Anti-GPR20 car and cells containing same

Abstract

Provided are: a CAR which targets GPR20 and exhibits a pharmacological activity against GIST cells; and cells each containing the CAR.　Specifically provided are: a CAR which contains an anti-GPR20 antigen-binding site containing specific CDRs; CAR-expressing cells each containing the CAR; and others.

Description

Anti-GPR20 CAR and cells containing same

[0001] The present invention relates to a chimeric antigen receptor (CAR) that recognizes GPR20 and a cell containing the same.

[0002] Cancer is a leading cause of death, and its incidence is expected to increase with the aging of the population, yet there is still an insufficient need for treatment. Conventional chemotherapeutic agents have side effects due to their low selectivity, which can cause damage to not only tumor cells but also normal cells, and the inability to administer sufficient amounts of drug can result in insufficient efficacy. For this reason, in recent years, more selective molecular targeted drugs, antibody drugs, and cell therapies have been developed that target molecules that exhibit characteristic mutations or high expression in cancer cells, or specific molecules involved in the carcinogenesis of cells.

[0003] Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that develop in the digestive tract and mesentery, from the esophagus to the rectum, with an annual incidence of 1 to 2 cases per 100,000 (Non-Patent Document 1). Approximately 86% of GISTs harbor activating mutations in the receptor tyrosine kinases KIT or PDGFRA, contributing to tumor cell proliferation. GIST treatment is primarily surgical resection, but tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib, and regorafenib are prescribed for unresectable, advanced, or metastatic GISTs (Non-Patent Document 2). While these TKIs often show remarkable efficacy against GISTs with the above mutations, they require continuous administration and are unable to completely eliminate GISTs. Ultimately, secondary mutations in the target KIT or PDGFRA, activating mutations in RAS and BRAF, and activation of other signaling pathways lead to drug unresponsiveness and progression of the disease. Furthermore, for wild-type GISTs, which do not have mutations in KIT or PDGFRA (a minority of cases), these TKIs show almost no therapeutic effect (Non-Patent Document 3). For this reason, the development of an effective treatment method for TKI-resistant GISTs has been desired.

[0004] GPR20 (G Protein-coupled receptor 20) is a seven-transmembrane protein consisting of 358 amino acids that belongs to the class A of the G protein-coupled receptor (GPCR) family, with the N-terminus extracellular and the C-terminus intracellular. The human GPR20 gene was first cloned in 1997 (Non-Patent Document 4), but the deduced amino acid sequence was partially different from that of a human GPR20 gene cloned by another researcher in 2008 (Non-Patent Document 5). The latter sequence, which is identical to the sequence registered in the NCBI human whole genome sequence analysis database, is currently published in a public database as the DNA sequence and amino acid sequence encoding human GPR20, and can be referenced by accession numbers such as NM_005293 and NP_005284 (NCBI).

[0005] GPR20 contains an amino acid sequence similar to that of GPCRs that recognize nucleotides or lipids, but its physiological function and in vivo ligand have not been identified. Experiments in which GPR20 was expressed in HEK293 cells have reported that GPR20 constitutively activates Gi-type trimeric G proteins in the absence of ligand stimulation (Non-Patent Document 5).

[0006] GPR20 messenger RNA (mRNA) expression has been confirmed in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, rectum, and leukocytes, with particularly high expression reported in the small intestine (Non-Patent Document 5). In the brain, expression has been reported in the thalamus, putamen, and caudate nucleus (Non-Patent Document 4). GPR20-deficient mice exhibit a hyperactivity disorder phenotype characterized by an increase in total movement distance in an open field test, suggesting that GPR20 is associated with spontaneous activity in the central nervous system (Patent Document 1). It has also been reported that GPR20 is highly expressed in GISTs (Non-Patent Document 6), and that GPR20 expression is regulated by ets variant 1 (ETV1), a transcription factor highly expressed in GISTs (Non-Patent Document 7).

[0007] Antibodies are expected to reduce side effects because they specifically bind to target antigens, and many antibody drugs have been developed against molecules that are highly expressed on the surface of cancer cells. However, their efficacy is limited. As a technology to improve efficacy while suppressing side effects, glycosylation technology that enhances antibody-dependent cellular cytotoxicity (ADCC) (Non-Patent Document 8) and antibody-drug conjugates (ADC) have been researched (Non-Patent Document 9), and some approved drugs exist.

[0008] Monoclonal antibodies against GPR20 such as 04-046, h046-H4e / L7, and 04-093 have been reported (Patent Documents 2 and 3), and antibody-drug conjugates in which antitumor compounds are bound to these antibodies have been evaluated in clinical trials, but their development has been discontinued due to insufficient efficacy (Non-Patent Document 10).

[0009] An example of an antibody-based modality is chimeric antigen receptor (CAR)-T cells, which recognize cancer cell-specific antigens. CARs are molecules that broadly contain three domains: an extracellular domain containing a binding domain for the target molecule, a transmembrane domain, and an intracellular domain that transmits stimuli into the cell. CARs are capable of specifically damaging cancer cells that express the target antigen (Non-Patent Document 11). As CAR-T cell therapy, CAR-T cells directed against CD19 have been approved for acute lymphoblastic leukemia and diffuse large B-cell lymphoma, and CAR-T cells directed against BCMA have been approved for multiple myeloma (Non-Patent Document 12).

[0010] The properties of antibodies suitable for CAR-T cells have not yet been elucidated. For example, in the case of CAR-T cells against GPRC5D, CAR-T cells have been generated based on multiple antibodies that bind to the antigen. However, it has been reported that some antibodies exhibit antigen-nonspecific cytotoxic activity, and that cytotoxic activity also varies depending on the hinge length (Non-Patent Document 13). With regard to affinity, while some reports have shown that antibodies with high affinity have improved cytotoxic activity (Non-Patent Document 14), others have shown that antibodies with low affinity have improved efficacy (Non-Patent Document 15). The properties of antibodies suitable for CAR-T cells cannot be predicted, and generating CAR-T cells that can be expected to have the desired specific efficacy requires a great deal of trial and error and ingenuity.

[0011] It is also known that CAR-T cells are unlikely to be effective against solid tumors. To date, there have been no reports that CAR-T cells targeting GPR20 actually exhibit efficacy against GIST cells.

[0012] US Patent Application Publication No. 2003 / 0018989 WO 2018-135501 WO 2018-181656

[0013] Nat Rev Cancer. 2011 Nov 17;11(12):865-78J Natl Compr Canc Netw. 2010 April; 8(0 2): S1-S44Drugs. 2015 Aug;75(12):1323-34Gene. 1997 Mar 10;187(1):75-81J Biol Chem. 2008 May 9;283(19):12747-55Cancer Res. 2001 Dec 15;61(24):8624-8Nature. 2010 Oct 14;467(7317):849-53J Biol Chem. 2002 Jul 26;277(30):26733-40Drug Discov Today. 2014 Jul;19(7):869-81Clin Cancer Res. 2023 Sep 15;29(18):3659-3667Cells. 2019 May 17;8(5):472Front Immunol. 2023 May 15:14:1188049Sci Transl Med. 2019 Mar 27;11(485):eaau7746Clin Cancer Res. 2013 Jun 15;19(12):3153-64.Nat Med. 2019 Sep;25(9):1408-1414

[0014] An object of the present invention is to provide a novel therapeutic method or drug that exhibits good efficacy against GIST cells, for which existing drug-conjugated anti-GPR20 antibodies have been insufficiently effective.

