Anti- LRRC15 chimeric antigens receptors and cells comprising the same

Abstract

The present invention provides a chimeric antigen-receptor targeting LRRC15, which exhibits drug efficacy against tumors associated with LRRC15 positive cells, and cells comprising the same.SOLUTION: To provide a chimeric antigen-binding receptor containing an anti- LRRC15 antigen-binding site containing a specific CDR, and to provide chimeric antigen-binding receptor-expressing cells containing the receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a chimeric antigen receptor (CAR) that recognizes LRRC15 and a cell containing the same. [Background technology]

[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] In solid tumors, cancer tissue is formed by the coexistence of fibroblasts, immune cells, blood vessels, and extracellular matrix around cancer cells. Fibroblasts, the most abundant cell in the stroma, play an important role in forming the tumor microenvironment. Fibroblasts are transformed into cancer-associated fibroblasts (CAFs) by factors secreted by cancer cells. These fibroblasts contribute to angiogenesis, cancer cell proliferation and invasion, cancer stem cell maintenance, drug resistance, and immunosuppression, and are closely involved in the progression and malignancy of cancer (Non-Patent Document 1). Therefore, development of cancer therapeutics targeting CAFs is underway, including CAF activation and functional inhibition, direct CAF depletion, and restriction of CAF-induced extracellular matrix remodeling (Non-Patent Document 2).

[0004] LRRC15 (leucine-rich repeat containing 15) is a single-pass transmembrane protein with 15 leucine-rich repeats (LRRs) that form the extracellular structural framework (Non-Patent Document 3). Human LRRC15 has been reported to exist in two isoforms: one containing 587 amino acids (NP_001128529.2) and another isoform with a truncated N-terminus containing 581 amino acids (NP_570843.2).

[0005] LRRC15 expression is very limited in normal tissues, but is expressed in some mesenchymal cells. It is known to be expressed in cancer cells of mesenchymal origin, such as sarcoma, melanoma, and glioblastoma. It is also expressed in several solid tumor tissues, such as lung cancer, pancreatic cancer, breast cancer, and head and neck cancer, but its expression is not in the cancer cells themselves, but is highly expressed in fibroblasts in the solid tumor tissue (Non-Patent Document 3).

[0006] LRRC15-positive CAFs are CAFs induced by TGFβ signaling. Clinical trials of immunotherapy in multiple cancer patients have shown that increased signaling associated with LRRC15-positive CAFs correlates with poor response to anti-PD-L1 therapy, and it has been reported that this influences the tumor microenvironment (Non-Patent Document 4). Furthermore, selective depletion of LRRC15-positive cells in a mouse model of pancreatic cancer resulted in a significant reduction in fibroblasts in the tumor and tumor regression. Furthermore, it has been reported that tumor regression was enhanced when combined with an anti-PDL1 antibody (Non-Patent Document 5).

[0007] Antibodies are expected to reduce side effects because they bind specifically 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. To improve efficacy while suppressing side effects, research has been conducted on glycosylation technology that enhances antibody-dependent cellular cytotoxicity (ADCC) (Non-Patent Document 6) and antibody-drug conjugates (ADC) (Non-Patent Document 7), and some drugs have been approved.

[0008] A monoclonal antibody against LRRC15, huM25, has been reported (Patent Document 1), and an antibody-drug conjugate formed by binding this antibody to an antitumor compound has been evaluated in clinical trials, but development has been discontinued (Non-Patent Document 8). In addition, a thorium conjugate containing an LRRC15-targeting moiety has been reported (Patent Document 2), and a multispecific binding compound that binds to LRRC15 and CD3 has been reported (Patent Document 3), but no drug targeting LRRC15 has yet demonstrated clinical efficacy.

[0009] One example of an antibody-based modality is chimeric antigen receptor (CAR)-T cells, which recognize cancer cell-specific antigens. CAR-T cells are molecules that contain three regions: an extracellular domain containing the binding domain for the target molecule, a transmembrane domain, and an intracellular domain that transmits stimuli into the cell. These molecules are capable of specifically damaging cells that express the target antigen (Non-Patent Document 9). As CAR-T cell therapy, CAR-T cells directed against CD19 have been approved for acute lymphoblastic leukemia and diffuse large B-cell lymphoma, while CAR-T cells directed against BCMA have been approved for multiple myeloma (Non-Patent Document 10).

[0010] To date, there have been no reports of CAR-T cells targeting LRRC15 showing efficacy against solid tumors. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US2017151343 [Patent Document 2] US2024108766 [Patent Document 3] US2023374130 [Non-patent literature]

[0012] [Non-Patent Document 1] Nat Rev Cancer. 2016,16(9),582-598 [Non-patent document 2] Front Pharmacol. 2023 May 14:14:1113378 [Non-patent document 3] Cancer Res. 2018 Jul 15;78(14):4059-4-72 [Non-patent document 4] Cancer Discov. 2020 Feb;10(2):232-253 [Non-Patent Document 5] Nature. 2022 Nov ;611(7934):148-154 [Non-patent document 6] J Biol Chem. 2002 Jul 26;277(30):26733-40 [Non-Patent Document 7] Drug Discov Today. 2014 Jul;19(7):869-81 [Non-patent document 8] Clin Cancer Res. 2021 Jul 1;27(13):3556-3566 [Non-Patent Document 9] Cells. 2019 May 17;8(5):472 [Non-Patent Document 10] Front Immunol. 2023 May 15:14:1188049 Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a chimeric antigen receptor (CAR) that targets LRRC15 and exhibits therapeutic efficacy against solid tumors, and cells containing the same.

[0014] As a result of intensive research aimed at solving the above problems, the present inventors discovered that CAR-T cells expressing a chimeric antigen receptor containing an anti-LRRC15 antigen-binding site on their cell surface exhibit a high tumor regression effect, and completed the present invention. That is, the present invention encompasses the following inventions. [1] A chimeric antigen receptor comprising antigen-binding sites for one or more antigens, wherein the chimeric antigen receptor comprises at least an antigen-binding site that specifically binds to LRRC15. [2] The chimeric antigen receptor according to [1], wherein the antigen-binding site that specifically binds to LRRC15 comprises a first variable region comprising amino acid sequences derived from CDRH1, CDRH2, and CDRH3 of a heavy chain variable region contained in a monoclonal antibody or scFv that specifically binds to LRRC15, and a second variable region comprising amino acid sequences derived from CDRL1, CDRL2, and CDRL3 of a light chain variable region contained in the monoclonal antibody or scFv. [3] The chimeric antigen receptor according to [1] or [2], wherein the antigen-binding site is one of the following: (1) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 40; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41 in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 containing an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added a light chain variable (VL) region comprising: (2) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46 in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47 in which one or several amino acids have been substituted, deleted, or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 containing an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added a light chain variable (VL) region comprising: (3) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49 in which one or several amino acids are substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 containing an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added a light chain variable (VL) region comprising: (4) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52 in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53 in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 containing an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added a light chain variable (VL) region comprising: (5) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55 in which one or several amino acids are substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 in which one or several amino acids have been substituted, deleted, or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 containing an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added a light chain variable (VL) region comprising: The chimeric antigen receptor comprising: [4] [3] The chimeric antigen receptor according to [3], wherein the antigen-binding site is one of the following: (1) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 40; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 a light chain variable (VL) region comprising: (2) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 a light chain variable (VL) region comprising: (3) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 a light chain variable (VL) region comprising: (4) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 a light chain variable (VL) region comprising: (5) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 The light chain variable (VL) region contains The chimeric antigen receptor comprising: [5] The chimeric antigen receptor according to any one of [1] to [4], wherein the antigen-binding site is one of the following: (1) a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 57; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (2) a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 59; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (3) a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (4) a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 61; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (5) a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 62; and A light chain variable (VL) region comprising an amino acid sequence that contains at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58. The chimeric antigen receptor comprising: [6] The chimeric antigen receptor according to any one of [1] to [5], wherein the antigen-binding site comprises only an antigen-binding site that specifically binds to LRRC15. [7] The chimeric antigen receptor according to any one of [1] to [5], comprising the antigen-binding site that specifically binds to LRRC15 and antigen-binding sites for one or more different antigens. [8] The chimeric antigen receptor according to [7], wherein the antigen-binding site for the different antigen is an antigen-binding site that binds to an antigen that is specifically expressed on the surface of cancer cells. [9] The chimeric antigen receptor according to [8], wherein the cancer cells are cancer cells contained in a solid cancer.

[10] A polynucleotide encoding the chimeric antigen receptor according to any one of [1] to [9].

[11] A vector comprising the polynucleotide according to

[10] , which is designed in such a manner that the chimeric receptor encoded by the polynucleotide can be expressed on the cell surface.

[12]

[11] The vector according to

[11] , wherein the chimeric antigen receptor according to [6] and a second chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells are designed in such a manner that they can be expressed on the cell surface as independent proteins.

[13] A method for producing a chimeric antigen receptor-expressing cell, comprising a step of introducing a gene into a cell in vitro using the polynucleotide according to

[10] or the vector according to

[11] or

[12] .

[14] A cell expressing the chimeric receptor according to any one of [1] to [9].

[15] The cell of claim

[14] , wherein the cell is an immune cell.

[16] The cell according to

[14] or

[15] , which expresses both the chimeric antigen receptor according to [6] and a second chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells.

[17] A pharmaceutical composition comprising the cells according to any one of

[14] to

[16] for treating a tumor associated with LRRC15-positive cells.

[18] The pharmaceutical composition of claim 17, wherein the tumor is a tumor containing LRRC15-positive cancer cells.

[19] The pharmaceutical composition according to

[17] , wherein the tumor is a solid cancer containing LRRC15-positive cancer stromal cells in the tumor tissue.

