 μL / min for 120 seconds, Running Buffer was run at a flow rate of 30 μL / min for 600 seconds, and Regeneration solution was run at a flow rate of 20 μL / min for 30 seconds. This series of four steps was measured for the h#3 antibody at seven concentrations of human claudin 18.2 protein ranging from 640 nM to 10 nM at a 2-fold common ratio.In the measurement of the h # 15 antibody, the h # 15 antibody prepared at 1 μg / mL was run at a flow rate of 10 μL / min for 25 seconds, Running Buffer at a flow rate of 30 μL / min for 720 seconds, Regeneration solution at a flow rate of 20 μL / min for 30 seconds, this series of three steps was repeated three times. Then, the h # 15 antibody prepared at 1 μg / mL was run at a flow rate of 10 μL / min for 25 seconds, purified human claudin 18.2 protein of the desired concentration at a flow rate of 30 μL / min for 120 seconds, Running Buffer at a flow rate of 30 μL / min for 600 seconds, Regeneration solution was run at a flow rate of 20 μL / min for 30 seconds. This series of four steps was performed for the h#15 antibody at seven concentrations of purified human claudin-18.2 protein, ranging from 640 nM to 10 nM at a 2-fold common ratio. For zolbetuximab measurements, zolbetuximab prepared at 1 μg / mL was run at a flow rate of 10 μL / min for 25 seconds, Running Buffer at a flow rate of 30 μL / min for 720 seconds, and Regeneration solution at a flow rate of 20 μL / min for 30 seconds. This series of three steps was repeated three times. Then, 1 μg / mL of zolbetuximab was prepared at a flow rate of 10 μL / min for 25 seconds, purified human claudin 18.2 protein of the desired concentration at a flow rate of 30 μL / min for 120 seconds, Running Buffer at a flow rate of 30 μL / min for 600 seconds, and Regeneration solution at a flow rate of 20 μL / min for 30 seconds. This series of four steps was measured with zolbetuximab at 8 concentrations of purified human claudin 18.2 protein ranging from 1280 nM to 10 nM at a 2-fold common ratio. As a result of the measurement, the binding and dissociation sensorgrams of both the h # 3 and h # 15 antibodies were curve-shaped, revealing that the human claudin 18.2 protein is difficult to dissociate from the h # 3 and h # 15 antibodies, and in particular hardly dissociates from the h # 15 antibody. In contrast, the sensorgram of the binding and dissociation of zolbetuximab was box-shaped, revealing that human claudin 18.2 protein dissociated from zolbetuximab very rapidly.These results indicated that the h#3 and h#15 antibodies bind more strongly to human claudin 18.2 protein than zolbetuximab. Furthermore, to quantify the difference in binding strength, KD values ​​were calculated using Cytiva's Biacore S200 Evaluation Software (29136649). Kinetics analysis was used to calculate the KD values ​​of the h#3 and h#15 antibodies, and affinity analysis was used to calculate the KD value of zolbetuximab. As a result, the KD values ​​of the h#3, h#15, and zolbetuximab were approximately 7 nM, 3 nM, and 215 nM, respectively. These results also indicated that the h#3 and h#15 antibodies bind more strongly to human claudin 18.2 protein than zolbetuximab.

Claims

1. An anti-human claudin 18.2 (hCLDN18.2) antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region according to any one of (a) to (f) below: (a) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 30, CDR2 consisting of the amino acid sequence of SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, CDR2 consisting of the amino acid sequence of SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, CDR2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 41; (c) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 45, CDR2 consisting of the amino acid sequence of SEQ ID NO: 46, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 48, CDR2 consisting of the amino acid sequence of SEQ ID NO: 49, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 53; (e) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 57, CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 59;or (f) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 60, CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 63, CDR2 consisting of the amino acid sequence of SEQ ID NO: 64, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 65; 2. The anti-hCLDN18.2 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region according to any one of the following (a) to (c): (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 67; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 69; or (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 70 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

71.

3. The monoclonal antibody of claim 1 or 2, which is a humanized antibody.

4. The anti-hCLDN18.2 antibody according to any one of claims 1 to 3, comprising a heavy chain and a light chain according to any one of (a) to (c) below: (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 72 and a light chain consisting of the amino acid sequence of SEQ ID NO: 73; (b) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 74 and a light chain consisting of the amino acid sequence of SEQ ID NO: 75; or (c) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 76 and a light chain consisting of the amino acid sequence of SEQ ID NO:

77.

5. A post-translationally modified anti-hCLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A fusion or complex of the anti-hCLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 5, or a cell expressing the anti-hCLDN18.2 antibody or its antigen-binding fragment of the present invention on the cell surface.

7. A pharmaceutical composition for treating cancer that expresses claudin 18.2, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, wherein the cancer expressing claudin 18.2 is gastric cancer, pancreatic cancer, lung cancer, or esophageal cancer.

9. A method for treating cancer that expresses claudin 18.2, comprising administering to a subject a monoclonal antibody or antigen-binding fragment thereof described in any one of claims 1 to 6.

10. The pharmaceutical composition according to claim 9, wherein the cancer expressing claudin 18.2 is gastric cancer, pancreatic cancer, lung cancer, or esophageal cancer.

11. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

12. An expression vector comprising the polynucleotide of claim 11.

13. A diagnostic agent for cancer expressing claudin 18.2, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

14. A kit for confirming the cross-reactivity of anti-human claudin antibodies, comprising a combination of an epithelial cell line in which all claudins have been knocked out and one or more epithelial cell lines that express 1 to 26 types of human claudins but do not express other claudins.

15. The kit according to claim 14, comprising an epithelial cell line in which all claudins have been knocked out, an epithelial cell line that expresses only one type of human claudin and no other claudins, and an epithelial cell line that expresses one or more human claudins other than the one type of human claudin and no other claudins.

Images