NZ757552B2 - Chimeric antigen receptor - Google Patents

Abstract

chimeric antigen receptor containing a target antigen binding domain, a transmembrane domain and a T cell activation signal transduction domain, wherein the target antigen is ganglioside GM2.

Description

[DESCRHDTION] [TITLE OF INVENTION] CHIMERIC ANTIGEN RECEPTOR ical Field] The present invention relates to a chimeric antigen receptor, a cell expressing a chimeric antigen receptor, a vector including a base sequence ng a chimeric antigen receptor, and the like.

Priority is claimed on Japanese Patent Application No. 61461, filed March 27, 2017, the content of which is incorporated herein by reference.

[Background Art] A chimeric antigen receptor (hereinafter, also referred to as "CAR") is an artificial chimeric protein in which a single-stranded antibody that recognizes a cell surface antigen of a cancer cell is fused with a signal transduction region that induces T cell activation. For example, by introducing a gene encoding a CAR into normal eral blood T cells having no tumor reactivity (peripheral blood T lymphocytes), it is possible to produce a large amount of CAR—expressing T cells (hereinafter also referred to as "CAR—T cells") capable of expressing a CAR. Such CAR—T cells have tumor reactivity, and thus can allow cancer cells to be ed t relying on interaction with the major histocompatibility x (MHC).

Regarding cancer immunotherapy by administration of CAR—T cells, more specifically, a therapy in which T cells are collected from a patient, a gene encoding a CAR is uced into such T cells, and the gene is amplified to be retransferred into the patient, clinical trials are currently in progress all over the world, and results showing efficacy in hematopoietic malignancies such as leukemia and lymphoma, and the like have been obtained.

However, in cancer immunotherapy using CAR—T cells, the current situation is that ive results are obtained only for hematopoietic malignancies, and effective s are not obtained for solid tumors. In order to develop an effective CAR for solid tumors, ion of target antigens is ant, and searches for target antigens applicable to solid tumors are required. id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"

[0005] Meanwhile, as factors in a case where CAR—T cell therapy is not effective against solid tumors, a low survival ratio of CAR-T cells in vivo, a low level of accumulation thereof in local tumors, activity tion thereof by suppressive s secreted by tumor cells and the like, and the like are conceivable. As a method for solving such a problem, a method has been reported in which a nucleic acid encoding an immune function-promoting factor of T cells is introduced into T cells together with a nucleic acid encoding a CAR (Patent Literature 1).

[Citation List] [Patent Literature] id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"

[0006] [Patent Literature 1] PCT International Publication No. WO2016/056228 [Summary of Invention] [Technical Problem] id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"

[0007] Since effects of CAR-T cells targeting an n expressed in a human solid tumor have not been confirmed in Patent Literature 1, CAR-T cells showing cy against such an antigen are required. [0007A] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

An objective of the t invention is to provide a novel CAR that targets a solid tumor antigen as a target antigen, and a CAR-T cell that is effective against solid tumors, or to at least provide the public with a useful . ion to Problem] In a particular aspect, the present invention es an isolated cell which expresses a chimeric antigen receptor, IL-7 and CCL19, the chimeric antigen receptor comprising: a target antigen-binding region; a transmembrane region; and a T cell activation signal transduction region, wherein the target antigen-binding region comprises a heavy-chain variable region and a light-chain variable region of an anti-ganglioside GM2 antibody, wherein the anglioside GM2 dy is an antibody selected from the [FOLLOWED BY PAGE 3a] group consisting of the following (a) to (c): (a) an antibody that comprises a heavy-chain le region comprising CDR1, CDR2, and CDR3 of a heavy-chain le region consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region comprising CDR1, CDR2, and CDR3 of a light-chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein CDR1s, CDR2s, and CDR3s of the heavy-chain variable region and the light-chain variable region are determined by the tion of Kabat, Chothia, AbM or contact; (b) an antibody that comprises a heavy-chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of a mutation in the heavy-chain le region and the light-chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, a, AbM or contact; and (c) an antibody that comprises a heavy-chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of a on in the heavy-chain variable region and the light-chain le region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, Chothia, AbM or t.

[FOLLOWED BY PAGE 3b] [0009B] In another particular aspect, the present invention provides a polynucleotide comprising a base sequence that encodes the chimeric antigen receptor, a base sequence that encodes IL7, and a base sequence that encodes CCL19, the chimeric antigen receptor comprising: a target antigen-binding region; a embrane region; and a T cell activation signal transduction region, n the target antigen-binding region comprises a heavy-chain viable region and a light-chain variable region of an anti-ganglioside GM2 antibody, wherein the anti-ganglioside GM2 antibody is an antibody selected from the group consisting of the following (a) to (c): (a) an antibody that comprises a heavy-chain variable region sing CDR1, CDR2, and CDR3 of a heavy-chain variable region consisting of the amino acid ce set forth in SEQ ID NO: 2, and a light-chain variable region comprising CDR1, DR2, and CDR3 of a light-chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein CDR1s, CDR2s and CDR3s of the heavy-chain le region and the light-chain le region are ined by the definition of Kabat, Chothia, AbM or contact; (b) an antibody that comprises a heavy-chain variable region ting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, n a location of mutation in the heavy-chain variable region and the light-chain [FOLLOWED BY PAGE 3c] variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, a, AbM or contact; and (c) an antibody that comprises a chain variable region consisting of an amino acid sequence having 90% or more ce identity to the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of on in the heavy-chain variable region and the light-chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, Chothia, AbM or contact. [0009C] The following aspects are also disclosed. (1) A chimeric antigen receptor including a target antigen-binding region; a transmembrane region; and a T cell activation signal transduction region, in which the target antigen is ganglioside GM2. (2) The chimeric antigen receptor according to (1), in which the target antigenbinding region includes a heavy-chain variable region and a light-chain variable region of an anti-ganglioside GM2 antibody. (3) The chimeric antigen receptor ing to (2), in which the anti-ganglioside GM2 antibody is an dy selected from the group consisting of the ing (a) to (a) an antibody that includes a heavy-chain variable region including CDR1, CDR2, and CDR3 of a heavy-chain variable region consisting of an amino acid sequence set forth in SEQ ID NO: 2, and a light-chain le region including CDR1, CDR2, and [FOLLOWED BY PAGE 3d] CDR3 of a light-chain variable region consisting of an amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2; (b) an antibody that includes a heavy-chain variable region ting of an [FOLLOWED BY PAGE 4] amino acid sequence in which one or several amino acids are mutated in the amino acid ce set forth in SEQ ID NO: 2, and a light—chain variable region consisting of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2; (c) an antibody that includes a heavy-chain variable region consisting of an amino acid sequence having 70% or more sequence ty to the amino acid sequence set forth in SEQ ID NO: 2, and a chain variable region ting of an amino acid sequence having 70% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to oside GM2. (4) The chimeric antigen receptor according to (3), in which the heavy—chain variable region includes the amino acid sequence set forth in SEQ ID NO: 2, and the light—chain variable region includes the amino acid sequence set forth in SEQ ID NO: 4. (5) The chimeric antigen receptor according to any one of (2) to (4), in which the anti—ganglioside GM2 antibody is a single—stranded antibody (scFv). (6) The chimeric antigen receptor according to (5), in which the scFv is a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence selected from the group consisting of SEQ ID N05: 10, 12, 14, and 16; (b) a ptide that consists of an amino acid sequence having 70% or more sequence ty to an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 12, 14, and 16, and that has a binding ability to ganglioside GM2; and (c) a polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a binding ability to ganglioside GM2. (7) The chimeric antigen receptor according to any one of (1) to (6), in which the transmembrane region is a transmembrane region of CD8. (8) The chimeric antigen receptor according to (7), in which the transmembrane region of CD8 includes a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more ce identity to the amino acid sequence set forth in SEQ ID NO: 20, and that has a embrane ability; and (c) a polypeptide that consists of an amino acid sequence in which one or l amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 20, and that has a transmembrane y. (9) The chimeric antigen receptor according to any one of (1) to (8), in which the T cell activation signal transduction region is a T cell activation signal uction region of CD3C. (10) The chimeric antigen receptor according to (9), in which the T cell activation signal transduction region of CD3C includes a ptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to the amino acid sequence set forth in SEQ ID NO: 28, and that has a T cell activation signal transduction ability; and (c) a polypeptide that ts of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 28, and that has a T cell activation signal transduction ability. (11) The chimeric antigen receptor according to (9) or (10), in which the T cell activation signal transduction region further includes at least one of a T cell activation signal transduction region of CD28 and a T cell activation signal transduction region of 4-1BB. (12) The chimeric antigen receptor according to (11), in which the T cell activation signal transduction region of CD28 es a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more ce identity (homology) to the amino acid sequence set forth in SEQ ID NO: 24, and that has a T cell activation signal transduction y; and (c) a polypeptide that ts of an amino acid sequence in which one or several amino acids are mutated in the amino acid ce set forth in SEQ ID NO: 24, and that has a T cell tion signal transduction ability. (13) The chimeric antigen receptor according to (11), in which the T cell activation signal transduction region of 4-1BB includes a polypeptide selected from the group consisting of the following (a) to (c): (a) a ptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to the amino acid sequence set forth in SEQ ID NO: 26, and that has a T cell activation signal transduction ability; and (c) a polypeptide that ts of an amino acid sequence in which one or several amino acids are mutated in the amino acid ce set forth in SEQ ID NO: 26, and that has a T cell activation signal transduction ability. (14) The chimeric antigen receptor according to any one of (11) to (13), in which the T cell activation signal transduction region includes the T cell activation signal transduction regions of CD28, 4-1BB, and CD3?, and is located in the order of CD28, 4- 1BB, and CD3? from an N-terminal side. (15) A cell which expresses the chimeric antigen or according to any one of (1) to (14). (16) The cell according to (15), which further expresses at least one of IL-7 or CCL19. (17) The cell according to (16), which expresses both IL-7 and CCL19. (18) The cell according to (16) or (17), in which the IL-7 includes a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more sequence identity to the amino acid ce set forth in SEQ ID NO: 59, and that has a T cell-immune-function-promoting function; and (c) a polypeptide that consists of an amino acid sequence in which one or l amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 59, and that has a T cell-immune-function-promoting function. (19) The cell ing to any one of (16) to (18), in which the CCL19 includes a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that includes an amino acid sequence set forth in SEQ ID NO: (b) a polypeptide that consists of an amino acid sequence having 70% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 61, and that has a T cell—immune—function—promoting function; and (c) a polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 61, and that has a T cell—immune—function—promoting function. (20) The cell ing to any one of (15) to (19), in which the cell is an immune cell. (21) The cell according to (20), in which the immune cell is a T cell. (22) A polynucleotide including a base sequence that encodes the chimeric antigen receptor according to any one of (1) to (14). (23) The polynucleotide according to (22), further including at least one of a base ce that encodes IL-7 or a base sequence that s CCL19. (24) The polynucleotide according to (23), including both the base ce that encodes IL—7 and a base sequence that encodes CCL19. (25) A vector ing a base sequence that encodes the chimeric antigen receptor according to any one of (1) to (14). (26) The vector ing to (25), further including at least one of a base sequence that s IL—7 or a base sequence that encodes CCL19. (27) The vector according to (26), including both the base sequence that encodes IL—7 and a base sequence that encodes CCL19. (28) A method for producing a cell expressing a chimeric antigen receptor, including introducing a polynucleotide or a vector that includes a base sequence encoding the chimeric antigen receptor according to any one of (1) to (14) into the cell. (29) The method for ing a cell expressing a chimeric antigen receptor according to (28), further including ucing a polynucleotide or a vector that includes at least one of a base sequence encoding IL-7 or a base sequence encoding CCL19 into the cell. (30) The method for producing a cell expressing a chimeric antigen receptor according to (29), further ing introducing a polynucleotide or a vector that includes both a base ce encoding IL-7 and a base sequence encoding CCL19 into the cell. (31) A pharmaceutical composition sing the cell according to any one of (15) to (20). (32) The pharmaceutical composition according to (31), which is a pharmaceutical composition for ng or preventing a tumor. [0009D] In the description in this specification reference may be made to subject matter which is not within the scope of the appended claims. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the appended claims. [0009E] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’ and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited to". tageous Effects of Invention] According to the present ion, a novel CAR that targets a solid tumor [FOLLOWED BY PAGE 9a] antigen as a target antigen, and a CAR-T cell that is effective against solid tumors are [Brief Description of Drawings] Fig. 1A is a schematic view showing an anti-GM2 CAR construct.