[20] The pharmaceutical composition according to

[19] , characterized in that the cell according to

[16] is used. [twenty one]

[19] The pharmaceutical composition according to

[19] , comprising cells expressing the chimeric antigen receptor according to [6], wherein the patient to whom the pharmaceutical composition is administered is undergoing cell therapy using cells expressing a chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells of the solid cancer. [twenty two] A method for treating a tumor associated with LRRC15-positive cells, the method comprising administering to a subject the pharmaceutical composition described in any one of

[17] to

[21] . [Effects of the Invention]

[0015] Immune cells expressing the chimeric antigen receptor of the present invention exhibit strong anti-tumor activity targeting LRRC15 due to the inclusion of an anti-LRRC15 antigen-binding site in the extracellular domain of the chimeric antigen receptor. [Brief explanation of the drawings]

[0016] [Figure 1-1] Figure 1-1 shows the results of flow cytometry in which the expression of CAR molecules in CAR-T cells using multiple anti-LRRC15 antibodies was confirmed by staining with biotinylated protein L. [Figure 1-2] Figure 1-2 shows the results of flow cytometry in which the binding ability of CAR-T cells using multiple anti-LRRC15 antibodies to the LRRC15 protein was confirmed by staining with histidine-tagged recombinant mouse LRRC15 protein. [Figure 2] Figure 2 shows the binding ability of various CAR-T cells to the LRRC15 protein, calculated from the measurement results in Figures 1-2. [Figure 3] Figure 3 shows the results of administering huM25 CAR-T cells to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells, and evaluating the effect on the area of ​​LRRC15-positive cells in the tumor using immunohistochemical staining. [Figure 4] Figure 4 shows the results of administering huM25 CAR-T cells to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells and evaluating their antitumor activity. [Figure 5] Figure 5 shows the results of evaluating the cytotoxic activity of CAR-T cells using multiple anti-LRRC15 antibodies against the Saos-2 cell line, a cancer cell line that expresses LRRC15. [Figure 6] Figure 6 shows the results of administering CAR-T cells utilizing multiple anti-LRRC15 antibodies to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells, and evaluating the anti-tumor activity. [Figure 7] Figure 7 shows the results of administering R024 H02L01 V2 CAR-T cells to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells, and evaluating the effect on the area of ​​LRRC15-positive cells in the tumor using immunohistochemical staining. [Figure 8] Figure 8 shows the results of evaluating the cytotoxic activity of R024 H02L01 V2 CAR-T cells against human head and neck cancer-derived cancer-associated fibroblasts (head and neck CAFs), which have high LRRC15 expression, and human lung cancer-derived cancer-associated fibroblasts (pulmonary CAFs), which have low LRRC15 expression. [Figure 9] Figure 9 shows the results of evaluating the antitumor activity of CD19 CAR-T cells and R024 H02L01 V2 CAR-T cells administered alone or in combination to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells. [Figure 10] Figure 10 shows the results of flow cytometry confirming the expression of CAR molecules in CAR-T cells in which both CAR sequences, a CAR utilizing an anti-CD19 antibody and a CAR utilizing an anti-LRRC15 antibody, were expressed on the same T cell using the P2A sequence. [Figure 11] Figure 11 shows the results of flow cytometry confirming the expression of CAR molecules in CAR-T cells in which both CAR sequences, one using an anti-CD19 antibody and the other using an anti-LRRC15 antibody, were expressed on the same T cell using an IRES sequence. [Figure 12]Figure 12 shows the results of evaluating the cytotoxic activity of CAR-T cells targeting two molecules, anti-CD19 and anti-LRRC15 antibodies, against the Saos-2 / Nuc-G cell line expressing LRRC15, the Raji / GFP cell line expressing CD19, and the Raji / GFP / hLRRC15 cell line expressing both molecules. DETAILED DESCRIPTION OF THE INVENTION

[0017] <Terminology> In the present invention, a "chimeric antigen receptor" refers to a protein having a structure in which three regions are arranged from the N-terminus to the C-terminus: an extracellular domain containing an antigen-binding site, a transmembrane region, and an intracellular domain containing an intracellular signal transduction region. A signal peptide sequence may also be bound to the N-terminus of the antigen-binding region. In the extracellular domain, a hinge region may be present between the antigen-binding region and the transmembrane region. Furthermore, in the intracellular domain, an intracellular costimulatory region may be present at the N-terminus of the intracellular signal transduction domain. The respective regions contained in the chimeric antigen receptor may be bound via any spacer consisting of 0 to 10 amino acids. Various known techniques employed in chimeric antigen receptor-expressing immune cell therapy (see, for example, Uckun et al., 2011, Brit. J. Hematol., 153:15-23; US 2012 / 0141505; and U.S. Patent Nos. 5,484,892; 5,573,924; 6,379,668; 7,744,877; 8,362,211; 9,023,999; 8,822,647; 9,328,156; and 9,034,324) can be applied to the present invention.

[0018] In the present invention, the term "antigen-binding site" refers to an amino acid sequence comprising a region contained in a protein that specifically binds to an antigen, such as an antibody, that contributes to specific binding to a target antigen (in the case of a typical IgG, the heavy and light chain variable regions, or the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 contained in the variable regions), and comprising a partial peptide that maintains a certain level of antigen-binding ability when expressed as a protein or peptide. Specific examples include a site comprising a variable region derived from a single-chain antibody, a site comprising a heavy chain variable region and a light chain variable region, a site comprising a structure similar to that of an scFv, a site comprising a structure similar to that of an scFab, etc.

[0019] As used herein, the term "transmembrane domain" refers to a region where, when a chimeric antigen receptor is expressed on a cell, the majority of the region is usually present in the cell membrane. It is not necessary that all of the amino acids contained in this region are present in the cell membrane; depending on the sequence employed and its surrounding amino acid sequences, a structure may be adopted in which a portion of the N-terminus of the sequence corresponding to the transmembrane domain is present outside the cell, and / or a portion of the C-terminus is present inside the cell.

[0020] As used herein, the intracellular domain refers to a domain that is generally most present intracellularly when a chimeric antigen receptor is expressed on a cell. It is not necessary for all of the amino acids contained in the domain to be present intracellularly; depending on the sequence employed and its surrounding amino acid sequences, a structure may be adopted in which a portion of the N-terminal side of the sequence corresponding to the intracellular domain is embedded in the cell membrane.

[0021] As used herein, the term "amino acid" includes all naturally occurring amino acids as well as modified amino acids. A "conservative amino acid variation" is one in which one amino acid residue is substituted with another amino acid residue without impairing the desired function or property of the protein. Such related amino acids can be conservatively substituted for each other if they belong to a group of amino acids with similar chemical and / or spatial properties of their side chains. The properties of such amino acids have been thoroughly analyzed and are well known in the fields of biochemistry and molecular biology.

[0022] As used herein, the term "polynucleotide" refers to a nucleic acid in which nucleoside or nucleotide monomers, each consisting of a natural base, sugar, and intersugar (backbone) linkage, are linked in a predetermined base sequence. The term also includes modified or substituted nucleic acids comprising non-natural monomers or portions thereof. Polynucleotides of the present invention may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixed pairs thereof, and include natural bases, including adenine, guanine, cytosine, thymidine, and uracil. These sequences may also contain modified bases. Examples of such modified bases include aza- and deaza-adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. Modified nucleotides include 5-methylcytidine, 5-methyluridine, pseudouridine, and the like. Polynucleotides of the present invention may be double-stranded or single-stranded, and may include sense and / or antisense strands. In the present invention, "immune cells" refer to blood cells that exert cytotoxic activity in the immune system's mechanism for eliminating non-self cells and abnormal cells, and examples thereof include T cells, NK cells, and macrophages. The immune cells of the present invention are not limited to cells contained in blood collected directly from a living body. Any cells that retain immune cell characteristics (e.g., expression of representative cell surface marker molecules, cytotoxic activity), such as immune cells induced to differentiate from pluripotent stem cells such as ES cells and iPS cells, can be used as the immune cells of the present invention, regardless of their origin or production method. Unless otherwise specified, the term "cell" herein may refer to a single cell or a cell population containing multiple cell types. In the present invention, "target cells" refer to cells against which chimeric antigen receptor-expressing immune cells exert their cytotoxic activity, and express on their cell surface a target antigen molecule that specifically binds to the antigen-binding site contained in the chimeric antigen receptor.

[0023] Examples of target cells for immune cells expressing anti-LRRC15 chimeric antigen receptors include LRRC15-positive cancer cells, LRRC15-positive cancer stromal cells, and LRRC15-positive cancer-associated fibroblasts. <Chimeric antigen receptors and antigen-binding sites contained therein> In one aspect, the present invention relates to a chimeric antigen receptor comprising antigen-binding sites for one or more antigens, wherein the chimeric antigen receptor comprises at least an antigen-binding site that specifically binds to LRRC15.

[0024] LRRC15 (leucine-rich repeat-containing protein 15) is a single-pass transmembrane protein containing 15 leucine-rich repeats. Its N-terminus is extracellular and its C-terminus is intracellular. While its detailed function remains unclear, it is known to bind to extracellular matrices, including collagen, and does not possess intracellular signaling domains (Cancer Res (2018) 78(14):4059-4072). Several species-specific LRRC15s are known, including human LRRC15 (also referred to as "hLRRC15"), cynomolgus monkey LRRC15 (also referred to as "cynoLRRC15"), mouse LRRC15 (also referred to as "mLRRC15"), and rat LRRC15 (also referred to as "ratLRRC15" or "rLRRC15"). Note that, throughout this specification, "LRRC15" may be used interchangeably with the LRRC15 protein. Furthermore, when the biological species of origin is not specified, it refers to human LRRC15.

[0025] Human LRRC15 is known to exist in two isoforms: one consisting of 587 amino acid residues (SEQ ID NO: 63; NP_001128529.2) and another containing 581 amino acid residues with an N-terminal truncation (SEQ ID NO: 64; NP_570843.2). However, both isoforms share the same amino acid sequence except for the signal peptide (the region from amino acid numbers 28 to 544 in SEQ ID NO: 63 and the region from amino acid numbers 22 to 538 in SEQ ID NO: 64).

[0026] LRRC15 also includes proteins that have the same biological activity as the above-mentioned human LRRC15 amino acid sequence, but in which one or more amino acid residues have been substituted, deleted, and / or added.