[0015] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that CAR-T cells expressing on their cell surface a CAR comprising an anti-GPR20 antigen-binding site containing a specific CDR exhibit strong cytotoxic activity against GPR20-positive cancer cell lines. Furthermore, the present inventors have found that CAR-T cells expressing on their cell surface a CAR comprising an anti-GPR20 antigen-binding site containing a specific CDR exhibit significantly greater tumor regression effects than drug-conjugated anti-GPR20 antibodies or the anti-tumor kinase inhibitor (also called tyrosine kinase inhibitor) ripretinib in a patient-derived GIST-implanted mouse model, thereby completing the present invention. That is, the present invention encompasses the following inventions. [1] An anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, wherein the antigen-binding site comprises: (1) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, with one or several amino acids substituted, deleted, or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, with one or several amino acids substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, with one or several amino acids substituted, deleted, or added; and the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, with one or several amino acids substituted, deleted, or added; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, with one or several amino acids substituted, deleted, or added; and (2) a light chain variable (VL) region comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26 in which one or several amino acids have been substituted, deleted, or added; or (3) a light chain variable (VL) region comprising the following CDRs: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69 in which one or several amino acids have been substituted, deleted, or added; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70 in which one or several amino acids have been substituted, deleted, or added; andthe anti-GPR20 chimeric antigen receptor (CAR), comprising: a heavy chain variable (VH) region comprising: an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71, with one or several amino acids substituted, deleted or added; and a light chain variable (VL) region comprising the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72, with one or several amino acids substituted, deleted or added; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73, with one or several amino acids substituted, deleted or added; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74, with one or several amino acids substituted, deleted or added. [2] The CAR according to [1], wherein the antigen-binding site comprises: (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) a light chain variable (VL) region comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:26; (d) a heavy chain variable (VH) region comprising the following CDRs: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:63; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:64; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:65; and the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:66; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:67; and(d) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74. [3] The CAR according to [1] or [2], wherein the antigen-binding site comprises the VH region and VL region described in (b) or the VH region and VL region described in (d). [4] The CAR according to any one of [1] to [3], wherein the antigen-binding site comprises: (a) a VH region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid numbers 124 to 246 in SEQ ID NO: 5, and a VL region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid numbers 1 to 108 in SEQ ID NO: 5; or (b) a VH region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid numbers 1 to 123 in SEQ ID NO: 7, and a VL region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid numbers 139 to 246 in SEQ ID NO: 7. [5] The CAR according to any one of [1] to [4], wherein the antigen-binding site has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, or at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7. [6] The CAR according to any one of [1] to [5], comprising, from the N-terminus, a signal peptide, the antigen-binding site, a hinge region, a transmembrane region, a costimulatory region, and an intracellular signal region, wherein: the signal peptide has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11;The CAR, wherein the hinge region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1; the transmembrane region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2; the costimulatory region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3; and the intracellular signal region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4. [7] A polynucleotide encoding the CAR according to any one of [1] to [6]. [8] A CAR gene expression vector comprising the polynucleotide according to [7]. [9] A method for producing a CAR-T cell, comprising a step of in vitro gene transfer into a cell using the polynucleotide according to [7] or the CAR gene expression vector according to [8].

[10] The method according to [9], wherein the cell is a T cell.

[11] A CAR-expressing cell expressing the CAR according to any one of [1] to [6].

[12] The CAR-expressing cell according to

[11] , which is a CAR-T cell.

[13] A pharmaceutical composition comprising the CAR-expressing cell according to

[11] or

[12] .

[14] The pharmaceutical composition according to

[13] , for use in cell therapy for treating a GPR20-positive tumor.

[15] The pharmaceutical composition according to

[14] , wherein the cell therapy is allogeneic cell therapy or autologous cell therapy.

[16] The pharmaceutical composition according to

[14] or

[15] , wherein the GPR20-positive tumor is a gastrointestinal stromal tumor (GIST).

[17] A method for treating a GPR20-positive tumor, comprising administering to a subject the CAR-expressing cell according to

[11] or

[12] or the pharmaceutical composition according to any one of

[13] to

[16] .

[18] The method according to

[17] , wherein the subject is a human.

[19] The method according to

[17] or

[18] , wherein the method is allogeneic cell therapy or autologous cell therapy.

[20] The method according to any one of

[17] to

[19] , wherein the GPR20-positive tumor is a gastrointestinal stromal tumor (GIST).

[21] Use of the CAR-expressing cell of

[11] or

[12] or the pharmaceutical composition of

[13] for cell therapy to treat a GPR20-positive tumor.

[22] Use of the CAR-expressing cell of

[11] or

[12] in the manufacture of a pharmaceutical composition for use in cell therapy to treat a GPR20-positive tumor.

[23] An anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, and which binds to an epitope to which at least one antigen-binding site selected from the group consisting of the following (a) to (d) binds; (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and (c) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable (VL) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68; or (d) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69;a heavy chain variable (VH) region comprising: an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:70; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:71; and a light chain variable (VL) region comprising the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:72; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:73; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:74.

[24] An anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, and which competes with at least one antigen-binding site selected from the group consisting of the following (a) to (d) for binding to human GPR20; (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and (c) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68; or (d)a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[25] The CAR according to any one of [1] to [6], wherein cells expressing the CAR have cytotoxic activity against GPR20-positive cancer cells.

[0016] The CAR of the present invention and CAR-expressing cells comprising the same exhibit strong cytotoxic activity against GPR20-positive cancer cells because they contain an anti-GPR20 antigen-binding site containing a specific CDR.

[0017] Figure 1 shows the evaluation of the cytotoxic activity of anti-GPR20 chimeric antigen receptor (CAR)-T cells (h046-L7 / H4e-BBz CAR-T and h046-H4e / L7-BBz CAR-T) against GIST cell lines. Figure 2 shows the evaluation of the cytotoxic activity of anti-GPR20 chimeric antigen receptor (CAR)-T cells (h046-H5b / L2-BBz CAR-T) against GIST cell lines. Figure 3 shows the evaluation of the cytotoxic activity of anti-GPR20 chimeric antigen receptor (CAR)-T cells (04-093-L / H-BBz CAR-T and 04-093-H / L CAR-T) against GIST cell lines. Figure 4 shows the evaluation of CAR-T cells using multiple anti-GPR20 antibodies in a mouse model transplanted with a GIST cell line. Figure 5 shows a comparison of the in vivo efficacy of anti-GPR20 chimeric antigen receptor (CAR)-T cells and a drug-conjugated anti-GPR20 antibody in a mouse model transplanted with patient-derived GIST-040 cells. Figure 6 shows a comparison of the in vivo efficacy of anti-GPR20 chimeric antigen receptor (CAR)-T cells and Ripretinib in a mouse model transplanted with patient-derived GIST-040 cells. Figure 7-1 shows a sequence listing of the sequences referred to herein. Figure 7-2 shows a sequence listing of the sequences referred to herein. Figure 7-3 shows a sequence listing of the sequences referred to herein. Figure 7-4 shows a sequence listing of the sequences referred to herein. Figure 7-5 shows a sequence listing of the sequences referred to herein. Figure 7-6 shows a sequence listing of the sequences referred to herein. Figure 7-7 shows a sequence listing of the sequences referred to herein. Figures 7-8 show a sequence listing of the sequences referred to herein. Figures 7-9 show a sequence listing of the sequences referred to herein. Figures 7-10 show a sequence listing of the sequences referred to herein.