Fig. 1B is a schematic view showing an IL-7/CCL19-expressing-anti-GM2 CAR vector and an anti-GM2 CAR-IL-7/CCL19-expressing T cell into which the vector has been introduced.

Fig. 2 shows results of checking an sion level of CAR in the anti-GM2 CAR-IL-7/CCL19-expressing T cells by flow cytometry. The left graph shows results of a non-transgenic T cell, and the right graph shows results of the anti-GM2 CAR-IL- [FOLLOWED BY PAGE 10] 7/CCLl9-expressing T cell.

Fig. 3 shows results of measuring concentrations of IL—7 and CCL19 in a culture supernatant of an anti—GM2 CAR—expressing T cell by ELISA. In the graphs, the term "GM2 CAR" ents an anti—GM2 CAR—IL—7/CCL19—expressing T cell, and the term "non—infection" represents a non—transgenic T cell (the same applies in the subsequent drawings).

Fig. 4 shows assay schedules of a tumor cytotoxicity assay and a co—culture assay using M2 CAR—IL—7/CCL19—expressing T cells.

Fig. 5A shows results of a chromium release assay using four types of anti—GM2 CAR—IL—7/CCL19—expressing T cells. Fig. 5A shows results of the assay in which malignant mesothelioma cell lines (Y—mes08A and MST0211H) are used as target cells.

In the graphs, each of "VLISVH," "VL25VH," "VH15VL," and "VHZSVL" ents results with the anti-GM2 CAR—IL—7/CCL19—expressing T cells including the corresponding sequences as scFV sequences of anti-GMZ CAR (the same applies in the subsequent graphs).

Fig. 5B shows results of a chromium release assay using four types of anti—GM2 CAR-IL—7/CCL19-expressing T cells. Fig. 5B shows results of the assay in which a cell lines (KMS—ll and KMS—ZSPE) are used as target cells.

Fig. 6A shows comparison results of um release assays of an anti—GM2 CAR—IL—7/CCL19—expressing T cell and a control CAR—T cell. Fig. 6A shows results of the assay in which a malignant mesothelioma cell line (Y—mesoSA) is used as a target cell. In the graphs, "FITC CAR—T" represents anti-FITC CAR-T cells used as a negative control (the same s in the subsequent graphs).

Fig. 6B shows comparison results of chromium release assays of an MZ —7/CCL19-expressing T cell and a control CAR—T cell. Fig. 6B shows results of the assay in which a a cell line (KMSl 1) is used as a target cell.

Fig. 6C shows comparison s of chromium release assays of an anti—GM2 CAR—IL—7/CCL19-expressing T cell and a control CAR—T cell. Fig. 6C shows results of the assay in which a colon cancer cell line ) is used as a target cell.

Fig. 7 shows results of a co—culture assay of a GM2—positive malignant mesothelioma cell line (Y—mesoSA) with anti-GM2 CAR—IL—7/CCL19-expressing T cells or control cells. In the , the term "tumor only" tes that only tumor cells are cultured (the same applies in the subsequent drawings).

Fig. 8 shows results of a co—culture assay of a GM2-positive malignant mesothelioma cell line (MST0221H) with anti—GM2 CAR—IL—7/CCL19—expressing T cells or control cells.

Fig. 9 shows results of a co-culture assay of a GM2-negative colon cancer cell line (SW480) with anti—GM2 CAR—IL—7/CCL19—expressing T cells or control cells.

Fig. 10 shows results of measuring IFN-y in a culture supernatant of a co-culture assay of each tumor cell line with anti—GM2 CAR—IL—7/CCL19-expressing T cells or control cells by ELISA.

Fig. 11A shows ssion of tumor growth when anti—GM2 CAR—IL— 7/CCL19—expressing T cells or ansgenic T cells are administered to immunodeficient mice to which MST0211H expressing Luciferase has been intrathoracically administered.

Fig. 11B shows progression of tumor growth when anti—GM2 CAR—IL— 7/CCL19-expressing T cells or non—transgenic T cells are administered to immunodeficient mice to which MST0211H expressing Luciferase has been intraperitoneally administered.

Fig. 12 shows analysis results of progression of tumor growth when anti—GM2 CAR-IL—7/CCL19-expressing T cells or non-transgenic T cells are administered to immunodeficient mice to which MST0211H expressing Luciferase has been horacically administered.

Fig. 13 shows is results of progression of tumor growth when anti-GM2 CAR—IL—7/CCL19—expressing T cells, anti-GM2 CAR—expressing T cells, or non— transgenic T cells are administered to deficient mice to which MST0211H sing Luciferase has been intrathoracically administered. In the figure, "x" indicates that a mouse died.

Fig. 14 shows s of analyzing effects on progression of tumor growth when anti—GM2 CAR—IL—7/CCL19—expressing T cells, anti—GM2 CAR—expressing T cells, or non—transgenic T cells are administered to immunodeficient mice to which MST0211H expressing Luciferase has been intrathoracically administered.

Fig. 15 shows results of analyzing effects on survival ratios of mice when anti- GM2 CAR—IL—7/CCL19—expressing T cells, anti-GM2 CAR—expressing T cells, or non— transgenic T cells are administered to deficient mice to which MST0211H expressing Luciferase has been intrathoracically administered.

[Description of Embodiments] Polypeptides, polynucleotides, s, and cells provided by the present invention may be in an isolated state. In other words, polypeptides, polynucleotides, s, and cells described in the present specification may be isolated polypeptides, isolated cleotides, isolated vectors, and isolated cells.

[Chimeric antigen receptor (anti—GM2 CAR)] In one embodiment, the present invention provides a chimeric antigen receptor including a target antigen-binding region; a embrane region; and a T cell activation signal uction region, in which the target antigen is ganglioside GM2.

In the present specification, the term "chimeric antigen receptor (CAR)" means a chimeric protein ing a target antigen—binding region, a transmembrane region, and a T cell activation signal transduction region. The chimeric protein means a protein including a sequence d from two or more kinds of heterologous proteins. A CAR is not limited to a CAR including only the above three regions, and includes a CAR including other regions. id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"

[0015] The CAR of the present embodiment includes a target antigen-binding region in which the target antigen is ganglioside GM2 (hereinafter also referred to as "GM2").

The term "target antigen—binding region" means an extracellular region that binds to a target antigen extracellularly when a CAR is expressed in a T cell. A CAR sed in a CAR—T cell transfers to a cell membrane to be in a state in which a target antigen—binding region d extracellularly and a T cell activation signal transduction region located intracellularly are connected through a transmembrane region that penetrates the cell membrane. When the CAR—T cell comes into contact with a cell having the target antigen as a membrane antigen, the target antigen—binding region binds to the target antigen, whereby the T cell tion signal is transmitted from the T cell activation signal transduction region to the inside of the T cell to activate the T cell.

In the CAR of the present embodiment, the target antigen to which the target antigen—binding region binds is GM2.

GM2 is a type of ganglioside that is a glycolipid having a sialic acid. A ganglioside is a molecule that constitutes a cell membrane of an animal, and is composed of a sugar chain that is a hydrophilic side chain, and sphingosine and a fatty acid which are hobic side chains. Gangliosides are classi?ed according to the binding type and number of bonds with sialic acid, and the presence or absence of g of N— acetylgalactosamine (GalNAc) and galactose (Gal) which bind to a non—reducing end.

GM2 is one of gangliosides having a sugar chain structure of GalNAcB1—4 (SAu2—3) GalBl—4Gchl—1 Ceramide. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"

[0018] The type and expression level of gangliosides vary depending on the cell type, organ type, animal type, and the like. It is known that expression of gangliosides changes quantitatively and qualitatively in the process of cancerous change of cells (Cancer Res 45: 2405-14(1985)). It has been reported that hardly any GM2 can be recognized as being sed in normal cells, but can be sed in tumors such as in lung cancer, neuroblastoma, glioma, melanoma, malignant elioma, and myeloma (Cancer Res 45: 2405—14 (1985); Cancer Res 50: 7444—9 (1990); Cancer Sci vol. 102 no. 12: 2157—2163; Cancer Sci vol. 106 no. 1: 102—107 (2015)).

The target antigen—binding region is not particularly limited as long as it can specifically bind to GM2, but ably includes an antigen—binding region of a monoclonal dy (hereinafter also referred to as an "anti—GM2 antibody") capable of specifically binding to GM2. The "antigen—binding region" of an antibody refers to a region involved in binding to an antigen in an antibody, and specifically refers to a region including a complementarity determining region (CDR). The antigen—binding region of an antibody includes at least one CDR of the antibody. In a preferred ment, the antigen—binding region of an antibody includes all six CDRs of the antibody. CDRs can be determined by any definition known for definition of CDRs, and it is possible to use, for example, the definition by Kabat, Chothia, AbM, cotact, and the like. Preferred examples of CDRs include CDRs d by Kabat.

An anti—GM2 antibody that can be used for the target antigen—binding region is not particularly limited, and may be a known antibody or a newly produced antibody.

When newly producing an anti—GM2 antibody, production of the anti—GM2 antibody may be med by a known method. For example, it is possible to use a method of immunizing an animal with GM2 to obtain a hybridoma, a phage display method, or the like.

An organism from which the anti-GM2 dy is derived is not ularly limited, but a human antibody is preferable. es of human anti—GM2 antibodies e an antibody having an amino acid sequence set forth in SEQ ID NO: 2 as a heavy—chain variable (VH) region and an amino acid sequence set forth in SEQ ID NO: 4 as a light—chain variable (VL) region, and the like. Amino acid sequences of CDRs 1 to 3 according to the definition of Kabat of the VH region consisting of the amino acid sequence set forth in SEQ ID NO: 2 are respectively shown as SEQ ID NOs: 63 to 65.

In on, amino acid sequences of CDRs 1 to 3 according to the definition of Kabat of the VL region consisting of the amino acid sequence set forth in SEQ ID NO: 4 are respectively shown as SEQ ID NOs: 66 to 68.

In a preferred embodiment, the target antigen—binding region can include the VH region and the VL region of the anti—GM2 antibody. For example, a polypeptide including a single—stranded dy (scFv) having the VH region and the VL region of the anti—GM2 antibody is a suitable example of the target antigen—binding region. The scFv is a polypeptide in which a VH region and a VL region of an antibody are linked via a e linker, and is generally used as a target antigen—binding region of a CAR.

In a case of using scFv, a peptide linker for linking the VH region and the VL region is not particularly limited, and peptide linkers generally used for scFv may be used. Examples of peptide linkers e a linker 15 (SEQ ID NO: 6), a linker 25 (SEQ ID NO: 8), and the like, but examples are not limited thereto.