[0027] As used herein, the term "an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or added" refers to an amino acid sequence in which one to several amino acids have been deleted, substituted, inserted, and / or added. "Several amino acids" refers to 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 amino acids.

[0028] The chimeric antigen receptor of the present invention comprises an antigen-binding site for one or more antigens, and at least an antigen-binding site that specifically binds to LRRC15. "Specifically binds to LRRC15" means specifically binding to at least human LRRC15 and does not preclude binding to other molecules. The anti-LRRC15 antibodies used in the following examples cross-link to human and mouse LRRC15, and such binding is also considered to be specific binding.

[0029] The chimeric antigen receptor of the present invention may be a receptor that contains only an antigen-binding site that specifically binds to LRRC15 (hereinafter, this may be referred to as a "monovalent LRRC15 chimeric antigen receptor"). The chimeric antigen receptor of the present invention may be a receptor that contains an antigen-binding site that specifically binds to LRRC15 and antigen-binding sites for one or more different antigens (hereinafter, this may be referred to as a "multivalent LRRC15 chimeric antigen receptor"). The ability of the antigen-binding site to bind to LRRC15 or different antigens can be analyzed by methods well known to those skilled in the art, including 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).

[0030] The antigen-binding site contained in the chimeric antigen receptor of the present invention that specifically binds to LRRC15 may be an amino acid sequence of a monoclonal antibody (mAb) capable of binding to LRRC15, antigen-binding fragments such as Fab and F(ab')2 of the mAb, a single-chain antibody, a single-chain variable fragment (scFv), or an scFab. The amino acid sequence of such an antigen-binding site can be obtained by well-known methods, such as sensitizing a non-human animal with a protein containing the extracellular domain of human LRRC15 as an antigen and obtaining a nucleotide sequence encoding the antibody or its variable region from the resulting immune cells. Alternatively, an scFv that binds to a target antigen can be obtained by phage display, in which a library of variable regions is expressed as scFv on the surface of phage and phages that bind to the antigen are selected (Nature Biotechnology (2005), 23, (9), pp. 1105-1116). The DNA sequence encoding the variable region that binds to the antigen can be determined by analyzing the genes of phages selected by binding to the antigen. Once the sequences of such heavy and light chain variable regions are known, the sequences of the CDRs contained in the variable regions can also be identified (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Natl. Rev. Biotechnology (2005) 23(9), pp. 1105-1116).

[0031] The antigen-binding site employed in the chimeric antigen receptor of the present invention may be an antigen-binding site designed based on the amino acid sequence of the variable region or CDR of the above-mentioned antibody or scFv. Alternatively, the amino acid sequence of the variable region or CDR may be modified to adjust the binding affinity with the antigen while maintaining the binding ability to LRRC15. A high binding affinity between a chimeric antigen receptor and a target antigen is not necessarily optimal, and the optimal binding affinity may vary depending on the target antigen and cell type. Therefore, an antigen-binding site with intentionally reduced binding affinity may be employed. The antigen-binding site employed in the chimeric antigen receptor of the present invention is preferably a single-chain antibody, scFv, or scFab, and more preferably an scFv.

[0032] For example, an scFv is a polypeptide in which the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody are linked via a peptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (eds. Rosenberg and Moore, Springer Verlag, New York, pp. 269-315 (1994)); Nature Biotechnology (2005), 23, pp. 1126-1136). In addition, a BiscFv, which is prepared by linking two scFvs via a peptide linker, can also be used as a bispecific antigen-binding site.

[0033] Methods for designing the amino acid sequence of 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). Chimeric antigen receptors that use scFvs as their antigen-binding sites can be produced by standard genetic engineering techniques. In these scFvs, the VH and VL domains are linked via a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA (1988), 85, pp. 5879-5883). In these scFvs, the order in which the heavy chain variable (VH) and light chain variable (VL) domains 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 light chain variable region of an scFv may be derived from the same antibody or from different antibodies.

[0034] The peptide linker sequence connecting the two variable regions can be any of a variety of known sequences, including artificial sequences, as linker sequences for single-chain antibodies and scFvs. Examples of artificial linker sequences include amino acid sequences rich in glycine and serine, such as the GS linker consisting of three amino acids of GSG (SEQ ID NO: 65) or four amino acids of GSGS (SEQ ID NO: 66), and a linker sequence represented by the formula (G4S)n. In the formula, n is 1 to 10, preferably n = 3 (GGGGSGGGGSGGGGS: SEQ ID NO: 6). The linker domain is also called a peptide linker, and known peptide linkers can be used as the extracellular linker domain. The sequence represented by the formula (G4S)n is called a G4S linker.

[0035] 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, any FRs derived from humans may be used without particular limitation. CDR-grafted variable regions or CDR-grafted scFvs in which the above-mentioned set of CDRs is grafted onto different FRs may also be used. Examples of CDR grafting include, but are not limited to, antibodies or antigen-binding fragments in which the CDRs in the variable region of an antibody or antigen-binding fragment are grafted onto a variable region derived from another immunoglobulin, antibodies in which the framework region sequence of the original antibody or antigen-binding fragment is also grafted onto a portion of the variable region in addition to the CDR, and antibodies in which one or more amino acids from any of the original antibodies or antigen-binding fragments are replaced with other amino acids.

[0036] In the above-mentioned CDR grafting, when an amino acid in a CDR is substituted with another amino acid, 1 to 6 amino acids selected from 1, 2, or 3 amino acids on the N-terminal and / or C-terminal side of the CDR region may be substituted, more preferably 1 to 4 amino acids selected from 2 amino acids on the N-terminal and / or C-terminal side, and even more preferably 1 or 2 amino acids selected from 1 amino acid on the N-terminal and / or C-terminal side.

[0037] Conventions for defining antibody CDRs and FRs include the definition by 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 by Al-Lazikani et al. (Chothia numbering scheme, J. Mol. Biol., 196:901-917 (1987)); the method by Martin et al., which is a compromise between Kabat and Chothia (AbM numbering scheme, Martin, ACR, Cheetham, JC and Rees, AR (1989) Proc. Natl. Acad. Sci. USA, 86, 9268-9272); and the definition by Lefranc et al. (IMGT numbering scheme, Developmental and Comparative Immunology, 27 (2003), 55-77).

[0038] Specifically, the chimeric antigen receptor of the present invention comprises two variable regions comprising the following specific CDRs as an antigen-binding site for LRRC15: (1) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, or the amino acid sequence in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS), or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added. a light chain variable (VL) region comprising: (2) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS), or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added. a light chain variable (VL) region comprising: (3) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS), or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added. a light chain variable (VL) region comprising: (4) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, or the amino acid sequence in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS), or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added. a light chain variable (VL) region comprising: (5) The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS), or an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence in which one or several amino acids have been substituted, deleted or added. The light chain variable (VL) region comprises:

[0039] 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, inserted, and / or added, as described above for the "amino acid sequence in which one or several amino acids have been substituted, deleted, or added," and which maintains a certain level of binding to LRRC15 when an scFv is formed. "Several amino acids" means 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 amino acids.

[0040] The chimeric antigen receptor of the present invention may comprise an antigen-binding site comprising the following variable regions (provided that, if there is a change in the amino acid sequence, it is a change in the FR region, and the sequence of the CDR is maintained): (1) a heavy chain variable (VH) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 57; and a light chain variable (VL) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 58; or (2) a heavy chain variable (VH) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 59; and a light chain variable (VL) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 58; or (3) a heavy chain variable (VH) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 60; and a light chain variable (VL) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 58; or (4) a heavy chain variable (VH) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 61; and a light chain variable (VL) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 58; or (5) a heavy chain variable (VH) region comprising an amino acid sequence that comprises 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 SEQ ID NO: 62; and A light chain variable (VL) region comprising 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 the amino acid sequence of SEQ ID NO: 58.

[0041] As used herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. It 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.). It can also be determined using genetic information processing software programs such as GENETYX Ver. 7 (Genetyx), DNASIS Pro (Hitachi Software), and Vector NTI (Infomax). Sequence identity between nucleotide sequences can also be determined in a similar manner.

[0042] Antigen-binding sites derived from single-chain antibodies consisting only of heavy chains can be produced in a similar manner to chimeric antigen receptors that use scFv as the antigen-binding site, using displayer libraries such as phage or yeast that carry genes encoding the variable regions of single-chain antibodies derived from camels, alpacas, llamas, etc. that bind to the antigen of interest, and incorporating them into an expression format suitable for a single variable region, according to publicly known methods or methods disclosed herein (see, for example, David R. Maass et al. J Immunol Methods, (2007 Jul 31); 324(1-2): 13-25; Lukas Roth et al., Methods Mol Biol., (2020); 2070: 173-189, etc.). <Transmembrane domain, intracellular signaling domain, hinge domain, and costimulatory domain> The chimeric antigen receptor of the present invention comprises, in addition to the antigen-binding site described above, a transmembrane region and an intracellular signaling region in the intracellular domain. The chimeric antigen receptor of the present invention may also comprise a hinge region and a costimulatory region in the intracellular domain. The transmembrane region and intracellular signaling region contained in the chimeric antigen receptor of the present invention are not particularly limited, as long as they are expressed on the surface of immune cells, transmit an antigen-binding signal into immune cells via the antigen-binding site, and exert cytotoxic activity against target cells expressing that antigen. Known configurations for each region can be combined, or partial sequences can be modified while retaining their functions, and these regions can be appropriately employed.

[0043] The transmembrane region of the chimeric antigen receptor of the present invention is not particularly limited as long as it penetrates the cell membrane. It may be derived from a natural membrane-bound protein or may be artificially designed. Examples include, but are not limited to, amino acid sequences derived from the transmembrane regions of T cell receptor α or β chains, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD27, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR. Artificially designed transmembrane regions have amino acid sequences primarily containing hydrophobic amino acids. For example, triplets of Phe, Trp, and Val may be found at each end of the synthetic transmembrane region. Examples of such polypeptides include those containing 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 the human CD8a-derived transmembrane domain, which contains the amino acid sequence of SEQ ID NO:3.