[0018] <Anti-GPR20 Chimeric Antigen Receptor (CAR)> A CAR is a receptor that contains an antigen-binding portion that recognizes an antigen specifically expressed in cancer cells, a hinge region, a transmembrane region, and an intracellular domain, and can specifically recognize and damage cancer cells that express a specific antigen.

[0019] The CAR of the present invention comprises, as its antigen-binding moiety, an antigen-binding site that specifically binds to GPR20, and is thereby able to specifically recognize and damage cancer cells expressing GPR20. <Antigen-binding site that specifically binds to GPR20> In the present invention, GPR20 is preferably human GPR20. The nucleotide sequence of human GPR20 cDNA is registered under accession number NM_005293 (NCBI). The amino acid sequence of human GPR20 is registered under accession number NP_005284 (NCBI). Note that, in the amino acid sequence of human GPR20, mutant proteins that have one or several amino acids substituted, deleted, or added and have biological activity equivalent to that of human GPR20 are also included in the human GPR20 referred to herein.

[0020] As used herein, the term "an amino acid sequence in which one or several amino acids have been substituted, deleted, or added" refers to an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, and / or added to the target amino acid sequence. "Several amino acids" means 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 amino acids.

[0021] The amino acid sequence of human GPR20 protein is set forth in SEQ ID NO: 135 in the Sequence Listing, and the extracellular region is composed of an extracellular domain (EC1) consisting of amino acid numbers 1 to 48, an extracellular domain (EC2) consisting of amino acid numbers 108 to 125, an extracellular domain (EC3) consisting of amino acid numbers 190 to 196, and an extracellular domain (EC4) consisting of amino acid numbers 260 to 275 of SEQ ID NO: 135 in the Sequence Listing.

[0022] The CAR of the present invention comprises an antigen-binding site that specifically binds to GPR20. The ability of the antigen-binding site to bind to GPR20 can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blot), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), or biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507).

[0023] The antigen-binding site contained in the CAR of the present invention may be a monoclonal antibody (mAb) capable of binding to GPR20, or may be prepared by modifying the sequence of the mAb while maintaining its binding to GPR20. Examples of the antigen-binding site include single-chain variable fragments (scFv), Fab, scFab, and F(ab') capable of binding to GPR20. ２ Antigen-binding regions of antibodies, including fragments, can also be used as antigen-binding sites in the present invention. "Antigen-binding region" refers to any fragment of an antibody that is capable of binding to a specific target of a given antibody.

[0024] The epitope of the antigen-binding site contained in the CAR of the present invention is not particularly limited as long as cells expressing the CAR have cytotoxic activity against GPR20-positive cancer cells, and may be, for example, the extracellular domain of GPR20, preferably EC1 and / or EC2.

[0025] As used herein, "epitope" refers to a partial peptide or partial three-dimensional structure of GPR20 to which the antigen-binding site of a CAR binds. The epitope, which is a partial peptide of GPR20, can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of GPR20 are prepared. A known oligonucleotide synthesis technique can be used to prepare the partial structures. For example, a series of polypeptides successively shortened to an appropriate length from the C-terminus or N-terminus of GPR20 can be prepared using genetic recombination techniques well known to those skilled in the art, and the reactivity of the antigen-binding site with these polypeptides can be examined to determine the approximate recognition site. After determining the reactivity of these polypeptides with the antigen-binding site, further shorter peptides can be synthesized and their reactivity can be examined to determine the epitope. Furthermore, when the antigen-binding site has a three-dimensional structure consisting of multiple extracellular domains in GPR20 as an epitope, the domain to which the antigen-binding site binds can be determined by modifying the amino acid sequence of a specific extracellular domain to alter the three-dimensional structure. The epitope can also be determined by identifying the amino acid residues of GPR20 adjacent to the antigen-binding site by X-ray structural analysis.

[0026] As used herein, "binding to the same epitope" means that the antigen-binding sites bind to a common epitope. If the second antigen-binding site binds to a partial peptide or partial steric structure to which the first antigen-binding site binds, it can be determined that the first and second antigen-binding sites bind to the same epitope. Furthermore, by confirming that the second antigen-binding site competes with the binding of the first antigen-binding site to the antigen (i.e., that the second antigen-binding site interferes with the binding of the first antigen-binding site to the antigen), it can be determined that the first and second antigen-binding sites bind to the same epitope, even if the sequence or structure of the specific epitope has not been determined. Therefore, by confirming that the second antigen-binding site competes with the binding of the first antigen-binding site to the partial peptide or partial steric structure to which the first antigen-binding site binds, it can be determined that the first and second antigen-binding sites bind to the same epitope of GPR20. A CAR comprising a first antigen-binding site and a CAR comprising an antigen-binding site that binds to the same epitope can be expected to have similar activity.

[0027] For example, scFv can be obtained by linking the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, eds., Springer Verlag, New York, pp. 269-315 (1994); Nature Biotechnology (2005), 23, pp. 1126-1136). In addition, a BiscFv fragment prepared by linking two scFvs with a polypeptide linker can also be used as a bispecific antigen-binding region.

[0028] Methods for producing scFvs are well known in the art (see, for example, U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and 5,455,030). In these scFvs, the VH and VL domains are linked via a linker, preferably a polypeptide linker, that does not form a conjugate (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, pp. 5879-5883). In these scFvs, the order in which the heavy chain variable (VH) domain and the light chain variable (VL) domain are linked is not particularly limited. That is, the C-terminus of the heavy chain variable (VH) region and the N-terminus of the light chain variable (VL) region may be linked via a linker (such a linking order may be expressed as HL or H / L), or the C-terminus of the light chain variable (VL) region and the N-terminus of the heavy chain variable (VH) region may be linked via a linker (such a linking order may be expressed as LH or L / H). The heavy chain variable region and the light chain variable region of the scFv may be derived from the same antibody or from different antibodies.

[0029] Antibodies generally contain six complementarity-determining regions (CDRs). The six CDRs are three in the VH region: HC-CDR1, HC-CDR2, and HC-CDR3, from the N-terminus to the C-terminus, and three in the VL region: LC-CDR1, LC-CDR2, and LC-CDR3, from the N-terminus to the C-terminus. Together, the six CDRs define the antibody paratope, which is the portion of the antibody that binds to the target antigen. The VH and VL regions contain framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. In the present invention, when the antigen-binding site is an scFv, the order in which the heavy and light chains are linked (HL / LH) is not important unless otherwise specified.

[0030] To define the CDRs and FRs of an antibody, several methods are used: the definition of Kabat et al. (Kabat numbering scheme, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); the definition of Al-Lazikani et al. (Chothia numbering scheme, J. Mol. Biol., 196:901-917 (1987)); the method of Martin et al. (AbM numbering scheme, a compromise between Kabat and Chothia; Martin, A. C. R., Cheetham, J. C. and Rees, A. R. (1989) Proc. Natl. Acad. Sci. USA, 86, 9268-9272); and the definition of Lefranc et al. (IMGT numbering scheme. Developmental and Comparative Immunology, 27 (2003), 55-77).

[0031] The antigen-binding site contained in the CAR of the present invention contains six specific CDRs, and therefore exhibits strong cytotoxic activity against GPR20-positive cancer cells when incorporated into the CAR.