The VH region and the VL region of the anti-GM2 antibody may be used as a VH region and a VL region used for scFv. red examples of anti—GM2 antibodies are as described above. In addition, a part of the sequence may be modified in the VH region and the VL region used for scFv as long as a binding ability to GM2 is retained.

For example, as scFv, the following examples can be preferably used. (1a) scFv that includes a VH region including CDRs 1 to 3 of the VH region consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a VL region including CDRs 1 to 3 of the VL region consisting of the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to GM2. (1b) scFv that includes the VH region consisting of the amino acid sequence set forth in SEQ ID NO: 2 and the VL region consisting of the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding y to GM2. (1c) scFv that includes a VH region consisting of an amino acid sequence in which one or l amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 2, and a VL region consisting of an amino acid sequence in which one or several amino acids are d in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to GM2. (1d) scFv that includes a VH region consisting of an amino acid sequence having 70% or more sequence identity (homology) to the amino acid sequence set forth in SEQ ID NO: 2, and a VL region consisting of an amino acid sequence having 70% or more sequence identity (homology) to the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding y to GM2.

In the above (1a), as sequences (framework sequences) other than CDRs, it is preferable to use framework sequences of known human antibodies. For example, it is possible to select sequences from framework sequences of amino acid sequences of human antibodies registered in known sequence ses such as GenBank, amino acid sequences selected from consensus sequences derived from each subgroup of human antibodies (Human Most Homologous Consensus ce; Sequences of ns of Immunological st by Kabat, E. A. et al., US Dept. Health and Human Services, 1991), and the like.

In the above (1c), the term "several" may refer to, for example, 2 to 30, preferably refers to 2 to 20, more preferably refers to 2 to 10, and still more preferably refers to 2 to 5. In addition, the term "mutated" may refer to any of deletion, tution, addition, and insertion, or a combination thereof. Furthermore, a location of mutation is preferably a region other than CDRs 1 to 3 (that is, a framework region).

In the above (1d), the sequence identity is not particularly limited as long as it is 70% or more, but is preferably 80% or more, is more preferably 85% or more, is even more preferably 90% or more, is still more preferably 95% or more, and is particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more. A sequence identity (or homology) between amino acid sequences is obtained as a proportion of matching amino acids to the entire amino acid sequence ing gaps in the obtained ent by juxtaposing two amino acid sequences while inputting gaps in portions corresponding to insertions and deletions so that the corresponding amino acids match most y. The sequence identity between amino acid sequences can be obtained using various types of homology search software known in the technical field. For example, a value of sequence ty of amino acid sequences can be obtained by ation based on an alignment obtained by the known homology search software BLASTP.

Specific examples of scFv’s include, for e, a polypeptide that es an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16; a polypeptide that consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16; a polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a binding ability to GM2; a polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a binding ability to GM2; and the like. Regarding the term "several" and the term ed," the same applies as described above. In addition, regarding the term "sequence identity," the same s as described above.

The term "transmembrane region" means a region that is present by penetrating a cell membrane and is linked to an extracellular region and an intracellular region when a CAR is expressed in a T cell. The transmembrane region is not particularly limited as long as it is a polypeptide having a function of penetrating a cell membrane. The embrane region may be derived from a natural protein or may be artificially designed. A transmembrane region derived from a natural protein can be obtained from any membrane—binding protein or transmembrane protein. In a red embodiment, the transmembrane region can transmit an activation signal to the T cell activation signal transduction region in response to binding of the target antigen to the target antigen— binding region. es of transmembrane regions include transmembrane regions of an or chain and a [3 chain of a T cell receptor, CD3C, CD28, CD38, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, and the like. Preferred es include a transmembrane region of CD8. An organism from which these proteins are derived is not particularly limited, but is preferably human. Amino acid sequences of these proteins are available from known ce ses such as GenBank. Examples of amino acid sequences of human CD8 include an amino acid ce registered as GenBank No: NM_001768.6, and the like, and examples of amino acid sequences of the transmembrane region include an amino acid sequence set forth in SEQ ID NO: 20.

In addition, the transmembrane region may be a mutant of the above—mentioned transmembrane region derived from a natural protein. Examples of mutants of a transmembrane region d from a natural protein include the following. (2a) A polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to an amino acid sequence (for e, SEQ ID NO: 20) of a transmembrane region derived from a natural protein, and that has a transmembrane ability. (2b) A polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence (for example, SEQ ID NO: ) of a transmembrane region derived from a natural protein, and has a transmembrane ability.

In the above (2a), the sequence identity is not particularly limited as long as it is 70% or more, but is ably 80% or more, is more ably 85% or more, is even more preferably 90% or more, and is particularly preferably 95% or more. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"

[0033] In the above (2b), the term "several" may refer to, for example, 2 to 10, preferably refers to 2 to 5, more preferably refers to 2 to 4, and still more preferably refers to 2 or 3. In addition, the term ed" may refer to any of deletion, substitution, addition, and insertion, or a combination thereof. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"

[0034] The term "T cell activation signal transduction region" means a region that is located intracellularly and its a T cell activation signal to the inside of the T cell when a CAR is expressed in the T cell. In a T cell, when an MHC—peptide x binds to a T cell receptor (TCR), a T cell activation signal is transmitted to the inside of the cell Via a TCR-CD3 x, and various phosphorylation signals are triggered (primary signal transduction). In addition, it is known that costimulatory molecules expressed on a cell surface of a T cell it costimulatory signals to the inside of the cell and support tion of the T cell by binding of each costimulatory molecule sed on a cell surface of an antigen presenting cell to a specific ligand (secondary signal transduction).

In the present specification, the term "T cell activation signal transduction" includes both the primary signal transduction and the secondary signal transduction mentioned above. The term "T cell activation signal transduction region" means an intracellular region ed in signal transduction of a protein involved in the primary signal transduction and the ary signal transduction.

The T cell activation signal transduction region is not ularly limited as long as it is a T cell activation signal transduction region of a protein involved in T cell activation signal uction. For example, an immunoreceptor tyrosine—based activation motif (ITAM) is known to be involved in primary signal transduction.

Accordingly, examples of T cell activation signal transduction regions include a T cell activation signal transduction region of a protein having an ITAM. Examples of proteins having an ITAM include CD3C, FcRy, FCRB, CD3y, CD35, CD38, CD5, CD22, CD79a, CD79b, CD66d, and the like. A T cell activation signal transduction region including an ITAM of these proteins is a preferred example of the T cell activation signal transduction region used for a CAR. More preferred examples thereof include a T cell activation signal transduction region of CD3C or the like.

In addition, costimulatory molecules are involved in secondary signal transduction as described above. Accordingly, es of T cell activation signal transduction regions also include a signal transduction region of costimulatory molecules.

Examples of costimulatory les include CD2, CD4, CD5, CD8, CD27, CD28, OXO40 (CD134), 4-1BB (CD137), ICOS, CD154, HVEM, GITR, Fc or— associated 7 chain, and the like. A T cell activation signal transduction region of these proteins is also a red example of the T cell activation signal transduction region used for a CAR. More preferred examples thereof include a T cell activation signal transduction region of CD28, 4—1BB, or the like.

An organism from which the above—mentioned ns are derived is not particularly limited, but is preferably human. Amino acid sequences of these proteins are available from known sequence databases such as GenBank. Examples of amino acid sequences of human CD3C include an amino acid sequence registered as GenBank No: 734.3, and the like, and examples of amino acid sequences of the T cell activation signal transduction region include an amino acid sequence set forth in SEQ ID NO: 28. In addition, examples of amino acid sequences of human CD28 include an amino acid sequence registered as GenBank No: NM_006139.2, and the like, and examples of amino acid sequences of the T cell activation signal transduction region include an amino acid sequence set forth in SEQ ID NO: 24. Furthermore, examples of amino acid ces of human 4—1BB include an amino acid sequence registered as GenBank No: NM_001561.5, and the like, and examples of amino acid sequences of the T cell activation signal transduction region include an amino acid sequence set forth in SEQ ID NO: 26. rmore, a T cell activation signal transduction region may be a mutant of the T cell activation signal transduction region derived from a natural protein as described above. Examples of mutants of an activation signal transduction region derived from a natural protein include the following. (3a) A polypeptide that consists of an amino acid sequence having 70% or more sequence identity ogy) to an amino acid ce (for e, SEQ ID N03: 24, 26, or 28) of a T cell activation signal transduction region derived from a natural protein, and that has a T cell activation signal transduction ability. (3b) A polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence (for example, SEQ ID NOS: 24, 26, or 28) of a T cell activation signal transduction region derived from a natural n, and has a T cell activation signal uction ability.

In the above (3a), the ce identity is not particularly limited as long as it is 70% or more, but is preferably 80% or more, is more preferably 85% or more, is even more preferably 90% or more, and is particularly preferably 95% or more.

In the above (3b), in a case of using a region of a protein involved in primary signal transduction, the term "several" may refer to, for example, 2 to 30, preferably refers to 2 to 20, more ably refers to 2 to 10, and still more preferably refers to 2 to 5. In addition, in a case of using a region of a costimulatory molecule, the term "several" may refer to, for example, 2 to 15, preferably refers to 2 to 10, more preferably refers to 2 to 5, and still more preferably refers to 2 or 3. In addition, the term "mutated" may refer to any of on, substitution, addition, and insertion, or a combination thereof. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"

[0041] The number of T cell activation signal uction regions included in the CAR of the present embodiment is not limited to one, and plural T cell activation signal transduction regions can be included. In this case, plural T cell activation signal transduction regions may be the same as or different with each other. In a preferred embodiment, the CAR includes two or more T cell activation signal transduction s.

In this case, the T cell activation signal transduction s included in the CAR are preferably a combination of a T cell activation signal uction region ed in primary signal transduction and a T cell activation signal transduction region ed in secondary signal transduction. Specific examples thereof include a combination of T cell activation signal transduction regions of CD3C and CD28, a combination of T cell activation signal transduction s of CD3C and 4—1BB, a combination of T cell activation signal transduction regions of CD3C, CD28, and 4-1BB, and the like.

In a case of combining the T cell activation signal transduction region involved in primary signal transduction with the T cell activation signal transduction region involved in secondary signal transduction, the T cell activation signal transduction region involved in primary signal transduction is preferably located at a C—terminal side. In the above—mentioned specific examples, it is preferable that the T cell activation signal transduction region of CD3§ be d on the C—terminal side of the T cell activation signal transduction region of CD28 or 4—lBB. In a case where both CD28 and 4—lBB are used, the regions may be located in any order, but examples of location include location in the order of CD28 and 4—1BB from an N terminal side.

In a case where only one T cell activation signal transduction region is used, it is preferable to use a T cell activation signal transduction region involved in primary signal transduction, and it is more preferable to use a T cell activation signal transduction region of CD3C.

The CAR of the t ment may include other regions in addition to the above regions. Examples of other regions include an extracellular hinge region, a cytoplasmic region, a spacer region, a signal peptide, and the like.

(Extracellular hinge region) The term "extracellular hinge region" means a region linking an extracellular target antigen-binding region and a transmembrane region. In a preferred embodiment, the CAR of the t embodiment includes an extracellular hinge region.