[0044] The intracellular signal region contained in the intracellular domain of the chimeric antigen receptor of the present invention may be any region, as long as it has the function of transmitting one or more signals that, when a target antigen binds to the antigen-binding site of the chimeric antigen receptor expressed in an immune cell, transmit a primary stimulus into the immune cell and initiate and / or transmit an intracellular signal that activates the effector function of the immune 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 amino acid sequence such as an intracellular signal region derived from human CD3ζ containing the amino acid sequence of SEQ ID NO: 5 or an intracellular signal region of human CD3δ.

[0045] The intracellular domain of the chimeric antigen receptor of the present invention may have, in addition to the intracellular signaling region, a costimulatory region to further enhance the signal. The costimulatory region may be any region capable of transmitting an auxiliary signal to activate the effector function of immune cells. The region may be any sequence derived from the intracellular domain or costimulatory region of a molecule commonly used in the field of CAR-T technology or a molecule expressed on the surface of T cells and known to enhance T cell activation. Examples of intracellular costimulatory regions include the intracellular domains of CD2, CD4, CD5, CD8α, CD8β, CD28, CD134, CD137 (4-1BB), ICOS, GITR, TNFR2, DR3, CD30, HVEM, CD27, OX40, and CD154, truncated fragments thereof comprising signaling motifs, or sequences derived from the costimulatory regions thereof. Specific examples include amino acids 236 to 351 of CD2 (NCBI RefSeq: NP_001758.2), 421 to 458 of CD4 (NCBI RefSeq: NP_000607.1), 402 to 495 of CD5 (NCBI RefSeq: NP_055022.2), 207 to 235 of CD8α (NCBI RefSeq: NP_001759.3), 196 to 210 of CD8β (GenBank: AAA35664.1), 180 to 220 of CD28 (NCBI RefSeq: NP_006130.1), 214 to 255 of CD137 (4-1BB, NCBI RefSeq: NP_001552.2), and OX40 of CD134 (NCBI RefSeq: NP_001552.2). Examples include the sequence of amino acids 241 to 277 of ICOS (NCBI RefSeq: NP_003318.1) and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP_036224.1).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 to an amino acid sequence such as a human 4-1BB-derived costimulatory region (SEQ ID NO: 4), a human CD28-derived costimulatory region, a human GITR-derived costimulatory region, a human TNFR2-derived costimulatory region, a human DR3-derived costimulatory region, a human CD30-derived costimulatory region, a human HVEM-derived costimulatory region, a human CD27 costimulatory region, or a human OX40-derived costimulatory region.

[0046] The chimeric antigen receptor of the present invention may comprise a hinge region. The hinge region may be an amino acid sequence that connects the antigen-binding site and the transmembrane domain to overcome steric hindrance and ensure access of the antigen-binding site to the target antigen. The hinge region may be a sequence derived from a linker or hinge region derived from a natural protein (e.g., hinge regions of CD8, CD28, etc., hinge regions derived from various IgGs, and hinge regions (short hinges) consisting of 4 to 8 amino acids, as described in known literature such as Stoiber et al., Cells. 2019 May 17;8(5):472), or an artificially designed sequence. The amino acid length of the linker region is, for example, 300 or less, 100 or less, or approximately 50 or less. Known hinge regions of proteins include amino acids 118 to 178 of human CD8α (NCBI RefSeq: NP_001759.3), amino acids 135 to 195 of human CD8β (GenBank: AAA35664.1), amino acids 315 to 396 of human CD4 (NCBI RefSeq: NP_000607.1), and amino acids 114 to 152 of human CD28 (NCBI RefSeq: NP_006130.1). 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 an amino acid sequence such as a human CD8a-derived hinge (SEQ ID NO: 2), a human CD8a-derived hinge, a human CD4-derived hinge, a human CD28-derived hinge, or an artificially designed flexible linker (SEQ ID NO: 67). <Signal peptide> The chimeric antigen receptor of the present invention may further comprise a signal peptide. Signal peptides are present at the N-terminus of many secretory proteins and membrane proteins, and are often approximately 15 to 30 amino acids in length, but are not limited thereto. The manner of linkage and type of sequence of such a signal peptide are not limited, so long as they do not inhibit the function of the chimeric antigen receptor and enable expression on the cell membrane. The linkage position is preferably the N-terminus of the chimeric antigen receptor. For example, the signal peptide may comprise an amino acid sequence that shares 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 the amino acid sequence of a signal peptide derived from human CD8a (SEQ ID NO: 1), a signal peptide derived from human immunoglobulin, or the like. <Other regions of the chimeric antigen receptor> The chimeric antigen receptor of the present invention may contain additional regions depending on the purpose, as long as the additional regions do not inhibit its function of exerting cytotoxicity against LRRC15-positive target cells. Examples of such additional regions include a detectable region and a spacer region for operably linking any of the regions in the chimeric antigen receptor.

[0047] The detectable region refers to a portion for detecting the chimeric antigen receptor of the present invention or the antigen-binding site contained in the chimeric antigen receptor by known methods, and examples thereof include fluorescent labels, luminescent labels, immunodetectable labels, and tag peptides. Fluorescent labels include fluorescent proteins such as green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and variants thereof. Luminescent labels include bioluminescent labels such as luciferase. Immunodetectable labels include haptens, peptides / polypeptides such as FLAG® tags, antibodies, and receptors.

[0048] 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. <Chimeric antigen receptor containing only an antigen-binding site that specifically binds to LRRC15> One embodiment of the chimeric antigen receptor of the present invention may be a chimeric antigen receptor that contains only the above-mentioned antigen-binding site that specifically binds to LRRC15 as the antigen-binding site. Such a chimeric antigen receptor that contains only the antigen-binding site that specifically binds to LRRC15 as the antigen-binding site can be used alone to treat tumors associated with LRRC15-positive cells, or can be used in combination with a second chimeric antigen receptor that contains an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells.

[0049] "Tumors associated with LRRC15-positive cells" are divided into two types: (a) tumors in which LRRC15-positive cancer cells are contained in the tumor tissue, and (b) tumors in which the cancer cells contained in the tumor tissue are LRRC15-negative but contain LRRC15-positive cells as cancer stromal cells.

[0050] In the case of tumors (a) above, monovalent LRRC15-CAR-expressing immune cells can attack the cancer cells themselves, thereby causing the tumor to regress, be removed, or be cured. In the tumor (b) described above, monovalent LRRC15-CAR-expressing immune cells attack LRRC15-positive cancer stromal cells (particularly cancer-associated fibroblasts), blocking the supply of growth stimulating factors and nutrients to the cancer cells and allowing the tumor to regress, be eliminated, or be cured. Furthermore, by combining this with immune cells expressing a second chimeric antigen receptor (CAR) containing an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells, the cancer cells themselves can also be directly attacked, resulting in further tumor treatment effects.

[0051] In the present invention, when a second chimeric antigen receptor containing an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells is used, the antigen specifically expressed on the surface of cancer cells may be any antigen that is specifically expressed on the surface of cancer cells compared to the surface of normal cells. Examples of such antigens include, but are not limited to, various cancer antigens such as HER2. The type of cancer cells is not particularly limited, but is preferably solid cancer. <Chimeric antigen receptor comprising an antigen-binding site that specifically binds to LRRC15 and an antigen-binding site for one or more different antigens> The chimeric antigen receptor of the present invention may be a chimeric antigen receptor that contains, as its antigen-binding site, an antigen-binding site that specifically binds to the above-mentioned LRRC15 and antigen-binding sites for one or more different antigens. Any different antigens may be used as long as they do not impair the cytotoxic activity against LRRC15-positive target cells. Examples of such different antigens include, but are not limited to, various cancer antigens such as HER2. The type of cancer cell is not particularly limited, but is preferably a solid cancer. <Polynucleotide encoding a chimeric antigen receptor> The present invention also relates to a polynucleotide encoding the chimeric antigen receptor described above. A polynucleotide encoding a chimeric antigen receptor refers to a polynucleotide containing a nucleotide sequence corresponding to the amino acid sequence of the chimeric antigen receptor described above. As used herein, "polynucleotide" refers to a polymer chain of nucleotides and can be DNA, RNA, or a combination thereof. The nucleic acid used may be a polynucleotide consisting of a nucleic acid molecule having the same chemical structure as natural DNA or RNA, or a polynucleotide to which various known modified nucleic acid molecules have been applied. Specifically, a polynucleotide encoding a chimeric antigen receptor is, for example, a polynucleotide containing a DNA or RNA sequence corresponding to the amino acid sequence of a chimeric antigen receptor. Such a polynucleotide may be in the form of an expression vector, DNA plasmid, or mRNA, which contains other genetic elements or chemical modifications for expressing the chimeric antigen receptor protein in host immune cells. When the polynucleotide encoding the chimeric antigen receptor of the present invention is introduced into immune cells as described below, it can express the chimeric antigen receptor on the cell surface and exert potent cytotoxic activity against the target LRRC15-positive target cells. <Expression vector> The present invention also relates to a vector containing a polynucleotide encoding the chimeric antigen receptor, designed in such a manner that the chimeric receptor encoded by the polynucleotide can be expressed on the cell surface. The vector of the present invention refers to a polynucleotide molecule modified to contain the above-described polynucleotide sequence and capable of expressing the chimeric antigen receptor intracellularly. Examples of vectors of the present invention include DNA vectors, RNA vectors, retroviral vectors, adenoviral vectors, adenovirus-associated vectors, Epstein-Barr (EB) virus vectors, and lentiviral vectors, as well as combinations thereof. For example, the vectors of the present invention can be constructed using standard techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[0052] The vector of the present invention may be a vector designed in such a manner that a chimeric antigen receptor comprising only an antigen-binding site that specifically binds to LRRC15 and a second chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells can each be expressed as independent proteins on the cell surface. <Method for producing chimeric antigen receptor-expressing cells> The present invention also relates to methods for producing chimeric antigen receptor-expressing cells, which comprise the step of transfecting cells with a polynucleotide encoding the chimeric antigen receptor or the chimeric antigen receptor gene expression vector.