[0032] Specifically, the CAR of the present invention comprises the following specific CDRs: (1) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, with one or several amino acids substituted, deleted, or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, with one or several amino acids substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, with one or several amino acids substituted, deleted, or added; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, with one or several amino acids substituted, deleted, or added; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, with one or several amino acids substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, with one or several amino acids substituted, deleted, or added; or (2) the following CDRs: a heavy chain variable (VH) region comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69, with one or several amino acids substituted, deleted or added; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70, with one or several amino acids substituted, deleted or added; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71, with one or several amino acids substituted, deleted or added; and a light chain variable (VL) region comprising the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72, with one or several amino acids substituted, deleted or added; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73, with one or several amino acids substituted, deleted or added; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74, with one or several amino acids substituted, deleted or added.Here, the "amino acid sequence in which one or several amino acids have been substituted, deleted, or added" refers to an amino acid sequence in which one to several amino acids have been deleted, substituted with other amino acids, inserted with other amino acids, and / or added with other amino acids, as described above for the "amino acid sequence in which one or several amino acids have been substituted, deleted, or added." "Several amino acids" means 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 amino acids.

[0033] The CAR of the present invention comprises the above-mentioned CDRs, and may comprise the following CDRs: (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; a heavy chain variable (VH) region comprising: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO:21; a HC-CDR2 comprising the amino acid sequence of SEQ ID NO:22; and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable (VL) region comprising the following CDRs: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:25; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:26; (c) a consensus (according to the AbM numbering scheme) derived from ScFvh046-H5b / L2: a heavy chain variable (VH) region comprising: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO:63; a HC-CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO:65; and a light chain variable (VL) region comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:66; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:67; and (d) a light chain variable (VL) region comprising the following consensus (according to the IMGT numbering scheme) CDRs derived from ScFvh046-H5b / L2: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:69; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:70;and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74. Furthermore, the antigen-binding site comprised in the CAR of the present invention may be an antigen-binding site that binds to the same epitope as the antigen-binding site comprising the CDRs. That is, it may be an antigen-binding site that recognizes the same partial peptide or partial three-dimensional structure of GPR20 as the partial peptide or partial three-dimensional structure recognized by the antigen-binding site comprising the CDRs, or it may be an antigen-binding site that competes for binding to GPR20 with the antigen-binding site comprising the CDRs.

[0034] Since 04-046, an anti-GPR20 antibody comprising the above CDR (a) or (b), has been suggested to bind to EC1 and EC2 in GPR20 (Cancer Discov. 2021 June 11(6):1508-1523), it is suggested that an antigen-binding site comprising the above CDR (a) or (b) or the above CDR (c) or (d) which is similar thereto also binds to EC1 and EC2.

[0035] Therefore, the CAR of the present invention may be an anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, and which binds to an epitope to which at least one antigen-binding site selected from the group consisting of the following (a) to (d) binds; (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and (c) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable (VL) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68; or (d) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a heavy chain variable (VH) region comprising: an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:70; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:71; and the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:72; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:73;and a light chain variable (VL) region comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74;

[0036] Furthermore, the CAR of the present invention may be an anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, and which competes with at least one antigen-binding site selected from the group consisting of the following (a) to (d) for binding to human GPR20: (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (c) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 64; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable (VL) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68; or (d) a light chain variable (VL) region comprising the following CDRs: a heavy chain variable (VH) region comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:69; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:70; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:71; and the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:72; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:73;and a light chain variable (VL) region comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74;

[0037] Furthermore, the antigen-binding site comprised in the CAR of the present invention is not particularly limited as long as cells expressing the CAR have cytotoxic activity against GPR20-positive cancer cells, and may comprise the above-described CDRs and include any of the following: (a) a VH region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 124 to 246 in SEQ ID NO: 5, and a VL region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 1 to 108 in SEQ ID NO: 5; or (b) a VH region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 1 to 123 in SEQ ID NO: 7, and a VL region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 139 to 246 in SEQ ID NO: 7.

[0038] Furthermore, the antigen-binding site comprised in the CAR of the present invention may have 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence of humanized antibody ScFvh046-L7 / H4e comprising the amino acid sequence of SEQ ID NO: 5, humanized antibody ScFvh046-H4e / L7 comprising the amino acid sequence of SEQ ID NO: 6, or humanized antibody ScFvh046-H5b / L2 comprising the amino acid sequence of SEQ ID NO: 7, respectively.

[0039] As used herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Percent identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, the various conditions (parameters) for identity searches using the BLAST program are described in Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available from the websites of NCBI and the DNA Data Bank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, MD 20894; Altschul et al.). Furthermore, genetic information processing software GENETYX Ver. The identity of a nucleotide sequence can also be determined using a program such as DNA ELISA Kit 7 (Genetics), DNASIS Pro (Hitachi Software), or Vector NTI (Infomax). The sequence identity of a nucleotide sequence can also be determined in a similar manner. <Hinge region, transmembrane region, and intracellular domain> The CAR of the present invention comprises a hinge region, transmembrane region, and intracellular domain in addition to the antigen-binding molecule described above.

[0040] The hinge region, transmembrane region, and intracellular domain contained in the CAR of the present invention may be any as long as they are expressed in a T cell, receive antigen information via an antigen-binding molecule, and can exert cytotoxic activity against cells expressing the antigen.

[0041] Specifically, the hinge region, transmembrane region, and intracellular domain comprised in the CAR of the present invention include a hinge region connected to the C-terminus of the antigen-binding molecule as the hinge region, a transmembrane region connected to the C-terminus of the hinge region as the transmembrane region, and a costimulatory region connected to the C-terminus of the transmembrane region as the intracellular domain, and an intracellular signal region.

[0042] The hinge region functions to connect the antigen-binding molecule and the transmembrane region to overcome steric hindrance and ensure access of the antigen-binding molecule to its target antigen. For example, the hinge region may be derived from a T cell receptor (TCR) or a TCR co-receptor, and may comprise an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with a human CD8a-derived hinge comprising the amino acid sequence of SEQ ID NO: 1.

[0043] The transmembrane domain may be any polypeptide that has the property of being present in the cell membrane. For example, the transmembrane domain may be a polypeptide that is naturally associated with an antigen-binding molecule and / or an intracellular domain. Examples of such polypeptides include those that contain an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% identical to the transmembrane domain of human CD8a, which contains the amino acid sequence of SEQ ID NO: 2.

[0044] The costimulatory region may be any region capable of transmitting an auxiliary signal for T cell activation. For example, the costimulatory region may be derived from a TCR co-receptor and may comprise an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with a human 4-1BB-derived costimulatory region comprising the amino acid sequence of SEQ ID NO:3.

[0045] The intracellular signal region may be any region that has the function of transmitting one or more signals that, upon binding between the antigen-binding molecule and a target antigen, transmit a primary stimulus into a T cell and initiate and / or transmit an intracellular signal that activates the immune cell effector function of the T cell. For example, the intracellular signal region may be derived from a TCR and may comprise an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to an intracellular signal region derived from human CD3z, which comprises the amino acid sequence of SEQ ID NO: 4. <Signal Peptide> The CAR of the present invention may further comprise a signal peptide at the N-terminus of the antigen-binding molecule.