The extracellular hinge region is not particularly limited as long as it can link a target antigen-binding region and a transmembrane . The extracellular hinge region may be derived from a natural protein or may be artificially designed. The extracellular hinge region can be composed of, for example, about 1 to 100 amino acids, and preferably about 10 to 70 amino acids. The extracellular hinge region is preferably a region that does not interfere with a binding ability to GM2 of the target n— binding region and does not interfere with signal transduction by the T cell tion signal transduction region.

Examples of extracellular hinge regions include extracellular hinge regions of CD8, CD28, CD4, and the like. In addition, a hinge region of an immunoglobulin (for example, lgG4 and the like) may be used. Preferred examples include an extracellular hinge region of CD8.

An organism from which the above—mentioned proteins are derived is not particularly limited, but is preferably human. Amino acid sequences of these proteins are available from known sequence databases such as GenBank. Examples of amino acid sequences of human CD8 include amino acid ces described above, and examples of amino acid sequences of the extracellular hinge region include an amino acid ce set forth in SEQ ID NO: 18.

In addition, the extracellular hinge region may be a mutant of the above- mentioned extracellular hinge region derived from a natural protein. Examples of mutants of an extracellular hinge region derived from a natural protein include the (4a) A ptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to an amino acid sequence (for example, SEQ ID NO: 18) of an extracellular hinge region derived from a natural protein. (4b) A polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence (for example, SEQ ID NO: 18) of an extracellular hinge region derived from a natural protein.

In the above (4a), the sequence identity is not particularly limited as long as it is 70% or more, but is preferably 80% or more, is more ably 85% or more, is even more preferably 90% or more, and is ularly preferably 95% or more.

In the above (4b), the term "several" may refer to, for example, 2 to 20, preferably refers to 2 to 15, more ably refers to 2 to 10, and still more preferably refers to 2 to 5. In addition, the term "mutated" may refer to any of deletion, substitution, addition, and insertion, or a ation thereof.

(Cytoplasmic region) A "cytoplasmic region" is a region adjacent to a cytoplasmic end of a embrane region in a transmembrane protein, and is a region consisting of about 3 to 50 amino acids. In a preferred embodiment, the CAR of the t embodiment includes a cytoplasmic region. The cytoplasmic region is not particularly limited as long as it is a region adjacent to a asmic side of the transmembrane region of the transmembrane protein. By linking the transmembrane region to the T cell activation signal transduction region via the cytoplasmic region, a structure of the transmembrane region can be ized. The cytoplasmic region may be derived from a l n or may be artificially designed. The cytoplasmic region may be composed of, for example, about 3 to 50 amino acids, preferably about 4 to 20 amino acids, and more preferably about 5 to 10 amino acids. The cytoplasmic region is preferably a region derived from the same protein as the case of the transmembrane . By using the cytoplasmic region derived from the same protein as the case of the transmembrane region, the structure of the transmembrane region can be kept more stable.

Examples of cytoplasmic regions include a asmic region of the proteins mentioned in the transmembrane region described above. Preferred examples include a cytoplasmic region of CD8. An organism from which these proteins are derived is not particularly limited, but is preferably human. Examples of amino acid sequences of the cytoplasmic region include an amino acid sequence set forth in SEQ ID NO: 22. id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"

[0053] In addition, the cytoplasmic region may be a mutant of the above-mentioned cytoplasmic region derived from a natural n. Examples of s of a cytoplasmic region derived from a natural protein include the following. (5a) A polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to an amino acid ce (for example, SEQ ID NO: 22) of a cytoplasmic region derived from a natural protein, and that has a transmembrane —stabling y. (5b) A polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence (for e, SEQ ID NO: 22) of a cytoplasmic region derived from a natural protein, and has a transmembrane region—stabling ability.

In the above (5a), the sequence identity is not particularly limited as long as it is 70% or more, but is preferably 80% or more, is more preferably 85% or more, is even more preferably 90% or more, and is particularly preferably 95% or more.

In the above (5b), the term "several" may refer to, for example, 2 to 5, preferably refers to 2 to 4, and more preferably refers to 2 or 3. In addition, the term "mutated" may refer to any of deletion, substitution, addition, and insertion, or a combination thereof.

(Spacer region) A "spacer region" is a short e that links two functional regions (domains) of a protein. In one , in the CAR of the present embodiment, each of the target antigen-binding region, the transmembrane region, the T cell activation signal transduction region, and the like described above may be linked via a spacer region.

The spacer region is not particularly limited, and a spacer region generally used for producing a chimeric protein may be used. A length of the spacer region may be 1 to 100 amino acids, and is preferably 10 to 50 amino acids. Examples of spacer regions include glycine—serine continuous ces and the like.

(Signal e) A "signal peptide" is a peptide that directs localization of a membrane protein or a secreted protein. In one aspect, the CAR of the present embodiment may include a signal peptide. The signal peptide is generally a peptide consisting of about 5 to 60 amino acids present at an N al of a membrane protein, and is removed in a d protein which has been completely localized.

The signal peptide used for the CAR of the present embodiment is preferably a signal peptide that directs localization of a protein to a cell ne, and is preferably a signal e of a membrane protein. es of signal peptides e signal peptides of 0L chain and a B chain of a T cell receptor, CD3C, CD28, CD38, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, an immunoglobulin heavy chain, an immunoglobulin light chain, and the like. Specific examples of amino acid sequences of the signal peptide include an amino acid sequence set forth in SEQ ID NO: 57.

In the CAR of the present embodiment, each of the above—mentioned regions can be located in the order of the target antigen—binding region, the transmembrane region, and the T cell activation signal transduction region from the N terminal. Each of these regions may be directly linked to each other, or may be linked via another region, a spacer sequence, or the like.

In a case Where the CAR of the t embodiment includes an extracellular hinge region, the extracellular hinge region is located between the target antigen—binding region and the transmembrane region. In addition, in a case Where the CAR of the present embodiment includes a cytoplasmic region, the cytoplasmic region is d between the transmembrane region and the T cell tion signal transduction region.

Furthermore, in a case Where the CAR of the present embodiment includes a signal peptide, the signal peptide is located at the N terminal of the CAR.

Specific examples of the CAR of the present embodiment include a CAR including a target antigen—binding region including scFv of an anti—GM2 antibody, a transmembrane region of CD8 (or a mutant thereof), a T cell activation signal transduction region of CD28 (or a mutant thereof), a T cell activation signal transduction region of 4-1BB (or a mutant thereof), and a T cell activation signal transduction region of CD3C (or a mutant thereof). More preferred es thereof include a CAR including a target antigen—binding region ing scFv of an anti—GM2 antibody, an extracellular hinge region of CD8 (or a mutant thereof), a transmembrane region of CD8 (or a mutant f), a cytoplasmic region of CD8 (or a mutant thereof), a T cell activation signal transduction region of CD28 (or a mutant f), a T cell activation signal transduction region of 4—1BB (or a mutant thereof), and a T cell tion signal transduction region of CDSC (or a mutant thereof).

Examples of such a CAR include a CAR including an amino acid sequence selected from the group consisting of SEQ ID N03: 40, 42, 44, and 46. In the amino acid ces set forth in SEQ ID NOs: 40, 42, 44, and 46, the sequences at positions 1 to 19 correspond to signal peptides. Accordingly, each of matured CARS in the above— mentioned examples includes the amino acid sequence at positions 20 to 874 of the amino acid sequence set forth in SEQ ID NO: 40, the amino acid sequence at positions to 884 of the amino acid sequence set forth in SEQ ID NO: 42, the amino acid sequence at positions 20 to 874 of the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence at positions 20 to 884 of the amino acid sequence set forth in SEQ ID NO: 46.

In a preferred embodiment, the CAR of the present embodiment consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 42, 44, and 46. In addition, a d CAR ts of an amino acid sequence selected from the group consisting of the amino acid sequence at ons 20 to 874 of the amino acid sequence set forth in SEQ ID NO: 40, the amino acid sequence at positions 20 to 884 of the amino acid sequence set forth in SEQ ID NO: 42, the amino acid sequence at positions 20 to 874 of the amino acid sequence set forth in SEQ ID NO: 44, and the amino acid sequence at ons 20 to 884 of the amino acid sequence set forth in SEQ ID NO: 46.

[Cell expressing anti—GM2 CAR (anti—GM2 CAR—expressing cell)] In one embodiment, the present invention provides a cell that expresses the CAR of the above embodiments.

The cell of the present embodiment expresses the CAR of the above embodiment (hereinafter also referred to as "anti—GM2 CAR"), and has the CAR on a cell e. In GM2-expressing cells, GM2 is ntly present on a cell surface.

When the cell of the present embodiment comes into contact with a GMZ—expressing cell, the cell binds to GM2 on the surface of the GM2—expressing cell Via the target antigen— binding region of the anti—GMZ CAR. ingly, the cell of the present embodiment is ted, and thereby release of cytolytic granules and production of cytokines are caused. These cytolytic granules and cytokines destroy the pressing cell.

The cell of the present ment is ably a mammalian cell, and may be, for example, a human cell or a cell of non—human mammalians such as mice, rats, cattle, sheep, horses, dogs, pigs, and monkeys, and is more preferably a human cell. The type of cells is not particularly limited, and examples include cells collected from blood, bone marrow ?uid, spleen, thymus, lymph nodes, and the like; immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites; and the like.

Preferable examples thereof include immune cells, and peripheral blood mononuclear cells separated from peripheral blood, and the like can be ably used. Among the cells contained in the peripheral blood mononuclear cells, effector cells are preferable, and T cells and their sor cells are particularly preferred cells. The type of T cells is not particularly limited, and T cells may be any T cells among (13 T cells, y?) T cells, CD8—positive T cells, cytotoxic T cells, CD4—positive T cells, helper T cells, memory T cells, naive T cells, tumor infiltrating T cells, natural killer T cells, and the like. Among these, CD8—positive T cells or cytotoxic T cells are more preferable.

It is preferable that, in addition to the anti—GM2 CAR, the cell of the present embodiment further express at least one of interleukin (lL)—7 or a chemokine (C—C motif) ligand 19 (CCL 19). In a preferred embodiment, the cell of the present embodiment is a cell that expresses (i) the anti-GM2 CAR, and (ii) at least one of IL-7 or CCL19. More preferably, the cell of the present embodiment is a cell that ses the anti—GM2 CAR, IL—7, and CCL19.

IL-7 is a ne essential for survival of T cells, and is produced by non— hematopoietic cells such as stromal cells in bone marrow, thymus, and lymphoid organs and tissues, but production thereof by T cells is hardly recognized.

Meanwhile, CCL19 is mainly produced from dendritic cells and macrophages in lymph nodes, and has a function of causing migration of T cells and B cell, and mature dendritic cells via its receptor, which is a CC chemokine receptor 7 (Chemokine (C—C motif) Receptor 7: CCR7).

An organism from which IL—7 and CCL19 are derived is not particularly limited, but is preferably human. Amino acid sequences of these proteins are available from known sequence databases such as k. For example, examples of amino acid sequences of human IL—7 include an amino acid ce registered as GenBank N0: NM_000880.3 (SEQ ID NO: 59), and the like. In addition, examples of amino acid sequences of human CCL19 e an amino acid sequence registered as GenBank No: NM_006274.2 (SEQ ID NO: 61), and the like. IL—7 and CCL19 have a signal peptide, and the signal peptide is removed from mature ns. For example, in the amino acid sequence set forth in SEQ ID NO: 59 of human IL—7, the sequence at positions 1 to 25 correspond to a signal peptide. Furthermore, for e, in the amino acid sequence set forth in SEQ ID NO: 61 of human CCL19, the sequence at positions 1 to 21 correspond to a signal peptide.