[0053] Gene transfer refers to the introduction of a polynucleotide, such as DNA or RNA, encoding a foreign gene into a host cell. In the present invention, it refers to the introduction of a polynucleotide encoding a chimeric antigen receptor gene or a chimeric antigen receptor gene expression vector containing such a polynucleotide into a host cell. Gene transfer can be carried out in vivo or in vitro. The method of gene transfer is not particularly limited, but includes methods such as infecting host cells with a chimeric antigen receptor gene expression vector, which is a viral vector; introducing polynucleotides, such as DNA plasmids or mRNA, encoding the chimeric antigen receptor into host cells by microinjection, electroporation, or incorporating them into nanoparticles; or introducing polynucleotides using transfection reagents such as lipofection; and introducing the chimeric antigen receptor gene into the host cell genome by genome editing using ZFN, TALEN, CRISPR-Cas9 systems, or combinations thereof.

[0054] The host cells used in the chimeric antigen receptor-expressing cells of the present invention or the method for producing the same are not particularly limited, and include prokaryotic host cells such as Escherichia coli, mammalian cells such as human, monkey, and mouse cells, plant cells, and eukaryotic host cells such as yeast, preferably human cells.

[0055] 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, 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 chimeric antigen receptor-expressing cells or cells differentiated from the prepared chimeric antigen receptor-expressing cells into a living organism is desired, a polynucleotide encoding the chimeric antigen receptor of the present invention can be introduced into the living organism itself or cells collected from a living organism of the same species.

[0056] Chimeric antigen receptor-expressing cells prepared using the methods of the present invention are cells expressing the chimeric antigen receptor of the present invention on their cell membrane. Such cells have the property of binding to the antigen LRRC15 at the antigen recognition site of the chimeric antigen receptor, and transmitting the binding signal to the intracellular signaling region, thereby activating the cells. Activation of the chimeric antigen receptor-expressing cells of the present invention varies depending on the type of host cell and the intracellular domain contained in the chimeric antigen receptor, and can be confirmed based on indicators such as cytokine release, increased cell proliferation rate, or changes in cell surface molecules.

[0057] When the host cell of the chimeric antigen receptor-expressing cell of the present invention is a T cell, such a cell is also called a CAR-T cell. When the host cell is a T cell, the cells activated by the above signal transduction release cytotoxic cytokines (such as tumor necrosis factor, lymphotoxin, etc.) and cytotoxic factors such as Granzyme B, and exhibit cytotoxic activity or cytolytic activity against target cells. For example, as described later, the chimeric antigen receptor-expressing cells can be used to treat tumors associated with LRRC15-positive cells.

[0058] The cytotoxic activity of the chimeric antigen receptor-expressing cells against target cells in vitro can be measured, for example, by measuring the growth inhibitory activity of the target cells. For example, in a culture system of a cancer cell line overexpressing LRRC15, the chimeric antigen receptor-expressing cells are added at various ratios, and the growth inhibitory activity of the cancer cells by the chimeric antigen receptor-expressing cells can be measured using a method such as measuring the survival rate of the cancer cells. <Method for treating tumors associated with LRRC15-positive cells, pharmaceutical composition for such treatment> The present invention also relates to the above chimeric antigen receptor-expressing cells, or a pharmaceutical composition containing the same, for use in cell therapy for treating tumors associated with LRRC15-positive cells.

[0059] The tumor associated with LRRC15-positive cells refers to a tumor containing cancer cells expressing the LRRC15 protein on the cell surface, and / or a tumor containing LRRC15-positive cancer stromal cells in the tumor tissue. In the present specification, "tumor" refers to a mass in which cells have proliferated progressively in a living body. Among tumors, in particular, those in which abnormal cells have spread to the surrounding area or metastasized to another organ or tissue are called "malignant tumors" or "cancers", and those derived from epithelial cells among them are called "carcinomas". However, in the present specification, "tumor", "cancer", and "carcinoma" may be used interchangeably.

[0060] Tumors associated with LRRC15-positive cells include tumors of mesenchymal origin, such as sarcoma, melanoma, and glioblastoma, which express the LRRC15 protein or LRRC15 gene, as well as solid cancers, such as lung cancer, pancreatic cancer, breast cancer, and head and neck cancer, which contain LRRC15-positive cancer stromal cells in their tumor tissue. It has been reported that cancer-associated fibers express LRRC15 on the cell surface of LRRC15-positive cancer stromal cells.

[0061] In the present invention, treatment of a tumor associated with LRRC15-positive cells means alleviating, improving, inhibiting the worsening or progression of, or eliminating one or more symptoms caused by the presence of a tumor associated with LRRC15-positive cells. The in vivo effect of chimeric antigen receptor-expressing cells on a tumor associated with LRRC15-positive cells can be measured, for example, by administering the chimeric antigen receptor-expressing cells to a tumor-bearing animal and measuring changes in tumor size, etc.

[0062] The method of the present invention for treating tumors associated with LRRC15-positive cells involves administering the chimeric antigen receptor-expressing cells to a subject with a tumor associated with LRRC15-positive cells, thereby achieving a specific cytotoxic effect on the tumor associated with LRRC15-positive cells in the subject. The method of the present invention includes cell therapy using chimeric antigen receptor-expressing cells derived from cells collected from the subject to be treated (i.e., autologous cell therapy), and cell therapy using chimeric antigen receptor-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, chimeric antigen receptor-expressing cells derived from collected host cells may be used as is, or stem cells such as iPS cells or ES cells may be differentiated into the desired host cells and then transformed into chimeric antigen receptor-expressing cells by introducing a chimeric antigen receptor gene.

[0063] The subject of the methods of the present invention may be any animal or human, preferably a mammal, more preferably a human. The present invention also relates to a pharmaceutical composition for treating tumors associated with LRRC15-positive cells, comprising the chimeric antigen receptor-expressing cells described above. The pharmaceutical composition of the present invention can comprise a therapeutically effective amount of the chimeric antigen receptor-expressing cells, as well as pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives, adjuvants, and one or more combinations thereof.

[0064] The pharmaceutical compositions of the present invention may be formulated to be suitable for injection or infusion into local tumor tissue, or intravenously, intraperitoneally, intrathecally or intracerebroventricularly. When the pharmaceutical composition of the present invention comprises chimeric antigen receptor-expressing cells that contain only the antigen-binding site that specifically binds to LRRC15 as the antigen-binding site, the patient to whom the pharmaceutical composition is administered may be a patient undergoing cell therapy using cells expressing a chimeric antigen receptor that contains an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells. Here, the cancer cells are preferably solid cancer cells that contain LRRC15-positive cancer stromal cells in the tumor tissue.

[0065] The present invention will be described in detail in the following examples, but the present invention is not limited to these examples. [Example]

[0066] Unless otherwise specified, genetic manipulation procedures in the following examples were carried out according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E. F., and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or other experimental manuals 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 (GenScript Japan Co., Ltd.) as needed. Example 1: Generation of cancer cell lines expressing human CD19 Example 1-1: Introduction of fluorescent proteins into cancer cell lines The human lung squamous cell carcinoma-derived cell line EBC-1 was infected with Incucyte Nuclight Red Lentivirus (puro) (Sartorius) according to the protocol. The human osteosarcoma-derived cell line Saos-2 was infected with Incucyte Nuclight Green Lentivirus (puro) (Sartorius) according to the protocol. After infection, cells were selected with antibiotics in medium supplemented with 1 μg / ml puromycin and 10% FBS, and the proliferated cells were used for the following evaluations. Example 1-2: Preparation of human CD19-expressing EBC-1 / NucR cells The EBC-1 / NucR cell line was transfected with CD19 OHu27366C pcDNA3.1(+) (GenScript Japan Co., Ltd.) as a CD19-expressing plasmid vector using LipofectAMINE LTX Reagent (Thermo Fisher Scientific). After transfection, the cells were selected with 600 μg / ml of Geneticin Selective Antibiotics (Thermo Fisher Scientific). The proliferated cells were then cloned by limiting dilution to generate EBC-1 / NucR / hCD19 cells. Example 2: Generation of CAR-T cells using multiple anti-LRRC15 antibodies Example 2-1: Construction of a CAR gene expression vector using multiple anti-LRRC15 antibodies CARs using huM25, a reported anti-LRRC15 antibody, were produced with reference to the amino acid sequences of the huM25 VH chain and VL chain described in WO2017 / 095805 (SEQ ID NOs: 16 and 17, respectively). CARs using hR024 were produced with reference to the amino acid sequences of the hR024 H02 VH chain, H09 VH chain, and L01 VL chain described in the specification attached to Japanese Patent Application No. 2024-002363 (SEQ ID NOs: 41, 35, and 65, respectively).

[0067] The structure of the anti-LRRC15 CAR consisted of, from the N-terminus, a signal peptide (sequence number 1), an anti-LRRC15 antibody scFv, a hinge region derived from human CD8α (sequence number 2), a transmembrane region derived from human CD8α (sequence number 3), a costimulatory region derived from human 4-1BB (sequence number 4), and a signal region derived from human CD3ζ (sequence number 5). The anti-LRRC15 antibody scFv sequences used were scFv huM25 (SEQ ID NO: 7), scFv R024 H02L01 (SEQ ID NO: 8), scFv R024 H02L01 V1 (SEQ ID NO: 9), scFv R024 H02L01 V2 (SEQ ID NO: 10), scFv R024 H02L01 V3 (SEQ ID NO: 11), scFv R024 H09L01 V4 (SEQ ID NO: 12), scFv R024 L01H02 (SEQ ID NO: 13), scFv R024 L01H02 V1 (SEQ ID NO: 14), scFv R024 L01H02 V2 (SEQ ID NO: 15), scFv R024 L01H02 V3 (SEQ ID NO: 16), and scFv R024 L01H09 V4 (SEQ ID NO: 17). In both cases, a 15-amino acid scFv linker sequence (SEQ ID NO: 6) consisting of three G4S repeats was used between the heavy and light chains, or between the light and heavy chains. The anti-LRRC15 CAR gene sequence was generated by gene synthesis and subsequent mutagenesis. The sequences used to generate the LRRC15 CAR and the generated anti-LRRC15 CAR sequences are shown in Table 1 as SEQ ID NOs: 18 to 28.