[0046] The signal peptide may be any signal peptide as long as it has the function of transporting and localizing the antigen-binding molecule to the surface of T cells. For example, the signal peptide may be derived from TCR and may comprise an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with a signal peptide derived from human CD8a containing the amino acid sequence of SEQ ID NO: 11. <Other Regions of CAR> The CAR of the present invention may comprise additional regions depending on the purpose, as long as the additional regions do not inhibit its function of exerting cytotoxic activity against GPR20-positive cancer cells. Examples of such additional regions include a detectable region and a spacer region for operably linking any of the regions in the CAR. The detectable region refers to a moiety for detecting the CAR of the present invention or the antigen-binding molecule contained in the CAR by a known method, and examples thereof include various radioactive labels, fluorescent labels, luminescent labels, immunodetectable labels, etc. Radioactive labels include iodine １２３ , iodine １２５ , iodine １２６ , iodine １３１ , iodine １３３ ,bromine ７７ , technetium ９９ｍ ,indium １１１ ,indium １１３ｍ ,gallium ６７,gallium ６８ ,ruthenium ９５ ,ruthenium ９７ ,ruthenium １０３ ,ruthenium １０５ ,mercury ２０７ ,mercury ２０３ ,rhenium ９９ｍ ,rhenium １０１ ,rhenium １０５ ,scandium ４７ , tellurium １２１ｍ , tellurium １２２ｍ , tellurium １２５ｍ ,thulium １６５ ,thulium １６７ ,thulium １６８ ,copper ６７ , fluorine １８ ,yttrium ９０ ,palladium １００ , bismuth ２１７ , and antimony ２１１ Examples of suitable labels include radioisotopes such as Fluorescent Labels (GFP, BFP, Y ...

[0047] The spacer region refers to a non-coding region for operably linking each region contained in the CAR, and its amino acid sequence can be determined arbitrarily by those skilled in the art. <Polynucleotide encoding a CAR> The present invention also relates to a polynucleotide encoding the above-mentioned CAR. The polynucleotide encoding a CAR refers to a polynucleotide comprising a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned CAR. As used herein, "polynucleotide" refers to a polymer chain of nucleotides and can be DNA, RNA, or a combination thereof. Specifically, the polynucleotide encoding a CAR can comprise, for example, a polynucleotide sequence comprising a DNA sequence or RNA sequence corresponding to the amino acid sequence of the CAR, or a cDNA sequence or mRNA sequence for expressing the CAR.

[0048] By introducing a polynucleotide encoding the CAR of the present invention into a cell as described below, the CAR can be expressed in the cell and exert strong cytotoxic activity against the target GPR20-expressing cancer cells. <CAR gene expression vector comprising a polynucleotide encoding a CAR> The present invention also relates to a CAR gene expression vector comprising a polynucleotide encoding the CAR. The vector of the present invention refers to a polynucleotide molecule modified to include the above-mentioned polynucleotide sequence and having the function of expressing a CAR in a cell. Examples of the vector of the present invention include DNA vectors, RNA vectors, retroviral vectors, adenoviral vectors, adenovirus-associated vectors, Epstein-Barr (EB) virus vectors, lentiviral vectors, and combinations thereof. For example, see Sambrook et al. The vector of the present invention can be prepared using standard techniques such as those described in "Molecular Cloning: A Laboratory Manual" (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). <Method for preparing a CAR-expressing cell> The present invention also relates to a method for preparing a CAR-expressing cell, the method comprising the step of introducing a polynucleotide encoding the CAR or the CAR gene expression vector into a cell.

[0049] Gene transfer refers to the introduction of foreign DNA encoding a gene into a host cell, and in the case of the present invention, refers to the introduction of a polynucleotide encoding a CAR gene or a CAR gene expression vector containing such a polynucleotide into a host cell. Gene transfer can be performed in vivo or in vitro. The method of gene transfer is not particularly limited, and examples include a method of infecting a host cell with a CAR gene expression vector, which is a viral vector; a method of introducing a CAR gene expression vector into a host cell by introduction using a transfection reagent such as microinjection, electroporation, or lipofection; and a method of introducing a CAR gene into the host cell genome by genome editing using ZFN, TALEN, the CRISPR-Cas9 system, or a combination thereof.

[0050] The host cells used in the CAR-expressing cell of the present invention or the method for producing the same are not particularly limited, and include prokaryotic host cells such as Escherichia coli, or eukaryotic host cells such as mammalian cells of human, monkey, mouse, etc., plant cells, and yeast. Human cells are preferred.

[0051] The type of cells used in the present invention is not particularly limited, and any type of cell can be used. For example, cells collected, isolated, or purified from body fluids, tissues, or organs, such as blood (peripheral blood, umbilical cord blood, etc.) or bone marrow, or cells differentiated from the above-mentioned cells, or pluripotent stem cells such as iPS cells and ES cells can be used (see, for example, Themeli et al. 2013). Specifically, for example, peripheral blood mononuclear cells (PBMCs), immune cells (including, for example, T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or hematopoietic cells (neutrophils, basophils)), or umbilical cord blood mononuclear cells can be used. For example, NK cells or T cells, T cell progenitors (hematopoietic stem cells, lymphocyte progenitors, etc.), or cell populations containing them can be used. Examples of T cells include CD8+ T cells, CD4+ T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell precursors include PBMCs. The above-mentioned cells may be collected from a living organism, obtained by expanding cells collected from a living organism, or established as a cell line. When transplantation of the prepared CAR-expressing cells or cells differentiated from the prepared CAR-expressing cells into a living organism is desired, a polynucleotide encoding the CAR of the present invention can be introduced into the living organism itself or cells collected from a living organism of the same species.

[0052] CAR-expressing cells prepared using the method of the present invention are cells expressing the CAR of the present invention on their cell membrane. Such cells have the property of binding to the antigen, GPR20, at the antigen recognition site of the CAR, and transmitting the binding signal to an intracellular signaling region, thereby activating the cells. Activation of the CAR-expressing cells of the present invention varies depending on the type of host cell and the intracellular domain contained in the CAR, and can be confirmed based on indicators such as cytokine release, increased cell proliferation rate, or changes in cell surface molecules.

[0053] When the host cell for the CAR-expressing cell of the present invention is a T cell, such a cell is also referred to as a CAR-T cell. When the host cell is a T cell, the cell activated by the above-mentioned signal transduction releases cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) and exerts cytocidal or cytotoxic activity against target cells. For example, as described below, the CAR-expressing cell can be used for cell therapy to treat GPR20-positive tumors.

[0054] The cytotoxic activity of CAR-expressing cells against target cells in vitro can be measured, for example, by measuring the cell growth inhibitory activity. For example, the cell growth inhibitory activity of CAR-expressing cells can be measured by adding CAR-expressing cells at various concentrations to a culture system of a cancer cell line overexpressing GPR20 and measuring the viability of the cancer cells. <Pharmaceutical composition comprising CAR-expressing cells for use in cell therapy to treat GPR20-positive tumors, and method for treating GPR20-positive tumors> The present invention also relates to the CAR-expressing cells or pharmaceutical compositions comprising the same for use in cell therapy to treat GPR20-positive tumors.

[0055] A GPR20-positive tumor refers to a tumor containing cells that express the GPR20 protein or GPR20 gene. As used herein, "tumor" refers to cells that grow progressively in a living organism. Among tumors, those in which abnormal cells spread to the surrounding area or metastasize to another organ or tissue are called "malignant tumors" or "cancers," and those derived from epithelial cells are called "cancers." However, as used herein, "tumor," "cancer," and "cancer" may be used interchangeably.

[0056] An example of a GPR20-positive tumor is a gastrointestinal stromal tumor (GIST) that expresses the GPR20 protein.

[0057] In the present invention, treatment of a GPR20-positive tumor refers to alleviating, improving, inhibiting the aggravation or progression of, or eliminating one or more symptoms caused by the presence of a GPR20-positive tumor. The in vivo effect of a CAR-expressing cell on a GPR20-positive tumor can be measured, for example, by administering the CAR-expressing cell to a tumor-bearing animal and measuring changes in tumor size, etc.