Furthermore, IL—7 and CCL19 may be mutants of the natural proteins described above. Examples of mutants of IL—7 include the following. (6a) A polypeptide that consists of an amino acid sequence having 70% or more sequence ty ogy) to an amino acid sequence of natural IL-7 (for example, SEQ ID NO: 59), and that has a T cell—immune—function-promoting function. (6b) A polypeptide that consists of an amino acid sequence in which one or several amino acids are mutated in the amino acid sequence of natural IL—7 (for example, SEQ ID NO: 59), and that has the T cell—immune—function-promoting function. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"

[0071] In addition, es of mutants of CCL19 include the following. (7a) A polypeptide that consists of an amino acid sequence having 70% or more sequence identity (homology) to an amino acid sequence of natural CCL19 (for example, SEQ ID NO: 61), and that has the T cell—immune—function-promoting function. (7b) A polypeptide that consists of an amino acid sequence in which one or l amino acids are mutated in the amino acid sequence of natural CCL19 (for example, SEQ ID NO: 61), and that has the T cell—immune—function—promoting function.

The term "T cell—immune—function—promoting function" means a function to maintain or e survival, growth, cytotoxic activity, migration activity, infiltration activity to tumor tissue, and the like of T cells.

In the above (6a) and (7a), the sequence ty is not particularly limited as long as it is 70% or more, but is preferably 80% or more, is more preferably 85% or more, is even more preferably 90% or more, and is ularly preferably 95% or more.

In addition, in the above (6b) and (7b), the term "several" may refer to, for example, 2 to 30, preferably refers to 2 to 20, more preferably refers to 2 to 10, and still more preferably refers to 2 to 5. In addition, the term "mutated" may refer to any of deletion, substitution, addition, and insertion, or a combination thereof.

In addition, mutants of IL-7 and CCL19 may be a mutant in which a signal peptide of these proteins is changed to another signal peptide, or may be a mutant in which a signal peptide is removed. Preferably, mutants of IL—7 and CCL19 have a signal peptide of secreted proteins and are secreted extracellularly.

In a case where the cell of the present embodiment is an isolated T cell, expression of at least one of IL—7 or CCL19 together with the anti—GM2 CAR promotes the immune function of the T cell, and thereby the T cell becomes excellent in cytotoxic activity against GM2—expressing cells, invasion to tumor tissue, and survival ability in a tumor microenvironment. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"

[0075] In addition, the cell of the present embodiment may express a e gene in addition to the anti—GM2 CAR. sion of the suicide gene in the cell of the present embodiment enables induction of apoptosis in the cell of the present embodiment as ary. The suicide gene is not particularly limited, and a known suicide gene can be used. es of suicide genes include a thymidine kinase (HSV—TK) gene of herpes simplex virus, an inducible caspase 9 gene, and the like. Cells sing HSV—TK can induce cell death by coexistence with ganciclovir. In addition, cells expressing inducible caspase 9 can induce cell death by coexistence with a chemical ion of dimerization (CID) such as AP1903. Amino acid sequences of the suicide genes are ble from known sequence ses such as GenBank. In on, sequences of commercially ble vectors including a suicide gene, and the like can also be used.

The cell of the present embodiment can also be said to be a cell (preferably a T cell) including the anti—GMZ CAR. Preferred examples of the cell of the t embodiment can be said to be a cell (preferably a T cell) including the anti—GM2 CAR, and at least one of lL-7 or CCL19. More preferred examples of the cell of the present embodiment can be said to be a cell (preferably a T cell) including the anti—GM2 CAR, IL—7, and CCL19. Even more preferred examples of the cell of the present embodiment can be said to be a cell (preferably a T cell) including the M2 CAR, IL—7, CCL19, and a suicide gene.

The cell of the present embodiment can be obtained by introducing a polynucleotide or a vector including a base sequence encoding the anti—GM2 CAR which will be described later into a cell. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"

[0078] [Polynucleotide ing base sequence encoding the anti—GM2 CAR] In one embodiment, the present invention es a polynucleotide including a base sequence that encodes the anti—GM2 CAR.

The polynucleotide of the present embodiment is not particularly limited as long as it es a base sequence encoding the anti—GM2 CAR. The anti—GM2 CAR is as described above in the n of [Chimeric antigen receptor (anti—GM2 CAR)]. The polynucleotide of the present embodiment preferably includes a base sequence encoding the amino acid sequence of the M2 CAR exemplified in the section of [Chimeric antigen receptor (anti—GM2 CAR)] described above. e of base sequences encoding a target antigen—binding region include a base sequence encoding scFv of an anti—GM2 antibody. More specifically, a polynucleotide including a base sequence encoding the amino acid ce set forth in SEQ ID NO: 2 and a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 4 can be exemplified. As the base sequence ng the amino acid ce set forth in SEQ ID NO: 2, a base sequence set forth in SEQ ID NOS: 1, 49, 51, or 53 can be exemplified. In addition, as the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 4, a base sequence set forth in SEQ ID NO: 3, 50, 52, or 54 can be exemplified.

These base sequences are preferably linked by a base sequence encoding a linker. The linker is as described in the section of "Chimeric antigen receptor" described above. For example, as a base sequence encoding the linker 15 described above, a base sequence set forth in SEQ ID NO: 5 or 55 can be exemplified. In addition, as a base sequence encoding the linker 25, a base sequence set forth in SEQ ID NO: 7 or 56 can be exemplified.

Specific examples of base sequences encoding scFv of an anti—GM2 antibody include a base sequence set forth in SEQ ID NO: 9, 11, 13, or 15, and the like.

A base sequence encoding the target n—binding region is preferably a codon—optimized base sequence ing to the species of cells to be introduced, and in a case of uction into human cells, a human—codon—optimized base ce is preferred.

In addition, base sequences encoding a transmembrane region and a T cell activation signal transduction region are available from known sequence databases such as GenBank. Furthermore, in a case where GM2 CAR includes other regions such as an ellular hinge region, base sequences encoding the other regions are also available from known ce databases such as GenBank.

For example, in a case where a transmembrane region of human CD8 is used as the transmembrane region, examples of base sequences encoding human CD8 e a base sequence registered as GenBank No: NM_001768.6, and the like. As base sequences encoding the transmembrane region, a base sequence set forth in SEQ ID NO: 19 can be exemplified.

Furthermore, for example, in a case where a T cell activation signal transduction region of human CD3C, human CD28, or human 4—1BB is used as the T cell tion signal uction region, examples of base sequences encoding human CD3C, human CD28, and human 4-1BB respectively include base sequences registered as GenBank Nos: NM_000734.3, NM_006139.2, and NM_001561.5, and the like. As respective base ces encoding the T cell activation signal transduction regions of human CD38; human CD28, and human 4—1BB, base ces set forth in SEQ ID NOs: 27, 23, and 25 can be exemplified.

Furthermore, for example, in a case where an extracellular hinge region of human CD8 is used as the extracellular hinge region, a base sequence set forth in SEQ ID NO: 17 can be exemplified as a base sequence encoding the extracellular hinge region. rmore, for example, in a case where a cytoplasmic region of human CD8 is used as the cytoplasmic region, a base sequence set forth in SEQ ID NO: 21 can be exemplified as a base sequence encoding the cytoplasmic region.

The base sequence encoding each of the above regions is not limited to known base sequences, and any sequence may be used as long as it is a base sequence encoding each of the above regions. Due to degeneracy of the c code, a plurality of codons corresponding to one amino acid are present. Accordingly, many base sequences encoding the same amino acid sequence are present. The base sequence encoding each of the above regions may be any of plural base sequences generated by degeneracy of the genetic code, as long as it is a base sequence encoding these regions.

A base sequence encoding each of the above—mentioned s is ably a codon—optimized base sequence according to the species of cells to be introduced, and in a case of introduction into human cells, a human—codon—optimized base sequence is preferred.

In addition, the base sequence encoding each of the above—mentioned regions may be a base sequence encoding a mutant of each region derived from a natural n.

Mutants of each region are as described above in the section of [Chimeric antigen receptor (anti—GM2 CAR)].

Respective base sequence encoding each region of the anti—GMZ CAR is preferably located in the order of the target antigen—binding region, the transmembrane region, and the T cell activation signal uction region from the 5’ side. In a case of using a signal peptide, an extracellular hinge region, or the like, it is preferable that the signal peptide be located at the 5’ side of the target antigen-binding , and the extracellular hinge region be located between the target antigen—binding region and the transmembrane region. The base sequences ng these regions may be directly linked, or may be linked via a base sequence encoding a spacer region. The spacer region is as described above in the section [Chimeric antigen receptor (anti—GM2 CAR)].

Specific examples of base sequences encoding the anti—GM2 CAR include a base sequence selected from the group ting of SEQ ID NOs: 39, 41, 43, and 45, and the like.

The polynucleotide of the present embodiment can be obtained by linking polynucleotides consisting of a base sequence encoding each region of the anti—GM2 CAR directly or Via a spacer sequence. The polynucleotides encoding each region of the anti—GM2 CAR may be obtained by al synthesis with a known method according to the base sequence of each region. In addition, by PCR, isothermal amplification, and the like using DNA extracted from T cells or the like, and cDNA obtained by reverse transcribing RNA extracted from T cells or the like as a template, polynucleotides ng each region may be ampli?ed and obtained. The polynucleotides encoding each region thus obtained may be subjected to modification such as substitution, deletion, addition, and insertion within a range not losing functions of each region after translation. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"

[0086] The polynucleotide of the present embodiment may include, in addition to the base sequence encoding the anti—GM2 CAR, regulatory sequences such as a promoter, an enhancer, a poly A addition signal, and a ator, base sequences encoding other proteins, and the like. id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"

[0087] Examples of other proteins include IL—7 and CCL19. Base ces encoding these proteins are available from known sequence databases such as GenBank. For example, in a case where human IL—7 is used, examples of base sequences encoding human IL—7 e a base sequence ered as GenBank No: 190.2 (SEQ ID NO: 58), and the like. In addition, in a case where human CCL19 is used, examples of base sequences encoding human CCL19 include a base sequence registered as GenBank No: NM_006274.2 (SEQ ID NO: 60), and the like.

Furthermore, the base sequences encoding these proteins are not limited to known base sequences, and any sequences may be used as long as they are base sequences encoding these proteins, and any of plural base sequences generated by degeneracy of the genetic code may be used. The base sequences encoding these ns are preferably a optimized base sequence according to the s of cells to be introduced, and in a case of introduction into human cells, a codon— optimized base sequence is preferred.

Furthermore, the base sequences encoding these proteins may encode mutants of natural IL—7 and natural CCL19. These mutants are as described above in the section [Cell expressing M2 CAR (anti-GM2 CAR-expressing cell)]. es of other proteins e a suicide gene. The suicide gene is as described above in the section [Cell expressing anti—GM2 CAR (anti—GM2 CAR— expressing cell)]. A base sequence encoding the suicide gene is available from known sequence databases such as k. In addition, sequences of commercially available vectors including a suicide gene can also be used.

In a case where the polynucleotide of the present embodiment includes a base sequence encoding another protein, a base sequence encoding a self—cleaving type peptide such as 2A peptide, an internal ribozyme entry site (IRES) sequence, and the like may be interposed between the base sequence encoding the anti—GM2 CAR and the base sequence encoding another protein. In addition, in a case where two or more other proteins are present, a leaving type peptide, an IRES, and the like may be interposed between the other proteins. By interposing these sequences, plural proteins can be expressed independently from one promoter.