[0068] A lentiviral plasmid vector for expressing anti-LRRC15 CAR was constructed. The CAR gene sequence, arranged as described above, was inserted into the Multiple Cloning Site of the pLVSINEF1α Pur Vector (Takara).

[0069] [Table 1-1]

[0070] [Table 1-2]

[0071] Table 1-3

[0072] Table 1-4

[0073] Table 1-5

[0074] Table 1-6

[0075] Table 1-7

[0076] Table 1-8

[0077] Table 1-9

[0078] Table 1-10

[0079] Table 1-11

[0080] Table 1-12

[0081] Table 1-13

[0082] Table 1-14

[0083] Table 1-15

[0084] Table 1-16

[0085] Table 1-17

[0086] Table 1-18

[0087] Example 2-2: Preparation of CAR-T cells using multiple anti-LRRC15 antibodies The Lentiviral High Titer Packaging Mix (Takara) and each lentiviral plasmid vector prepared in Example 2-1 were transfected into the Lenti-X 293T Cell Line (Takara) using TransIT-293 Transfection Reagent (Mirus Bio) according to the protocol for the Lentiviral High Titer Packaging Mix with pLVSIN series (Takara). The medium was replaced one day later. After two days, the culture supernatant was collected, and after medium replacement, the cells were cultured for another day. The virus contained in the culture medium was concentrated using a Lenti-X concentrator (Takara) according to the protocol. For viral infection, plates coated with 4 μg / mL CD3 antibody (clone name: OKT3) and 20 μg / mL RetroNectin were prepared according to the protocol for RetroNectin (Takara). Next, human T cells were prepared from human peripheral blood mononuclear cells using the EasySep Human T Cell Isolation Kit (STEMCELL TECHNOLOGIES) at a concentration of 1 x 10 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). 6 The cells were suspended at 1000 x g / mL and added to a retronectin-coated plate along with virus concentrate. After centrifugation at 1000 x g for 60 minutes, the cells were cultured in a CO2 incubator. After one day of culture, virus concentrate was added again, the cells were centrifuged at 1000 x g for 60 minutes, and the culture was continued in a CO2 incubator. After three days, T cells were collected from each well and seeded onto a G-Rex Well Plate (WILSONWOLF). Growth medium was added, and the cells were cultured for an additional six or seven days to prepare CAR-expressing T cells (CAR-T cells).

[0088] Table 2 shows the correspondence between each antibody scFv used and the CAR-T cells produced.

[0089] [Table 2]

[0090] Example 3: Evaluation of LRRC15 CAR-T cells Example 3-1: Confirmation of expression of various anti-LRRC15 CARs on T cells and confirmation of ligand binding ability For each CAR-T cell prepared in Example 2-2, Biotinylated protein L (Thermo Fisher) was diluted in FCM buffer (Phosphate-buffered saline containing 5% FBS), added to the cells, and incubated at 4°C for 30 minutes. After washing with FCM buffer, Brilliant Violet 421 Streptavidin (BioLegend) was diluted in FCM buffer and incubated at 4°C for 30 minutes. After washing with FCM buffer, the cells were suspended in FCM buffer and measured using flow cytometry (BD FACSCantoII) to confirm CAR expression.

[0091] In parallel, for each of the CAR-T cells prepared in Example 2-2, histidine-tagged recombinant mouse LRRC15 protein was diluted in FCM buffer, added to the cells, and allowed to react at 4°C for 30 minutes. After washing with FCM buffer, His-Tag(D3I1O)XP Rabbit mAb (Cell Signaling TECHNOLOGY) was diluted in FCM buffer, added to the cells, and allowed to react at 4°C for 30 minutes. After washing with FCM buffer, Allophycocyanin (APC) AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch) was diluted in FCM buffer and allowed to react at 4°C for 30 minutes. After washing with FCM buffer, the cells were suspended in FCM buffer and measured using flow cytometry (BD FACSCantoII) to confirm the binding of CAR molecules using various scFv sequences to the LRRC15 protein.

[0092] After exporting each measurement result as an FCS file, FlowJo software was used to calculate the CAR positive rate and binding to LRRC15 protein from each fluorescence. As shown in Figure 1-1, all CAR-T cells expressed CAR molecules, and as shown in Figures 1-2 and 2, it was confirmed that the binding ability to LRRC15 protein differed depending on the scFv sequence used. Example 3-2: Evaluation of the effect of huM25 CAR-T cells on the area of ​​LRRC15-positive cells in tumors using a mouse model transplanted with a cancer cell line To evaluate the in vivo effect of huM25 CAR-T cells, huM25 CAR-T cells were administered to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells prepared in Examples 1-2, and the effect on LRRC15-positive cells in the tumor was evaluated. EBC-1 / NucR / hCD19 cells were cultured at 2 × 10 in PBS containing 50% Matrigel (Corning). 7 The solution was adjusted to 0.1 cells / mL and 0.1 mL was subcutaneously implanted into NOG mice (female or male, 4-6 weeks old) (Day 0). The long and short diameters (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the following formula.

[0093] Estimated Tumor Volume (mm 3 ) = Mean estimated tumor volume for each individual: Estimated tumor volume (mm 3 ) = 1 / 2 × [tumor long diameter] × [tumor short diameter] × [tumor short diameter] On day 14, tumors were divided into groups based on tumor volume, with n = 4 in each group, and huM25 CAR-T cells were administered at 5x10 7 cells, 1.5x10 7 cells, and 5x10 6 The solution was adjusted to cells / mL, and 0.2 mL was administered into the tail vein.

[0094] Fourteen days after administration of CAR-T cells, tumors were excised from two individuals, and changes in the area of ​​LRRC15-positive cells in the tumor were examined using the method described below. The excised tumors were immersed in Mildform 10N (FUJIFILM) for one day, after which formalin-fixed, paraffin-embedded (FFPE) specimens were prepared. Thin sections were prepared from the FFPE specimens and stained with HE staining and anti-LRRC15 antibody. The stained specimens were scanned using a Nanozoomer S360 (Hamamatsu Photonics). The LRRC15-positive area was analyzed using the Area Quantification module in image analysis software (HALO 2.2.1870, Indica Labs). The results are shown in Figure 3.

[0095] It was confirmed that administration of huM25 CAR-T cells reduced the area occupied by LRRC15-positive stromal cells in the tumor in a mouse xenograft model using EBC-1 / NucR / hCD19 cells. Example 3-3: Evaluation of antitumor activity of huM25 CAR-T cells using a mouse model transplanted with a cancer cell line The huM25 CAR-T cells were administered to immunodeficient mice transplanted with the EBC-1 / NucR / hCD19 cells prepared in Example 1-2, and the ability to inhibit tumor growth was evaluated. EBC-1 / NucR / hCD19 cells were cultured at 2 × 10 in PBS containing 50% Matrigel (Corning). 7 The solution was adjusted to 0.1 mL / mL and subcutaneously transplanted into NOG mice (female or male, 4-6 weeks old) at 0.1 mL (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 same method as in Example 3-2.

[0096] On Day 14, tumors were divided into groups based on tumor volume, with n = 5 in each group, and huM25 CAR-T cells were administered at 5x10 7 cells, 1.5x10 7 cells, and 5x10 6 The solution was adjusted to cells / mL, and 0.2 mL was administered into the tail vein.

[0097] After CAR-T cell administration, tumor diameter was measured over time, and antitumor activity was evaluated based on changes in estimated tumor volume. The results are shown in Figure 4. Administration of huM25 CAR-T cells was shown to exhibit significant antitumor activity in a mouse xenograft model using EBC-1 / NucR / hCD19 cells. Example 3-4: Evaluation of the cytotoxic activity of CAR-T cells using multiple anti-LRRC15 antibodies against LRRC15-expressing cancer cell lines The CAR-T cells prepared in Example 2-2 using multiple anti-LRRC15 antibodies were evaluated for their cytocidal activity against the Saos-2 cell line, a cancer cell line that expresses LRRC15.

[0098] 3000 Saos-2 cells (target cells) were seeded in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) in a 96-well cell culture plate. Then, 1000, 3000, and 9000 anti-LRRC15 antibody-activated CAR-T cells (effector cells) were seeded in AIM-V medium containing 5% FBS and 100 U / mL IL-2, along with untransfected T cells at the same total number. After 2 days of coculture, effector cells were removed by washing each well several times with PBS. Target cells adherent to the bottom of the well were detected using the CellTiter Glo Luminescent Cell Viability Assay (Promega). Cell viability (%) was calculated using the following formula:

[0099] Viability (%) = 100 × ((chemiluminescence measurement value after co-culture with LRRC15 CAR-T on day 2) / (chemiluminescence measurement value after co-culture with non-transfected T cells on day 2)) The results are shown in Figure 5. CAR-T cells using anti-LRRC15 antibodies exhibited cytotoxic activity against Saos-2 cells expressing LRRC15, depending on the number of co-cultured effector cells. Example 3-5: Evaluation of antitumor activity of CAR-T cells using multiple anti-LRRC15 antibodies in a mouse model transplanted with cancer cell lines In the same manner as in Example 3-3, EBC-1 / NucR / hCD19 cells were transplanted into immunodeficient mice, and on Day 14, the mice were divided into groups based on tumor volume so that each group had n = 3. CAR-T cells against various LRRC15 were transplanted into the mice at 5 x 10 6 The solution was adjusted to cells / mL, and 0.2 mL was administered into the tail vein.

[0100] CAR-T cells using multiple anti-LRRC15 antibodies prepared in Example 2-2 were administered, and their antitumor activity was evaluated. The results are shown in Figure 6. Administration of CAR-T cells utilizing various LRRC15 antibodies demonstrated significant antitumor activity in a mouse xenograft model using EBC-1 / NucR / hCD19 cells. Example 3-6: Evaluation of the effect of R024 H02L01 V2 CAR-T cells on the area of ​​LRRC15-positive cells in tumors using a mouse model transplanted with a cancer cell line R024 H02L01 V2 CAR-T cells were administered in the same manner as in Example 3-2, and the effect on the area of ​​LRRC15-positive cells in the tumor was evaluated. The results are shown in Figure 7.