[0058] The cell therapy of the present invention for treating GPR20-positive tumors results in a cytotoxic effect specific to the GPR20-positive tumor in a subject by administering the CAR-expressing cells to the subject having the GPR20-positive tumor. The cell therapy of the present invention includes cell therapy using CAR-expressing cells derived from cells collected from the subject to be treated (i.e., autologous cell therapy), and cell therapy using CAR-expressing cells derived from cells collected from an individual other than the subject to be treated (i.e., allogeneic cell therapy). In both autologous and allogeneic cell therapy, CAR-expressing cells derived from collected host cells may be used as is, or CAR-expressing cells derived from stem cells such as iPS cells or ES cells differentiated into desired host cells and then introduced with a CAR gene may be used.

[0059] The subject of the cell therapy of the present invention may be any animal or human, preferably a mammal, more preferably a human.

[0060] The pharmaceutical composition of the present invention can comprise, in addition to a therapeutically effective amount of CAR-expressing cells, a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, adjuvant, and one or more combinations thereof.

[0061] The pharmaceutical compositions of the present invention may be formulated to be suitable for injection or infusion into a tumor or blood.

[0062] The present invention will be described in detail in the following examples, but the present invention is not limited to these examples.

[0063] Unless otherwise specified, genetic manipulation procedures in the following examples were performed according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E.F., and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) or other experimental manuals commonly used by those skilled in the art, or, when commercially available reagents or kits were used, according to the instructions provided with the commercially available products. Gene synthesis and vector construction were outsourced to Synthesis and Cloning (FASMAC Corporation, Thermo Fisher Scientific) as needed. Example 1: Preparation of CAR-T cells using multiple anti-GPR20 antibodies Example 1-1: Preparation of CAR gene expression vector using multiple anti-GPR20 antibodies CAR-T cells were prepared using the humanized antibody h046-H4e / L7, humanized antibody h046-H5b / L2 (see WO18135501 A1), and rat antibody 04-093 (see WO18181656 A1), which have been reported as anti-GPR20 antibodies, and it was verified whether they could be expressed on a membrane as CAR.

[0064] The anti-GPR20 CAR construct used consisted of, from the N-terminus, a signal peptide, anti-GPR20 antibody ScFv, a human CD8a-derived hinge region (SEQ ID NO: 1), a human CD8a-derived transmembrane region (SEQ ID NO: 2), a human 4-1BB-derived costimulatory region (SEQ ID NO: 3), and a human CD3z-derived intracellular signal region (SEQ ID NO: 4).The anti-GPR20 antibody ScFv used was humanized antibody ScFvh046-L7 / H4e (SEQ ID NO: 5), humanized antibody ScFvh046-H4e / L7 (SEQ ID NO: 6), humanized antibody ScFvh046-H5b / L2 (SEQ ID NO: 7), rat antibody ScFv04-093L / H (SEQ ID NO: 8), or rat antibody ScFv04-093H / L (SEQ ID NO: 9). In all cases, a 15-aa ScFv linker sequence (SEQ ID NO: 10) with three G4S repeats was used between the heavy chain and light chain, or between the light chain and heavy chain. The human CD8a signal peptide sequence (SEQ ID NO: 11) was used as the signal peptide. To confirm CAR expression, a FLAG tag sequence (SEQ ID NO: 12) was placed at the N-terminus of the anti-GPR20 antibody ScFv via a 4-amino acid spacer sequence (SEQ ID NO: 13). The anti-GPR20 CAR gene was prepared by gene synthesis.

[0065] A lentiviral plasmid vector for expressing anti-GPR20 CAR was constructed. The gene sequence arranged as described above was introduced into the Multiple Cloning Site of pLVSIN EF1a IRES ZsGreen1 (Takara). To confirm gene introduction, the ZsGreen1 region following the IRES was removed, and BFP (SEQ ID NO: 14) was introduced into the same site. The lentiviral plasmid vectors for expressing CAR derived from each anti-GPR20 antibody ScFv of humanized antibody ScFvh046-L7 / H4e, humanized antibody ScFvh046-H4e / L7, humanized antibody ScFvh046-H5b / L2, rat antibody ScFv04-093L / H, and rat antibody ScFv04-093H / L, and BFP, were designated pLVSIN-EF1a- They were designated h046-L7 / H4e-BBz-IRES-BFP, pLVSIN-EF1a-h046-H4e / L7-BBz-IRES-BFP, pLVSIN-EF1a-h046-H5b / L2-BBz-IRES-BFP, pLVSIN-EF1a-04-093-L / H-BBz-IRES-BFP, and pLVSIN-EF1a-04-093-H / L-IRES-BFP.

[0066]

[0067]

[0068]

[0069]

[0070] The sequences of each CDR in the anti-GPR20 antibody ScFvs ScFvh046-L7 / H4e, ScFvh046-H4e / L7, and ScFvh046-H5b / L2 are shown in Table 2, with AbM numbering, IMGT numbering, Kabat numbering, and Chothia numbering.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Example 1-2: Generation of CAR-T cells using multiple anti-GPR20 antibodies According to the pLVSIN EF1a IRES ZsGreen1 protocol, each lentiviral plasmid vector prepared in Example 1-1 and Lentiviral High Titer Packaging Mix (Takara) were transfected into Lenti-X 293T Cell Line (Takara) using TransIT-293 Transfection Reagent (Takara). After two days, the culture supernatant was collected, and after a medium change, the cells were cultured for another day, and the supernatant was collected. The virus contained in the culture medium was concentrated using a Lenti-X concentrator (Takara) according to the protocol. For viral infection, plates were prepared by coating with 4-10 μg / mL of CD3 antibody (clone name: OKT3) and 20-100 μg / mL of RetroNectin according to the RetroNectin (Takara) protocol. Next, 0.1-2 × 10 human peripheral blood mononuclear cells were cultured in AIM-V medium (Thermo Fisher Scientific) containing 5% fetal bovine serum (FBS) (hereafter referred to as basal medium) supplemented with 100-200 U / mL IL-2 (hereafter referred to as growth medium). ６The cells were suspended at 1000 x g for 30-60 minutes, and then added to a retronectin-coated plate together with the virus concentrate. The plate was then centrifuged at 1000 x g for 30-60 minutes, and then transferred to an incubator for culture. The growth medium was replaced with fresh medium every 2-3 days, and culture was continued for 7-14 days to prepare T cells expressing anti-GPR20-CAR (anti-GPR20 CAR-T cells). CAR-T cells prepared using pLVSIN-EF1a-h046-L7 / H4e-BBz-IRES-BFP, pLVSIN-EF1a-h046-H4e / L7-BBz-IRES-BFP, pLVSIN-EF1a-h046-H5b / L2-BBz-IRES-BFP, pLVSIN-EF1a-04-093-L / H-BBz-IRES-BFP, and pLVSIN-EF1a-04-093-H / L-IRES-BFP were designated h046-L7 / H4e-BBz CAR-T, h046-H4e / L7-BBz CAR-T, and h046-H5b / L2-BBz, respectively. The anti-GPR20 antibody ScFvs, plasmid vectors, and CAR-T cells prepared were designated 04-093-L / H-BBz CAR-T, 04-093-L / H-BBz CAR-T, or 04-093-H / L CAR-T. Table 3 shows the correspondence between the anti-GPR20 antibody ScFvs, plasmid vectors, and prepared CAR-T cells used.