Examples of 2A peptides include 2A peptides of picornavirus, rus, insect virus, aphthovirus, trypanosoma virus, and the like. As a specific e, an amino acid ce of 2A peptide (F2A) of picornavirus is shown in SEQ ID NO: 62. A base sequence encoding 2A peptide is preferably a codon—optimized base ce according to the species of cells to be introduced, and in a case of introduction into human cells, a human—codon—optimized base sequence is preferred. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"

[0091] In on, the polynucleotide of the present embodiment may be a polynucleotide having regulatory sequences such as a promoter, an enhancer, a poly A addition signal, and a terminator for each of protein—coding sequences of the base sequence ng the anti-GM2 CAR and the base sequence encoding other proteins.

Furthermore, the polynucleotide may be a polynucleotide in which some protein—coding sequences independently have regulatory sequences, and the other—protein—coding sequences linked via 2A peptide, IRES, or the like have common regulatory sequences. r including base sequence encoding anti—GM2 CAR] In one embodiment, the present invention es a vector including a base sequence that encodes the anti—GM2 CAR.

The polynucleotide of the ment may be in a form of a vector. The type of vector is not particularly limited, and a commonly used expression vectors and the like can be used. The vector may be linear or circular, and may be a non—viral vector such as a plasmid, may be a viral vector, or may be a transposon vector. es of vectors include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and the like.

Examples of viral vectors include Sendai virus vectors, retrovirus (including lentivirus) vectors, Adenovirus vectors, associated virus vectors, herpes virus vectors, vaccinia virus vectors, pox virus vectors, polio virus s, sindbis virus vectors, rhabdovirus vectors, xovirus vectors, orthomyxovirus vectors, and the like. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"

[0095] es of plasmid vectors include plasmid vectors for animal cell expression, such as pAl-ll, pXTl, pRc/CMV, pRc/RSV, and pcDNAl/Neo.

An episomal vector is a vector capable of extrachromosomal autonomous replication. Examples of episomal vectors include vectors ning sequences which are necessary for autonomous replication and are derived from EBV, SV40, and the like as vector elements. ic examples of vector elements necessary for autonomous replication include a replication start point, and a gene encoding a protein that binds to a vector at the ation start point to control replication. Examples f include oriP, Which is a replication start point, and EBNA—l gene in a case of EBV, and ori, which is a replication start point, and a SV40LT gene in a case of SV40.

Examples of artificial chromosome vectors include Yeast artificial chromosome (YAC) vectors, Bacterial artificial chromosome (BAC) vectors, Pl—derived artificial chromosome (PAC) vectors, and the like.

Preferred examples of the vector of the present embodiment e viral vectors, and more red examples thereof include retrovirus vectors. Examples of the retrovirus vectors include a pMSGVl vector (Tamada k et al., Clin Cancer Res 18: 6436—6445 (2012)) and a pMSCV vector (manufactured by Takara Bio Inc.). By using a retrovirus vector, a gene in the vector is incorporated into the genome of a host cell, and thereby the gene can be stably expressed in the host cell for a long time.

In addition to the base sequences described above in the section [Polynucleotide including base sequence encoding anti—GM2 CAR], the vector of the present embodiment may include a replication start point; a base ce ng a protein that binds to the vector at the replication start point to control replication; a base sequence encoding a marker gene such as a drug resistance gene and a reporter gene; and the like.

The base sequence encoding the M2 CAR is preferably located within the vector so as to be expressed under the control of an appropriate promoter. In addition, in a case Where the base sequences encoding other ns are included, these base sequences are preferably located within the vector so as to be expressed under the control of an appropriate promoter. es of promoters include an SRa er, an SV40 early stage promoter, an LTR of retrovirus, a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, a herpes simplex virus ine kinase (HSV—TK) promoter, an EFla promoter, a othionein promoter, a heat shock promoter, and the like. In addition, an enhancer of an IE gene of human CMV may be used together with the promoter. As an example, a CAG promoter (including a cytomegalovirus enhancer, a chicken B-actin promoter, and a poly A signal site of a B—globin gene), and the like can be ned. Furthermore, as described above in [Polynucleotide including base sequence encoding anti—GM2 CAR], transcriptions thereof may be performed under control of a common promoter by locating a base sequence encoding a self—cleaving type peptide or an IRES between each of protein—coding sequences.

In a preferred embodiment, in addition to the base sequence encoding the anti- GM2 CAR, the vector of the present embodiment further includes at least one of a base sequence encoding IL—7 and a base sequence encoding CCL19. In a more preferred embodiment, in addition to the base sequence encoding anti-GM2 CAR, the vector of the present embodiment further includes a base ce encoding IL—7 and a base sequence ng CCL19.

The vector of the present embodiment ably includes a base sequence encoding the anti—GM2 CAR functionally linked to an appropriate promoter. More preferably, the vector of the present embodiment includes a base sequence in which the base sequence encoding the anti—GM2 CAR, the base sequence encoding IL—7, and the base ce encoding CCL19 are linked via a base sequence ng a self—cleaving type peptide or an IRES. The base sequence is onally linked to an appropriate promoter. The phrase "functionally linked to a promoter" means that a base sequence is linked downstream of a promoter so as to be expressed under the l of the promoter.

In the above example, location order of the base sequence encoding the anti—GM2 CAR, the base sequence encoding IL—7, and the base ce encoding CCL19 is not particularly limited, and may be any location order.

[Method for producing cell expressing anti—GM2 CAR] In one embodiment, the present invention provides a method for producing a cell expressing the anti-GM2 CAR, the method including introducing a polynucleotide or vector including a base sequence encoding the anti—GM2 CAR into a cell.

The cell sing the anti—GM2 CAR of the above embodiment nafter also referred to as "anti—GM2 CAR—expressing cell") can be obtained by introducing a polynucleotide or a vector including the base sequence encoding the anti—GM2 CAR of the above ment into a cell. The polynucleotide or the vector introduced into a cell is retained in the cell in a state capable of expressing the anti—GM2 CAR. The phrase "state capable of expressing" means a state in which the base sequence encoding the anti—GM2 CAR can be transcribed and translated.

A method for introducing a polynucleotide or a vector into a cell is not particularly d, and known methods can be used. Examples thereof include a viral infection method, a lipofection method, a microinjection , a calcium phosphate method, a DEAE—dextran method, an electroporation method, a method using transposon, a particle gun method, and the like.

In addition, in a case where the vector is a retrovirus vector, appropriate ing cells may be selected based on LTR sequences and packaging signal sequences included in the vector, and irus particles may be prepared by using the same. Examples of packaging cells include PG13, PA317, GP+E—86, GP+envAm—12, Psi—Crip, and the like. In addition, 293 cells or 293T cells with high transfection efficiency can be used as packaging cells. Since various retrovirus vectors and packaging cells that can be used for packaging the vector are widely cially available, these commercially ble products may be used. For example, it is possible to use GP2-293 cells (manufactured by Takara Bio Inc.), Plat-GP cells (manufactured by Cosmo Bio Co., Ltd.), PG13 cells 0686 manufactured by ATCC), PA317 cells 078 ctured by ATCC), and the like, and a commercially available kit such as Retrovirus packaging Kit Eco (manufactured by Takara Bio Inc.) may be used.

In a case where other foreign proteins such as IL-7, CCL19, and a suicide gene are expressed in the anti-GM2 CAR-expressing cells, base sequences encoding these other proteins may be incorporated into a vector including a base sequence encoding the anti-GM2 CAR, or may be incorporated into another . In a case where base sequences encoding other proteins are included in the other vector, the vector can be introduced into a cell simultaneously or separately with the vector including the base sequence encoding the anti-GM2 CAR.

In addition, the anti-GM2 CAR-expressing cell may be produced by incorporating a polynucleotide including the base sequence encoding the anti-GM2 CAR in the genome of a cell so that the polynucleotide can be expressed under the control of an appropriate er, by using known gene-editing techniques and the like.

Examples of gene-editing techniques include techniques using endonucleases such as zinc finger nuclease, transcription activation-like effector nuclease (TALEN), red Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system, and pentatricopeptide repeat (PPR). In a case where other foreign proteins are expressed in the anti-GM2 CAR-expressing cell, similarly, a polynucleotide ing the base ce encoding the other foreign n may be incorporated into the genome of the cell so that the polynucleotide can be expressed under the control of an appropriate er, by using gene-editing techniques and the like. For example, a method of orating a polynucleotide including a base sequence encoding the anti—GM2 CAR (or other proteins) functionally linked to an appropriate promoter into a ding region and the like in a cell genome; a method of incorporating a polynucleotide including a base sequence encoding the M2 CAR (or other proteins) downstream of an endogenous promoter in a cell genome; and the like are i?ed. Examples of endogenous ers include promoters of TCROL and TCRB, and the like.

After introducing a polynucleotide or a vector including a base sequence encoding the anti—GM2 CAR into a cell, expression of the anti—GM2 CAR in the cell can be confirmed by a known method such as ?ow cytometry, RT—PCR, rn blotting, Western blotting, ELISA, and ?uorescent immunostaining. In addition, expression of other foreign proteins such as IL—7 and CCL19 can also be similarly confirmed by known methods. id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"

[0109] [Pharmaceutical composition including anti—GM2 CAR—expressing cell] In one embodiment, the present ion provides a pharmaceutical composition including the anti—GM2 CAR—expressing cell.

The anti—GM2 pressing cell show specific cytotoxic activity against GMZ—expressing cells. Accordingly, the anti—GMZ CAR—expressing cell can be used to treat or prevent a disease involving a GM2—expressing cell. Since GM2 is expressed in a wide range of tumor cells including lung , neuroblastoma, glioma, melanoma, malignant mesothelioma, myeloma, and the like, the pharmaceutical composition including the anti—GM2 CAR—expressing cell can be used as a pharmaceutical composition for treating or preventing tumors. The tumor may be a tumor generated from any of bone tissue, cartilage , fat tissue, muscle tissue, vascular tissue, and hematopoietic . Examples of tumors e cancer such as glioma, melanoma, malignant mesothelioma, lung cancer, pancreatic cancer, head and neck cancer, liver cancer, uterine cancer, bladder cancer, biliary cancer, esophageal , testicular tumor, thyroid cancer, brain cancer, prostate cancer, colon cancer, kidney cancer, ovarian cancer, breast cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, anaplastic cancer, large cell cancer, small cell cancer, skin cancer, vaginal cancer, neck cancer, spleen cancer, a cancer, bronchial cancer, small intestine cancer, stomach cancer, gallbladder cancer, and testicular cancer; sarcoma such as osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant hemangioendothelioma, malignant schwannoma, and soft tissue sarcoma; blastoma such as neuroblastoma, hepatoblastoma, medulloblastoma, nephroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumor; blood cancer such as lymphoma, leukemia, and myeloma; and the like, but examples are not limited thereto.

In ular, the pharmaceutical composition of the present embodiment is suitable as a pharmaceutical composition for treating or ting tumors expressing GM2. es of tumors expressing GM2 include lung cancer, neuroblastoma, glioma, melanoma, ant mesothelioma, a, and the like, but examples are not limited thereto. Whether or not a tumor expresses GM2 can be confirmed by, for example, a known method using an anti—GM2 antibody or the like. Examples of known methods include ?ow cytometry, ELISA, immunostaining, ?uorescent immunostaining, and the like. Cells (preferably T cells) that express any one or both of IL—7 and CCL19 in addition to the anti—GM2 CAR exert strong cytotoxic activity t even solid tumors as long as they are tumors expressing GM2. For this , the pharmaceutical composition of the present embodiment including cells (preferably T cells) sing the anti—GM2 CAR, and any one or both of IL—7 and CCLl9 can be particularly preferably used for solid tumors expressing GM2. Accordingly, the ceutical composition for treating or preventing solid tumors, which includes a cell (preferably T cell) expressing the anti—GM2 CAR, and any one or both of IL—7 and CCL19, is a preferred example of the pharmaceutical composition of the present ment. The term "solid tumor" means a tumor other than blood cancer arising from hematopoietic tissues, and includes epithelial cell cancers and non—epithelial cell cancers.