[0101] It was confirmed that administration of R024 H02L01 V2 CAR-T cells reduced the area occupied by LRRC15-positive stromal cells in the tumor in a mouse xenograft model using EBC-1 / NucR / hCD19 cells. Example 3-7: Evaluation of the cytotoxic activity of R024 H02L01 V2 CAR-T cells against human tumor-derived stromal cells The R024 H02L01 V2 CAR-T cells prepared in Example 2-2 were evaluated for their cytotoxic activity against human head and neck cancer-derived cancer-associated fibroblasts (BioIVT) with high LRRC15 expression and human lung cancer-derived cancer-associated fibroblasts (BioIVT) with low LRRC15 expression.

[0102] Twenty thousand cancer-associated fibroblasts (target cells) were seeded in a 96-well cell culture plate in DMEM medium supplemented with 10% FBS. After one day of culture, R024H02L01 V2 CAR-T cells (effector cells) were seeded at 2,000, 6,000, and 20,000 CAR-positive cells, respectively, and non-transfected T cells were seeded at the same total number in AIM-V medium containing 5% FBS and 100 U / mL IL-2. After three days of coculture, each well was washed with PBS, and cells attached to the bottom of the well were detached and collected using TrypLE (Thermo Fisher Scientific). An equal volume of FCM Buffer containing 2 μg / mL 7-Aminoactinomycin D was added to each well to stain dead cells, and the cells were analyzed using a MACSQUANT Analyzer 10 (Miltenyi Biotech) flow cytometer. After measurement, the target cell fraction was selected on the FSC / SSC plot, and the cell group that was negative for 7-aminoactinomycin D staining was selected as live target cells. Viability (%) was calculated using the following formula.

[0103] Viability (%) = 100 × ((Number of target cells after 3 days of co-culture with effector cells) / (Number of target cells after 1 day of culture with target cells alone)) The results are shown in Figure 8. The cytocidal activity against human head and neck cancer-derived cancer-associated fibroblasts with high LRRC15 expression was dependent on the number of R024 H02L01 V2 CAR-T cells generated. However, no cytocidal activity was observed against human lung cancer-derived cancer-associated fibroblasts with low LRRC15 expression. Example 4: Generation of CAR-T cells against CD19 Example 4-1: Construction of a CAR gene expression vector using an anti-CD19 antibody The sequence of FMC63, reported as an anti-CD19 antibody, was referenced (Ian C et al., Molecular Immunology, Vol. 34, No. 16-17). As shown in Table 3, the amino acid sequences of the VH chain and VL chain of FMC63 are SEQ ID NO: 29 and SEQ ID NO: 30, respectively. The anti-CD19 CAR structure consisted of, from the N-terminus, a signal peptide (SEQ ID NO: 1), anti-CD19 antibody FMC63 scFv (SEQ ID NO: 31), a hinge region derived from human CD8α (SEQ ID NO: 2), a transmembrane region derived from human CD8α (SEQ ID NO: 3), a costimulatory region derived from human 4-1BB (SEQ ID NO: 4), and a signal region derived from human CD3ζ (SEQ ID NO: 5). The anti-CD19 CAR gene sequence was generated by gene synthesis (SEQ ID NO: 32). A lentiviral plasmid vector for expressing anti-CD19 CAR was constructed. The CAR gene sequence, arranged as described above, was introduced into the Multiple Cloning Site of the pLVSINEF1α Pur Vector (Takara).

[0104] [Table 3-1]

[0105] [Table 3-2]

[0106] [Table 3-3]

[0107] Example 4-2: Construction of CD19 CAR and LRRC15 CAR gene expression vectors Using the CAR sequence using huM25 prepared in Example 2-1 and the CD19 CAR sequence prepared in Example 4-1, vectors for expressing both CARs were constructed.

[0108] A self-cleaving peptide P2A sequence (SEQ ID NO: 33) was placed between both CAR gene sequences, and the placed gene sequence was introduced into the Multiple Cloning Site of pLVSINEF1α Pur Vector (Takara).

[0109] In addition, an internal ribosome entry site (IRES) (SEQ ID NO: 34) was placed between both CAR gene sequences, and the placed gene sequence was introduced into the Multiple Cloning Site of pLVSINEF1α Pur Vector (Takara). Example 4-3: Construction of a CAR gene expression vector targeting two molecules using anti-CD19 antibody and anti-LRRC15 antibody The CAR, which targets two molecules using anti-CD19 and anti-LRRC15 antibodies, consisted of an N-terminal signal peptide (SEQ ID NO: 1), anti-CD19 antibody scFv FMC63 (SEQ ID NO: 31), a 25-amino acid linker sequence consisting of five G4S repeats (SEQ ID NO: 35), anti-LRRC15 antibody scFv R024 H02L01 V2 (SEQ ID NO: 10), a human CD8α hinge region (SEQ ID NO: 2), a human CD8α transmembrane region (SEQ ID NO: 3), a human 4-1BB costimulatory region (SEQ ID NO: 4), and a human CD3ζ signal region (SEQ ID NO: 5). The CAR gene sequence arranged in this manner was introduced into the Multiple Cloning Site of the pLVSINEF1α Pur Vector (Takara). Example 4-4: Preparation of CD19 CAR-T and CAR-T cells targeting CD19 and LRRC15 Following the protocol for the Lentiviral High Titer Packaging Mix with pLVSIN series (Takara), the lentiviral plasmid vectors prepared in Examples 2-1, 4-1, 4-2, and 4-3 and the Lentiviral High Titer Packaging Mix (Takara) were transfected into the Lenti-X 293T Cell Line (Takara) using TransIT-293 Transfection Reagent (Mirus Bio). After one day, the medium was replaced. After two days, the culture supernatant was collected, and after a medium replacement, the supernatant was collected after another day of culture. The virus contained in the culture medium was concentrated using a Lenti-X concentrator (Takara) according to the protocol. For viral infection, plates coated with 4 μg / mL CD3 antibody (clone name: OKT3) and 20 μg / mL Retronectin were prepared according to the protocol for Retronectin (Takara). Next, human peripheral blood mononuclear cells were cultured at a concentration of 1x10 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). 6 The cells were suspended at 1000 x g / mL and added to a retronectin-coated plate along with virus concentrate. After centrifugation at 1000 x g for 60 minutes, the plate was cultured in a CO2 incubator. After one day of culture, virus concentrate was added again, the plate was centrifuged at 1000 x g for 60 minutes, and the culture was continued in a CO2 incubator. After three days, T cells were collected from each well, seeded into a cell culture flask, growth medium was added, and the plate was cultured for an additional six or seven days to prepare CAR-expressing T cells (CAR-T cells). The names of the CAR-T cells produced are listed in Table 4.

[0110] [Table 4]

[0111] Example 5: Evaluation of CAR-T cells targeting CD19 and LRRC15 Example 5-1: Examination of antitumor activity by combining CD19 CAR-T cells and LRRC15 CAR-T cells CD19 CAR-T cells and R024 H02L01 V2 CAR-T cells were administered alone or in combination to immunodeficient mice transplanted with EBC-1 / NucR / hCD19 cells prepared in Examples 1-2, and tumor growth inhibition was evaluated. EBC-1 / NucR / hCD19 cells were cultured at 2 × 10 in HBSS containing 0.1% bovine serum albumin (BSA). 7 The tumor was then prepared at a concentration of 1x10 cells / mL and 0.1 mL of the solution was subcutaneously implanted into NOG mice (female, 6 weeks old) (Day 0). The long and short diameters (mm) of the tumor were measured over time using an electronic digital caliper, and the estimated tumor volume was calculated using the same method as in Example 3-2. On Day 12, the tumors were divided into groups based on tumor volume, with each group consisting of n = 4 or 5. CD19 CAR-T cells were used, with CAR-positive cells at 1x10 5 cells / mL, 3x10 5 R024 H02L01 V2 CAR-T cells were cultured at 1x10 CAR-positive cells / mL. 7 cells / mL, 3x10 7 The solution was adjusted to 0.1 cells / mL with HBSS containing 0.1% BSA, and non-transfected T cells were added to the same total number, followed by administration of 0.1 mL into the tail vein.

[0112] Antitumor activity was evaluated by measuring tumor volume over time after CAR-T cell administration, and the results are shown in Figure 9. Compared to administration of CD19 CAR-T cells or R024 H02L01 V2 CAR-T cells alone, their combined administration demonstrated significant antitumor activity in a mouse xenograft model using EBC-1 / NucR / hCD19 cells. Example 5-2: Evaluation of CAR-T cells expressing both CARs using anti-CD19 antibodies and anti-LRRC15 antibodies The expression of CD19 CAR and LRRC15 CAR was examined using the CAR-T cells prepared in Example 4-4.

[0113] For each of the prepared CAR-T cells, histidine-tagged recombinant mouse LRRC15 protein was diluted in FCM buffer and added to the cells for 30 minutes at 4°C. After washing with FCM buffer, His-Tag(D3I1O)XP Rabbit mAb (Cell Signaling Technology) was diluted in FCM buffer and added to the cells for 30 minutes at 4°C. After washing with FCM buffer, Allophycocyanin (APC) AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch) and FITC-Labeled Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Y45) (Acro) were diluted in FCM buffer and added to the cells for 30 minutes at 4°C. After washing with FCM buffer, the cells were suspended in FCM buffer and analyzed by flow cytometry (BD The expression of CD19 CAR and LRRC15 CAR was confirmed by measurement using FACSCantoII.

[0114] The results are shown in Figures 10 and 11. It was confirmed that both CAR sequences could be expressed on the same T cells using the P2A and IRES sequences. Example 5-3: Construction of GFP or human LRRC15 / GFP gene expression vector GFP or human LRRC15 / GFP gene expression vectors were constructed using the GFP sequence (SEQ ID NO: 36) and the human LRRC15 sequence (SEQ ID NO: 37).