[0083] If necessary, the collected cells were cryopreserved using CELLBANKER1 (Takara). When using frozen cells for experiments, they were thawed and then incubated in a growth medium for one day before use.

[0084] Example 1-3: Confirmation of expression of anti-GPR20 CAR on T cells A solution prepared by diluting an APC-labeled anti-FLAG tag antibody (BioLegend) in FACS buffer (phosphate buffered saline containing 5% FBS) was added to each CAR-T cell, and the resulting solution was added to the cells and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were suspended in FACS buffer containing 7-aminoactinomycin D and measured using flow cytometry (MACSQUANT Analyzer 10 or BD FACSCanto II). After outputting the data as an FCS file, the CAR-positive rate was calculated from the APC fluorescence in live cells using FLOW JO software. As a result, as shown in Table 4, CAR expression on the membrane was confirmed for all anti-GPR20 CARs.

[0085] Example 2: Evaluation of cytotoxic activity of CAR-T cells using multiple anti-GPR20 antibodies Example 2-1: Establishment of cancer cell lines GIST-T1 nucGFP and GIST-T1 GPR20 KO nucGFP for evaluation of cytotoxic activity GIST-T1 (Cosmo Bio) was used as a GPR20-positive GIST cell line. DMEM medium supplemented with 10% FBS was used as the medium. Cells in which the GPR20 gene of GIST-T1 was knocked out using CRISPR / Cas9 were generated as a GPR20-negative GIST cell line. Knockout was performed using Lipofectamine CRISPRMAX Transfection Reagent (Thermo Fisher Scientific), GPR20 TrueGuide Synthetic gRNA (Thermo Fisher Scientific), and TrueCut Cas9 Protein v2 according to the protocol. GPR20 knockout GIST-T1 cells were cultured for approximately one month, and rat antibody 04-093 was suspended in FACS buffer at 10 μg / mL, added to the cells, and incubated at 4°C. After washing with FACS buffer, the cells were stained with Alexa Fluor 488 Signal-Amplification Kit for Mouse Antibodies, and the Alexa Fluor 488-negative fraction was sorted single-cell using a cell sorter SH800S (Sony). After culturing the sorted cells, the proliferated cells were suspended in FACS buffer with humanized antibody h046-H4eL7 at 10 μg / mL, added to the cells, and incubated at 4°C. APC anti-human IgG Fc Antibody (Biolegend) was diluted 100-fold with FACS buffer, added to the cells, and incubated at 4°C. The cells were washed with FACS buffer, suspended in FACS buffer containing DAPI, and confirmed to be GPR20 knockout cells using flow cytometry (MACSQUANT Analyzer 10). These cells were designated GIST-T1 GPR20KO.

[0086] To evaluate cytotoxic activity in Incucyte S3 (Sartorius), GIST-T1, GIST-T1 GPR20 KO, or GPR20-negative cells, AsPC-1, were infected with Incucyte Nucleight Green Lentivirus (puro) (Sartorius) according to the protocol. After infection, cells were selected with antibiotics in DMEM medium supplemented with 0.25 μg / ml puromycin and 10% FBS (GIST-T1, GIST-T1 GPR20 KO) or in RPMI medium supplemented with 1 μg / ml puromycin and 10% FBS (AsPC-1). The proliferated cells were used for the cytotoxic activity evaluation described below. Example 2-2: Evaluation of cytotoxic activity using Incucyte In the evaluation of cytotoxic activity, anti-GPR20 CAR-T cells were co-cultured with cancer cells GIST-T1, GIST-T1 GPR20 KO, or AsPC-1, and cancer cell proliferation was evaluated using Incucyte S3. A solution of an APC-labeled anti-FLAG tag antibody diluted 100-fold with FACS buffer was added to the anti-GPR20 CAR-T cells, and the cells were added and incubated at 4°C. After washing with FACS buffer, the cells were suspended in FACS buffer containing 1-10 μg / mL of 7-aminoactinomycin D and measured using flow cytometry (MACSQUANT Analyzer 10 or BD FACSCanto II). After outputting as an FCS file, the CAR-positive rate was calculated from the APC fluorescence in live cells using FLOW JO software. The number of anti-GPR20 CAR-T cells or cancer cells was counted using a cell analyzer NC-250 (Chemometec). For co-culture to evaluate cytotoxicity, 1 x 10 cancer cells were cultured in a 96-well microplate. ４ cells / well, 3x10 CAR positive cells ３ (E / T=0.3), 1x10 ４ (E / T=1), 3x10 ４The cells were seeded at 3 (E / T = 3) cells / well and observed over time using a fluorescence microscope using Incucyte S3 under culture conditions of 37°C and 5% CO2. Survival (%) was calculated using the following formula. Calculation formula: Survival (%) = 100 × ((cancer cell count in co-cultured CAR-T cells on day 2) / (cancer cell count in co-cultured CAR-T cells on day 0)) / ((cancer cell count in untreated CAR-T cell control on day 2) / (cancer cell count in untreated CAR-T cell control on day 0)) As a result, h046-L7 / H4e-BBz CAR-T, h046-H4e / L7-BBz CAR-T, and h046-H5b / L2-BBz CAR-T showed significant cytotoxic activity against GPR20-positive GIST-T1 ( Figures 1 and 2 ). On the other hand, 04-093-L / H-BBz CAR-T and 04-093-H / L CAR-T did not exhibit cytotoxic activity against GPR20-positive GIST-T1 (Figure 3). This indicates that h046-L7 / H4e-BBz CAR-T, h046-H4e / L7-BBz CAR-T, and h046-H5b / L2-BBz CAR-T are CAR-T cells that exhibit activity among CAR-T cells that use a GPR20 antibody. Example 3: Evaluation of CAR-T cells in a mouse model transplanted with a cancer cell line using multiple anti-GPR20 antibodies To evaluate the efficacy of GPR20 CAR-T cells in vivo, h046-L7 / H4e-BBz CAR-T and h046-H5b / L2-BBz CAR-T, which were anti-GPR20 CAR-T cells that showed antigen-dependent activity in vitro, anti-GPR20 CAR-T cells were administered to immunodeficient mice transplanted with a cancer cell line, and the ability to inhibit tumor growth was evaluated. The human GIST cancer line GIST-T1 (Cosmo Bio) was diluted at 5 x 10 in PBS containing 50% Matrigel (Corning). ７ The medium was adjusted to give a concentration of 1000 cells / mL, and 0.1 mL of the medium was subcutaneously transplanted into NOG or NSG mice (female or male, 4-6 weeks old) on Day 0. The major and minor axes (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0087] Estimated Tumor Volume (mm3) = average value of estimated tumor volume for each individual: Estimated tumor volume (mm3) for each individual = 1 / 2 × [tumor major axis] × [tumor minor axis] × [tumor minor axis] On Day 20, mice were divided into groups based on tumor volume so that each group had n = 4. One day later, anti-GPR20 CAR-T cells were injected into the mice at a concentration of 4 × 10 6 The solution was adjusted to give cells / mL, and 0.2 mL was administered into the tail vein.

[0088] As a result, CAR-T cells using humanized ScFv h046-L7 / H4e and humanized ScFv h046-H5b / L2 completely shrank the tumor (the mean values ​​of Tumor Growth Inhibition (TGI, calculated using the formula shown below) on Day 49 compared with the estimated tumor volume at the time of grouping were 100% and 100%, respectively), demonstrating the strong pharmacological efficacy of anti-GPR20 CAR-T cells ( Figure 4 ).