The pharmaceutical composition of the t embodiment may include other components such as a pharmaceutically acceptable carrier, in addition to the anti—GM2 CAR-expressing cell. es of other components include, in addition to a pharmaceutically acceptable carrier, a T cell activating factor such as cytokines, an immunostimulant, an immune checkpoint inhibitor, cells expressing other CAR, an anti— in?ammatory agent, and the like, but es are not limited thereto. Examples of pharmaceutically able carriers include a cell culture medium, a logical salt solution, a phosphate buffer solution, a citrate buffer solution, and the like.

The pharmaceutical ition of the t embodiment can be administered by a known method, but preferably can be administered to a patient by injection or infusion. An administration route is preferably intravenous administration, but is not limited thereto, and administration may be performed by injection into a tumor, or the like.

The pharmaceutical composition of the present embodiment may include a eutically effective amount of the anti—GMZ CAR-expressing cells. The term "therapeutically effective " means an amount of an agent ive for treating or preventing a disease. The therapeutically effective amount may vary depending on a disease state, age, sex, body weight, and the like of a subject for administration. In the pharmaceutical composition of the present embodiment, the above-mentioned therapeutically effective amount of the anti—GM2 CAR-expressing cells may be, for example, an amount that enables the anti—GM2 CAR—expressing cells to suppress growth of tumors.

A dose and an administration interval of the pharmaceutical composition of the present embodiment can be appropriately selected depending on age, sex, body weight, and the like of a t for stration; the type, degree of progression, symptoms, and the like of a disease; an administration method; and the like. As a dose, a therapeutically effective amount can be administered, and examples thereof include 1 x 104 to 1 x 1010 cells, preferably 1 x 105 to 1 x 109 cells, and more preferably 5 x 106 to 5 x 108 cells as the number of cells to be administered per administration.

An administration interval of the pharmaceutical composition of the present embodiment may be, for example, every week, every 10 to 30 days, every month, every 3 to 6 months, every year, or the like. In addition, since the anti—GM2 CAR—expressing cells can be autonomously proliferated in the body of a subject for administration, they may be administered only once. atively, the number of the anti—GM2 CAR— expressing cells in a body may be monitored after stration, and an administration period may be determined according to the result. id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116" id="p-116"

[0116] In addition, the pharmaceutical composition of the present embodiment can be used in combination with other ncer . Examples of other anticancer agents include alkylating drugs such as cyclophosphamide, antimetabolites such as pentostatin, molecularly targeted drugs such as mab, kinase inhibitors such as imatinib, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporin, anti-cancer antibiotics such as idarubicin, plant alkaloids such as irinotecan, platinum preparations such as cisplatin, hormone therapy drugs such as tamoxifen, immunoregulatory drugs such as nivolumab and pembrolizumab, and the like, but examples are not limited thereto. id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117" id="p-117"

[0117] In addition, in other aspects, the present invention es 1) use of the anti— GM2 CAR-expressing cell in production of a pharmaceutical composition for ng or preventing a tumor; 2) a method for treating or preventing a GM2—expressing tumor, the method including administering the anti-GM2 CAR-expressing cell to a subject (for example, a patient suffering from a tumor expressing GM2, a t who has undergone surgical removal of a tumor, and the like); 3) the M2 CAR—expressing cell for use in ent or prevention of a tumor; and 4) use of the anti—GMZ CAR-expressing cell for treating or preventing a tumor.

Furthermore, in another aspect, the present invention provides a kit for producing the anti—GM2 CAR—expressing cell, the kit including the vector of the above— bed embodiment. The kit is not particularly limited as long as it includes the vector of the above-described embodiment, and the kit may include ctions for producing the anti—GM2 CAR—expressing cell, a reagent used to introduce the vector into a cell, and the like.

[Examples] Hereinafter, the present ion will be described by examples, but the present invention is not limited by the following es.

[Example 1] Preparation of anti—GM2 CAR—expressing T cells expressing IL—7 and CCL 19 tion of T cell immune function—promoting factor) At least hundreds of molecules capable of controlling T cell function are present in a living body. The inventors of the present invention have selected IL-7 and CCL19 among a vast number of combinations as an immune on-promoting factor for enhancing an antitumor effect in CAR—T cells.

The above-mentioned IL-7 is a ne essential for survival of T cells, and is produced by non—hematopoietic cells such as stromal cells present in bone , thymus, lymph organs and tissues, and the like. Meanwhile, an ability to produce IL-7 is hardly recognized in T cells.

In addition, the above—mentioned CCL19 is mainly produced from dendritic cells and macrophages in lymph nodes, and has a function of causing migration of T cells and B cell, and mature dendritic cells via its receptor, CCR7. id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122" id="p-122"

[0122] (ScFv ces of anti—GM2 CAR) Sequences of anti—GM2 scFvs were designed based on sequences of known GM2 antibodies. In order to compare the order of VL and VH and types of linkers, each DNA fragment of VL—linker 15—VH (SEQ ID NO: 9: VL15VH), VL—linker 25—VH (SEQ ID NO: 11: VL25VH), VH—linker 15—VL (SEQ ID NO: 13: VH15VL), and VH—linker 25— VL (SEQ ID NO: 15: VH 25VL) was sized. In the following examples, VL15 VH was used as the anti—GM2 scFv sequence, unless otherwise specified.

(Preparation of M2 CAR—expressing vector expressing IL—7 and CCL19) First, chemical synthesis was performed on a DNA fragment of ILF2A— CCL19 (SEQ ID NO: 33: IL—7—F2A—CCL19) encoding human IL—7 (no stop codon), and subsequent F2A and human CCL19. Next, using the existing mouse anti—CD20 CAR— IL—7/CCL19 vector obtained by inserting a uct consisting of a mouse anti-CD20 scFv, a mouse CD8 transmembrane , a mouse CD28—4-1BB—CD3Q intracellular signal motif, and a mouse IL—7—F2A—mouse CCL19 into a pMSGVl retrovirus sion vector (Tamada k et al., Clin Cancer Res 18: 6436—6445 (2012)), a region of the mouse ILF2A-CCL19 in the vector was replaced with the synthesized human ILF2A- CCL19 DNA fragment (SEQ ID NO: 33) by restriction enzyme (NsiI and SalI) treatment and on. Furthermore, chemical synthesis was performed on a human anti-CD20 CAR DNA fragment (SEQ ID NO: 37: anti—CD20 CAR) consisting of a human anti— CD20 scFv, a human CD8 transmembrane region, and a human CD281BB-CD3Q intracellular signal motif, and a mouse D20 CAR region in the vector was replaced with this DNA fragment by restriction enzyme (NcoI and E001) treatment and ligation.

Finally, the DNA fragments (SEQ ID NOs: 9, 11, 13, and 15) encoding human anti—GM2 scFv were chemically synthesized, and the anti—CD20 scFv in the vector was replaced with this DNA fragment by restriction enzyme (NcoI and NotI) treatment and ligation.

The uct of the anti—GM2 CAR DNA fragment is shown in Fig. 1A, and a location drawing of the obtained vector is shown in Fig. 1B.

(Preparation of anti—GM2 CAR—expressing vector expressing IL—7/CCL19 and HSV—tk) In CAR-T cell therapy, a strong immune response to a target antigen may cause systemic side effects such as cytokine release syndrome. In order to cope with such a problem, a CAR uct was produced in which a herpes virus-derived thymidine kinase gene, HSV-tk, was introduced as a suicide gene. When this construct was transfected and the HSV-tk was expressed in CAR-T cells, addition of ganciclovir, which is a cytomegalovirus therapeutic drug, induces apoptosis of the CAR-T cells to kill them, and ganciclovir administration allows control of CAR-T cells in the body.

First, 2A-CCL19-HSV-tk DNA fragment (SEQ ID NO: 35: ILF2ACCL19-HSV-tk ) was chemically synthesized. Next, the region of ILF2A-CCL19 in the anti-GM2 pressing vector (SEQ ID NO: 39) expressing IL-7 and CCL19 was replaced with the synthesized ILF2A-CCL19-HSV-tk DNA nt (SEQ ID NO: 35) by restriction enzyme (NsiI and SalI) treatment and ligation, and thereby an anti-GM2 CAR expression vector (SEQ ID NO: 47) sing CL19 and HSV-tk was produced.

(Production of retrovirus into which IL-7/CCL19 expressing-anti-GM2 CAR vector had been introduced) A retrovirus was produced for gene transfection into T cells. Using Lipofectamine 3000 (manufactured by Life Technology Inc.), the above-mentioned IL- 7/CCL19 expression-anti-GM2 CAR vector and p-Ampho plasmid (manufactured by Takara Bio Inc.) was transfected into a GP2-293 packaging cell line (manufactured by Takara Bio Inc.), and thereby a retrovirus into which the IL-7/CCL19 expressing-anti-GM2 CAR vector had been introduced was produced. The atant containing the retrovirus was recovered 48 hours after the ection.

As a culture solution of the GP2-293 cells, DMEM to which 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin were added was used. In addition, as a culture solution of T cells used in Examples to be described later, GT-T 551 containing 2.0% human AB type serum (manufactured by , 1% Penicillin-Streptomycin (manufactured by Wako Pure Chemical Industries, Ltd.), and 2.5 µg/ml amphotericin B (manufactured by Bristol-Myers ) was used.

(Genetic transduction of T cells) Peripheral blood mononuclear cells were collected from the blood of healthy donors, and were cultured with 2 × 106 IL-2 (200 IU/ml: manufactured by ech) in a 5% CO2 incubator at 37°C for 3 days on a plate on which an anti-CD3 onal antibody (5 µg/ml) and RetroNectin (registered trademark: manufactured by Takara Bio Inc., 25 µg/ml) were layered to activate T cells. On the second day after the start of culture, 500 µl/well of the supernatant containing the retrovirus into which the IL-7/CCL19 expressing- anti-GM2 CAR vector ed above was introduced was added to a surface-untreated 24-well plate that was coated in advance with 25 µg/ml of ectin (manufactured by Takara Bio Inc.), and thereby a retrovirus preload plate was produced by centrifugation at 2000 g for 2 hours. A total of two plates was produced, and after the completion of centrifugation, the plates were washed with 1.5% BSA/PBS and stored at 4°C until being used. On the third day of culture, activated cells were recovered from the plate and prepared as cell suspension (1 × 105 ml). A first retrovirus infection was performed by adding 1 ml per well of this cell suspension to the retrovirus preload plate, and culturing in a 5% CO2 incubator at 37°C for 24 hours in the presence of IL-2 (a final concentration of 200 IU/ml). On the next day (culture day 4), a second retrovirus infection was performed by transferring the cell solution of each well to a stored second virus preload plate, centrifuging at 500 g for 1 minute, and culturing at 37°C for 4 hours. After 4 hours of culture at 37°C, 1 ml of the cell suspension of each well was transferred to a new 12-well cell culture plate, d 4-fold with a fresh culture solution (GT-T551) containing IL-2 (200 IU/ml), and cultured at 37°C in a 5% CO2 incubator. The culture was performed up to day 7 from the start day of culturing the peripheral blood mononuclear cells, and thereby T cells (anti-GM2 -7/CCL19-expressing T cells) into which the IL-7/CCL19 expressing-anti-GM2 CAR vector had been introduced were obtained (Fig. 1B). In addition, at the same time, as a CAR-negative cell control, non-transgenic cells, which activated peripheral blood mononuclear cells obtained from the same healthy human donor in the same manner but which were not infected with retrovirus, were produced. id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128" id="p-128"

[0128] [Example 2] CAR expression measurement by flow cytometry (flow cytometric analysis) Analysis of an expression level of CAR that recognizes GM2 as an antigen was performed by two-color flow tric analysis. The produced M2 CAR-IL- 9-expressing T cells were reacted with biotinylated protein L (manufactured by GenScript), allophycocyanin (APC)-labeled streptavidin (manufactured by Affymetrix), and APC-labeled anti-CD8 monoclonal antibody actured by Affymetrix), and ng was performed. EC800 (manufactured by Sony) was used for Flow cytometry and FlowJo software (manufactured by Tree Star) was used for data analysis.