[0115] The human LRRC15 / GFP gene expression vector contained, from the N-terminus, human LRRC15, the self-cleaving peptide T2A sequence (SEQ ID NO: 38), and the GFP gene sequence. The GFP or human LRRC15 / GFP gene sequence was prepared by gene synthesis. The lentiviral plasmid vector for expressing GFP or human LRRC15 / GFP was prepared by inserting the above gene sequence into the Multiple Cloning Site of the pLVSIN EF1α Pur Vector (TAKARA). Example 5-4: Preparation of Raji / GFP cells and Raji / GFP / hLRRC15 cells The human Burkitt's lymphoma-derived cell line Raji was infected with lentivirus and transduced with GFP or GFP and human LRRC15 to generate Raji / GFP and Raji / GFP / hLRRC15 cells. Lentivirus was prepared according to the protocol of the Lentiviral High Titer Packaging Mix with pLVSIN series (Takara). The lentiviral plasmid vectors prepared in Example 5-3 and the Lentiviral High Titer Packaging Mix (Takara) were transduced into the Lenti-X 293T Cell Line (Takara) using TransIT-293 Transfection Reagent (Mirus Bio). The lentivirus was then collected from the culture supernatant. The virus in the culture medium was concentrated using a Lenti-X concentrator (Takara) according to the protocol. To infect Raji cells with lentivirus, cell culture plates were coated with 20 μg / mL Retronectin according to the protocol of Retronectin (Takara). Next, Raji cells were cultured at 1x10 in RPMI medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (FBS) and supplemented with penicillin-streptomycin (Thermo Fisher Scientific). 6 The cells were suspended at a concentration of 1000 cells / mL, added to a retronectin-coated plate together with the virus concentrate, and centrifuged at 1000 g for 60 minutes. After centrifugation, the cells were cultured in a CO2 incubator to prepare Raji / GFP and Raji / GFP / hLRRC15 cells. The proliferated cells were cloned by limiting dilution to generate Raji / GFP and Raji / GFP / hLRRC15 cells. Example 5-5: Examination of anti-tumor activity by combining CAR-T cells targeting two molecules using anti-CD19 antibody and anti-LRRC15 antibody Among the CAR-T cells prepared in Example 4-4, CD19 CAR-G4S-R024 H02L01 V2 CAR-T) was evaluated for its cytotoxic activity against Saos-2 / NucG cells, a cancer cell line that expresses LRRC15, Raji / GFP cells, a cancer cell line that expresses CD19, or Raji / GFP / hLRRC15 cells that express LRRC15 and CD19. The R024 H02L01 V2 CAR-T cells prepared in Example 2-2 and the CD19 CAR-T cells prepared in Example 4-4 were used as control cells.

[0116] Cytotoxicity of Saos-2 / NucG cells was evaluated using the Incucyte live cell analysis system (Sartorius). 10,000 Saos-2 / NucG cells (target cells) were seeded in McCoy's 5A medium supplemented with 15% fetal bovine serum (FBS) in a 96-well cell culture plate. Then, CAR-T cells (effector cells) were added to AIM-V medium containing 5% FBS and 100 U / mL IL-2 at CAR-positive cell counts of 3,000 (E / T = 0.3), 10,000 (E / T = 1), or 30,000 (E / T = 3). The number of target cells was measured over a 5-day period using an Incucyte S3 system at 37°C and 5% CO2. The viability (%) of target cells was calculated using the following formula: calculation formula Viability (%) = 100 × ((Number of target cells after 4 days of co-culture with effector cells) / (Number of target cells after 4 days of culture with target cells alone)) The cytotoxicity of Raji cells was evaluated using a MACSQUANT Analyzer 10 (Miltenyi Biotech) flow cytometer. 10,000 Raji / GFP or Raji / GFP / hLRRC15 cells (target cells) were seeded in 96-well cell culture plates in RPMI medium supplemented with 10% FBS. Subsequently, CAR-T cells (effector cells) were seeded in AIM-V medium containing 5% FBS and 100 U / mL IL-2 at CAR-positive cell counts of 100 (E / T = 0.01), 1,000 (E / T = 0.1), 5,000 (E / T = 0.5), 10,000 (E / T = 1), or 20,000 (E / T = 2), respectively, and co-cultured at 37°C and 5% CO2. One day after co-culture, an equal volume of FCM Buffer containing 2 μg / mL of 7-aminoactinomycin D was added to each well to stain dead cells, and measurements were performed using a MACSQUANT Analyzer 10. After measurements, cell fractions were selected on an FSC / SSC plot, and the group of cells that were negative for 7-aminoactinomycin D staining and positive for GFP were selected as target cells. The viability (%) of the target cells was calculated using the following formula. calculation formula Viability (%) = 100 × ((Number of target cells after 1 day of co-culture with effector cells) / (Number of target cells after 1 day of culture with target cells alone)) The results are shown in Figure 12. The generated CD19 CAR-G4S-R024 H02L01 V2 CAR-T cells targeting CD19 and LRRC15 had cytocidal activity against LRRC15-expressing cancer cell lines, CD19-expressing cancer cell lines, and cancer cell lines expressing both LRRC15 and CD19.

Claims

1. A chimeric antigen receptor comprising antigen-binding sites for one or more antigens, wherein the chimeric antigen receptor comprises at least an antigen-binding site that specifically binds to LRRC15.

2. The chimeric antigen receptor according to claim 1, wherein the antigen-binding site that specifically binds to LRRC15 comprises a first variable region comprising amino acid sequences derived from CDRH1, CDRH2, and CDRH3 of a heavy chain variable region contained in a monoclonal antibody or scFv that specifically binds to LRRC15, and a second variable region comprising amino acid sequences derived from CDRL1, CDRL2, and CDRL3 of a light chain variable region contained in the monoclonal antibody or scFv.

3. 3. The chimeric antigen receptor of claim 1 or 2, wherein the antigen-binding site is: （１） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 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: 40; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41 in which one or several amino acids have been substituted, deleted, or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added. or a light chain variable (VL) region comprising: （２） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 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: 46; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47 in which one or several amino acids have been substituted, deleted, or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added. or a light chain variable (VL) region comprising: （３） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 49; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50 in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added. or a light chain variable (VL) region comprising: （４） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 51 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 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: 52; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53 in which one or several amino acids have been substituted, deleted or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added. or a light chain variable (VL) region comprising: （５） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 54 in which one or several amino acids have been substituted, deleted or added; HC-CDR2 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 55; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56 in which one or several amino acids have been substituted, deleted, or added; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42 in which one or several amino acids have been substituted, deleted or added; LC-CDR2 comprising an amino acid sequence consisting of Trp-Ala-Ser (WAS) in which one or several amino acids have been substituted, deleted, or added; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 in which one or several amino acids are substituted, deleted, or added. a light chain variable (VL) region comprising: The chimeric antigen receptor comprising:

4. 4. The chimeric antigen receptor of claim 3, wherein the antigen-binding site is: （１） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 40; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or a light chain variable (VL) region comprising: （２） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 45; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:47; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or a light chain variable (VL) region comprising: （３） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 48; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:49; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or a light chain variable (VL) region comprising: （４） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:51; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 52; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:53; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 or a light chain variable (VL) region comprising: （５） The following CDRs: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:54; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:55; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; a heavy chain variable (VH) region comprising: The following CDRs: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 42; LC-CDR2 comprising the amino acid sequence Trp-Ala-Ser (WAS); and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44 a light chain variable (VL) region comprising The chimeric antigen receptor comprising:

5. 5. The chimeric antigen receptor of claim 1, wherein the antigen-binding site is: （１） a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO:57; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or （２） a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO:59; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or （３） a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or （４） a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 61; and a light chain variable (VL) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 58; or （５） a heavy chain variable (VH) region comprising an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 62; and A light chain variable (VL) region comprising an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

58. The chimeric antigen receptor comprising:

6. The chimeric antigen receptor according to any one of claims 1 to 5, comprising only an antigen-binding site that specifically binds to LRRC15 as the antigen-binding site.

7. The chimeric antigen receptor according to any one of claims 1 to 5, comprising the antigen-binding site that specifically binds to LRRC15 and antigen-binding sites for one or more different antigens.

8. The chimeric antigen receptor according to claim 7 , wherein the antigen-binding site for the different antigen is an antigen-binding site that binds to an antigen that is specifically expressed on the surface of cancer cells.

9. The chimeric antigen receptor according to claim 8, wherein the cancer cells are cancer cells contained in a solid cancer.

10. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10, the vector being designed in such a manner that the chimeric receptor encoded by the polynucleotide can be expressed on the cell surface.

12. 12. The vector according to claim 11, wherein the chimeric antigen receptor according to claim 6 and a second chimeric antigen receptor comprising an antigen-binding site that binds to an antigen that is specifically expressed on the surface of a cancer cell are designed in a manner such that they can be expressed on the cell surface as independent proteins.

13. A method for producing a chimeric antigen receptor-expressing cell, comprising a step of in vitro gene transfer into a cell using the polynucleotide of claim 10 or the vector of claim 11 or 12.

14. A cell expressing the chimeric receptor of any one of claims 1 to 9.

15. The cell of claim 14 , wherein the cell is an immune cell.

16. The cell according to claim 14 or 15, which expresses both the chimeric antigen receptor of claim 6 and a second chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of a cancer cell.

17. A pharmaceutical composition comprising the cells according to any one of claims 14 to 16 for treating a tumor associated with LRRC15-positive cells.

18. The pharmaceutical composition of claim 17, wherein the tumor is a tumor containing LRRC15-positive cancer cells.

19. The pharmaceutical composition of claim 17, wherein the tumor is a solid cancer containing LRRC15-positive cancer stromal cells in the tumor tissue.

20. The pharmaceutical composition according to claim 19, characterized in that the cell according to claim 16 is used.

21. 20. The pharmaceutical composition according to claim 19, comprising cells expressing the chimeric antigen receptor of claim 6, wherein the patient to whom the pharmaceutical composition is administered is undergoing cell therapy with cells expressing a chimeric antigen receptor comprising an antigen-binding site that binds to an antigen specifically expressed on the surface of cancer cells of the solid cancer.

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