[0089] TGI (%) = 100 × (estimated tumor volume at time of grouping - estimated tumor volume at time of TGI calculation) / estimated tumor volume at time of grouping Example 4: Evaluation of CAR-T cells using anti-GPR20 antibody in a mouse model transplanted with patient-derived cancer cells Example 4-1: In vivo comparison of efficacy with drug-conjugated anti-GPR20 antibody in a mouse model transplanted with patient-derived cells GIST-040 GIST040 (obtained from the National Institutes of Biomedical Innovation, Health and Nutrition) was passaged and maintained by subcutaneously transplanting tumors excised from patients with gastrointestinal stromal tumors into immunodeficient mice as blocks. Tumor blocks were cut into approximately 3 mm cubes and transplanted subcutaneously into the right flank of female nude mice (Day 0), and on Day 57, the mice were grouped based on tumor volume. On the day of grouping, the anti-GPR20 CAR-T cell group contained 5 × 10 CAR-positive cells. ６ cells / mL (low dose) or 5 x 10 ７The concentration was adjusted with PBS to 1000 cells / mL (high dose), and 0.2 mL was administered intravenously into the tail vein. For comparison, the anti-GPR20 antibody-drug conjugate h046-H4e / L7-ADC1 (see WO18135501 A1) and the vehicle (formulation buffer) used to dissolve h046-H4e / L7-ADC1 were used. The anti-GPR20 antibody-drug conjugate group and the vehicle group were administered intravenously at a dose of 10 mg / kg on days 57 and 71. h046-H4e / L7-ADC1 was prepared by the method described in Example 7)-14 of WO18135501 A1.

[0090] As a result, two doses of the anti-GPR20 antibody-drug conjugate had a weak inhibitory effect on tumor growth (the mean TGI on Day 101 was -390%). On the other hand, a single dose of CAR-T cells using humanized ScFv h046-L7 / H4e demonstrated a strong tumor regression effect (the mean TGI on Day 101 was 65% at the low dose and 92% at the high dose), with complete regression achieved in 2 / 4 of the mice at the high dose (Figure 5). Example 4-2: Comparison of in vivo efficacy with ripretinib in a mouse model transplanted with patient-derived cells GIST-040 GIST040 (obtained from the National Institutes of Biomedical Innovation, Health and Nutrition), which was also used in Example 4-1, was transplanted subcutaneously into the right flank of female nude mice (Day 0), and on Day 55, the mice were divided into groups based on tumor volume. One day later, the anti-GPR20 CAR-T cell group contained 5x10 CAR-positive cells. ６ cells / mL (low dose) or 2.5 x 10 ７ The solution was prepared at 0.2 mL / mL (high dose) and administered intravenously via the tail vein. Ripretinib (Deciphera Pharmaceuticals), approved for GIST treatment, and the vehicle (NSC3) used to dissolve ripretinib were used for comparison. The ripretinib and vehicle groups were orally administered 1 mg / head daily (excluding weekends) for a total of 19 doses starting from Day 56.

[0091] As a result, the tumor growth inhibitory effect of Ripretinib was partial (mean TGI on Day 98 was -92%). On the other hand, CAR-T cells using humanized ScFv h046-L7 / H4e showed a strong tumor regression effect after a single administration (mean TGI on Day 98 was 72% at the low dose and 100% at the high dose), with complete regression achieved in 4 / 4 mice at the high dose (Figure 6).

Claims

1. An anti-GPR20 chimeric antigen receptor (CAR) comprising an antigen-binding site that specifically binds to GPR20, wherein the antigen-binding site comprises: (1) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, with one or several amino acids substituted, deleted, or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, with one or several amino acids substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, with one or several amino acids substituted, deleted, or added; and the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, with one or several amino acids substituted, deleted, or added; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, with one or several amino acids substituted, deleted, or added; and (2) a light chain variable (VL) region comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26 in which one or several amino acids have been substituted, deleted, or added; or (3) a heavy chain variable (VH) region comprising the following CDRs: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69 in which one or several amino acids have been substituted, deleted, or added; an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70 in which one or several amino acids have been substituted, deleted, or added; and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71 in which one or several amino acids have been substituted, deleted, or added; and the following CDRs: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 72 in which one or several amino acids have been substituted, deleted, or added; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73 in which one or several amino acids have been substituted, deleted, or added; and The anti-GPR20 chimeric antigen receptor (CAR), comprising: a light chain variable (VL) region including an LC-CDR3 comprising an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence of SEQ ID NO:

74.

2. The CAR according to claim 1, wherein the antigen-binding site comprises: (a) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:63; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:64; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:65; and a light chain variable (VL) region comprising the following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:66; LC-CDR2 comprising the amino acid sequence of SEQ ID NO:67; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:68; or (d) a heavy chain variable (VH) region comprising the following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:69; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:70; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:71; and a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:72; the CAR, comprising a light chain variable (VL) region comprising: an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

74.

3. The CAR of claim 1 or 2, wherein the antigen-binding site comprises the VH and VL regions described in (b) or the VH and VL regions described in (d).

4. The CAR according to any one of claims 1 to 3, wherein the antigen-binding site comprises: (a) a VH region comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 124 to 246 of SEQ ID NO:5, and a VL region comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5; or (b) a VH region comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 1 to 123 of SEQ ID NO:7, and a VL region comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in amino acid numbers 139 to 246 of SEQ ID NO:

7.

5. A CAR according to any one of claims 1 to 4, wherein the antigen-binding site comprises at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5, at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6, or at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

7.

6. The CAR according to any one of claims 1 to 5, comprising, from the N-terminus, a signal peptide, the antigen-binding site, a hinge region, a transmembrane region, a costimulatory region, and an intracellular signal region, wherein: the signal peptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11; the hinge region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1; the transmembrane region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2; the costimulatory region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3; and the intracellular signal region comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

4.

7. A polynucleotide encoding the CAR according to any one of claims 1 to 6.

8. A CAR gene expression vector comprising the polynucleotide of claim 7.

9. A method for producing a CAR-expressing cell, comprising a step of in vitro gene transfer into a cell using the polynucleotide of claim 7 or the CAR gene expression vector of claim 8.

10. The method of claim 9, wherein the cell is a T cell.

11. A CAR-expressing cell that expresses the CAR according to any one of claims 1 to 6.

12. The CAR-expressing cell of claim 11, which is a CAR-T cell.

13. A pharmaceutical composition comprising the CAR-expressing cell of claim 11 or 12.

14. A pharmaceutical composition comprising the CAR-expressing cells of claim 13 for use in cell therapy to treat GPR20-positive tumors.

15. The pharmaceutical composition of claim 14, wherein the cell therapy is an allogeneic cell therapy or an autologous cell therapy.

16. The pharmaceutical composition according to claim 14 or 15, wherein the GPR20-positive tumor is a gastrointestinal stromal tumor (GIST).

17. A method for treating a GPR20-positive tumor, the method comprising administering to a subject a CAR-expressing cell described in claim 11 or 12 or a pharmaceutical composition described in any one of claims 13 to 16.

18. The method of claim 17, wherein the GPR20-positive tumor is a gastrointestinal stromal tumor (GIST).

19. Use of the CAR-expressing cell of claim 11 or 12 or the pharmaceutical composition of claim 13 for cell therapy to treat GPR20-positive tumors.

20. Use of the CAR-expressing cell of claim 11 in the manufacture of a pharmaceutical composition for use in cell therapy to treat GPR20-positive tumors.

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