The results are shown in Fig. 2. The left graph shows results of cells into which no CAR gene was transfected and the right graph shows results of the anti-GM2 CAR-IL- 7/CCL19-expressing T cells. The numerical values in the graphs represent percentages of the tive populations. As shown in Fig. 2, about 68% of CAR expression was med in the anti-GM2 CAR-IL-7/CCL19-expressing T cells. id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"

[0130] [Example 3] Production of IL-7 and CCL19 (Measurement of IL-7 and CCL19 concentrations in culture supernatant of anti-GM2 CARIL-7 /CCL19-expressing T cells) A e supernatant of the anti-GM2 CAR-IL-7/CCL19-expressing T cells or non-transgenic cells on day 7 of the culture described above was recovered, and production of IL-7 and CCL19 by the anti-GM2 -7/CCL19-expressing T cells was examined using a cially available ELISA kit (manufactured by Peprotech, and R & D systems, respectively).

[Results] The results are shown in Fig. 3. As shown in Fig. 3, in the culture supernatant of the anti-GM2 CAR-IL-7/CCL19-expressing T cells (GM2 CAR), 300 pg/ml or more of IL- 7 and 2000 pg/ml or more of CCL 19 were detected. Based on these results, it was confirmed that the anti-GM2 CAR-IL-7/CCL19-expressing T cells express IL-7 and CCL19, and the expressed IL-7 and CCL19 are extracellularly secreted. On the other hand, in the culture supernatant (non infection) of the control non-transgenic T cells, amounts of both IL-7 and CCL19 were below a detection limit (Not ed).

[Example 4] GM2 sion in each tumor cell (Flow cytometric analysis) Malignant mesothelioma cell lines Y-meso8A and MSTO211H, myeloma cell line KMS- 11, KMS-28PE and colon cancer cell line SW480 were stained with an anti-GM2 antibody and a control anti-DNP dy which were labeled with Alexa 488, and expression of GM2 in each tumor cell was measured by ?ow cytometric analysis. For both Alexa 488—labeled anti—GM2 antibody and Alexa 488—labeled anti—DNP antibody, the staining was performed at 10 ug/sample.

Expression of GM2 was not observed in the colon cancer cell line SW480, but expression of GM2 was confirmed in the malignant mesothelioma cell lines Y—meso8A and MSTOleH, and myeloma cell lines KMS—ll and KMS—28 PE.

[Example 5] Cytotoxicity assays (51Cr release assay 1 by anti—GM2 CAR—lL—7/CCLl9—expressing T cells) Fig. 4 shows test schedules of production of anti—GM2 CAR-IL-7/CCL19— sing T cells, a tumor cytotoxicity assay, and a co-culture assay. As shown in Fig. 4, the CAR—IL—7/CCL19-expressing T cells were recovered on day 8, and cytotoxic ty of the CAR-IL—7/CCL19 expressing—T cells against tumor cells was evaluated by using a standard 4 hour 51Cr release assay.

Various tumor cells expressing human GM2 were used as target cells. The tumor cell line was cultured at 37°C for 1 hour in the presence of 100 uCi Naz 51CrO4, and then washed 3 times, and 5 x 103 cells per well were added to a 96 well V—bottom plate (manufactured by Nunc). fter, as or T cells, four types of anti—GM2 CAR—IL—7/CCL19—expressing T cells with different combinations of VH and VL, and a linker in scFv of anti—GM2 CAR, or ansgenic T cells were added, and co—culture was performed with the target cells at 37°C for 4 hours. An effector/target ratio (E/T ratio) was adjusted within a range of 2.5, 5, 10, 20, and 40. Maximum release and spontaneous release of the target cells were ed by culturing the target cells in a 10% Triton—X (manufactured by Sigma—Aldrich)—containing culture solution, or in only a culture solution. 51Cr release of the supernatant was measured with a TopCount scintillation counter actured by PerkinElmer). A percentage of cytotoxic activity was calculated by the equation: cytotoxic activity (%) = [(assay e — spontaneous release)/(maximum release — spontaneous e)] x 100.

[Results] The s are shown in Figs. 5A and 5B. Fig. 5A shows the s in which malignant mesothelioma cell lines (Y—mes08A and MST0211H) were used as target cells, and Fig. 5B shows the results in which myeloma cell lines (KMS—ll and KMS— 28PE) were used as target cells. In the graphs, each of "VL15VH," H," "VH15VL," and "VH25VL" represents the anti—GM2 CAR—lL—7/CCL19—expressing T cells including the corresponding sequences as scFv sequences of anti-GM2 CAR. As shown in Figs. 5A and B, the anti—GM2 CAR—IL—7/CCL19—expressing T cells exhibited cytotoxicity against the tumor cell lines by any combination of VH, VL, and a linker.

On the other hand, the control non—transgenic T cells (non infection) showed almost no cytotoxic activity against the tumor cell lines. In Figs. 5A and SE, a lateral axis of the graphs represents a ratio of effector (T cell) to target (tumor cell) in an E/T ratio, and a vertical axis represents cytotoxic ty (%). (51Cr release assay 2 by anti—GM2 CAR—lL—7/CCL19—expressing T cells) To examine GM2 specificity of cytotoxic activity by the anti—GM2 CAR—IL— 7/CCL19-expressing T cells, using the anti-GM2 CAR—IL—7/CCL19-expressing T cells, anti—FITC CAR—T cells that recognize FITC as a control for CAR—T cells, and non— transgenic T cells, cytotoxic activity of each cell against a GM2—positive tumor cell line and a GMZ—negative tumor cell line was ed and examined. For the anti—GM2 CAR-IL-7/CCL19-expressing T cells, cells containing VL15VH as scFv sequence were used.

[Results] The results are shown in Figs. 6A to 6C. Fig. 6A shows the s in which the malignant mesothelioma cell line (Y-MESO8A) was used as a target cell, Fig. 6B shows the results in which the myeloma cell line ) was used as a target cell, and Fig. 6C shows the results in which the colon cancer cell line (SW480) was used as a target cell.

As shown in Figs. 6A to 6C, no significant cytotoxic activity of anti-FITC CAR-T cells (FITC CAR-T) was recognized against any target cells, which was almost the same level of that of the non-transgenic T cells (non infection). On the other hand, anti-GM2 CAR-IL-7/CCL19-expressing T cells (GM2 CAR-T) exhibited cytotoxicity against GM2-expressing cells (Y-MESO8A and KMS11), but did not exhibit cytotoxicity against cells not expressing GM2 ). Based on the above description, it was confirmed that anti-GM2 CAR-IL-7/CCL19-expressing T cells induce cytotoxic ty specifically against GM2. In Figs. 6A to 6C, a lateral axis of the graphs represents a ratio of or (T cell) to target (tumor cell) in an E/T ratio, and a vertical axis represents cytotoxic activity (%).

(Co-culture assay) As shown in Fig. 4, anti-GM2 CAR-IL-7/CCL19-expressing T cells were recovered on day 7, and, on a 24-well cell culture plate, were co-cultured with a GM2- positive tumor cell line or a GM2-negative tumor cell line in a 37°C incubator after adjusting an effector:tumor cell ratio to 1:1 to 1:3. Cytotoxic activity was observed microscopically 2 or 3 days after the start of co-culture, and IFN-? produced in the culture supernatant was measured using a commercially available IFN-y ELISA kit (manufactured by BioLegend). As controls for anti-GM2 CAR—IL-7/CCL19—expressing T cells, anti—FITC CAR-expressing T cells and non—transgenic T cells were used.

[Results] The results are shown in Figs. 7 to 9. Figs. 7 and 8 show the results in which the malignant elioma cell line (Y—mesoSA, MST0221H) was used as a target cell, and Fig. 9 shows the s in which the colon cancer cell line (SW480) was used as a target cell. As shown in Figs. 7 to 9, in ture of the l anti-FITC CAR— expressing T cells (FITC CAR—T) or non—transgenic T cells (non—infection) with target tumor cells, all target tumor cells were observed to grow by the same level as in tumor— only wells.

On the other hand, in the anti—GM2 CAR-IL—7/CCL19-expressing T cells (GM2 CAR—T), differences in tumor growth were observed depending on the type of target tumor cell. In co—culture with GM2—negative target cells (SW 480: Fig. 9), target tumor cells grew by the same level as in tumor—only wells. On the other hand, when co— cultured with GMZ—positive tumor cells (Y—mesoSA: Fig. 7 and 1H: Fig. 8), the number of tumor cells clearly decreased as compared to the tumor—only wells and the wells of co—culture with control cells.

Based on these results, it was confirmed that anti—GM2 CAR—IL—7/CCL19— expressing T cells damages tumor cells in an antigen—specific manner, as in the 51Cr release assay. In addition, as shown in Fig. 10, in IFN—y ELISA using a supernatant after co—culture, tion of IFN—y was confirmed only in the co—culture supernatant of the anti-GM2 CAR-IL—7/CCL19-expressing T cells and the GM2-positive target cells (MST0221H and Y—mesoSA).

[Example 6] Therapeutic effect in tumor model (Administration of anti—GM2 CAR-lL-7/CCL19—expressing T cells to X-ray irradiated mice) lD/ILngKO (NSG) mice, which are immunodeficient mice, were irradiated with 2 Gy X-ray, and then inoculated with 1 x 104 luciferase—expressing MST0211H intraperitoneally or intrathoracically. After one day, 2.5 x 107 anti—GM2 CAR—IL—7/CCL19—expressing T cells (1 X 107 cells for cells in which CAR expression was confirmed) or the same number of non—transgenic T cells as a control were stered intravenously to this intraperitoneally tumor model group (n = 2) or intrathoracic tumor model group (n = 3). The day of cell administration was considered day 0, and a tumor volume (2 luminescence intensity due to luciferase ty) was evaluated over time using IVIS imaging system (manufactured by Perkin Elmer).

[Results] Results of changes in tumor volume of mice are shown in Figs. 11A and 11B.

Fig. 11A shows a result in an intrathoracic tumor model, and Fig. 11B shows a result in an intraperitoneal tumor model. As shown in Fig. 11A, in the intrathoracic tumor model, tumor growth was med in the group (non ion) to which non—transgenic T cells were administered, but no apparent tumor growth was confirmed in the group (GM2 CAR—T) to which anti—GM2 CAR—lL—7/CCL19—expressing T cells were administered. In addition, as shown in Fig. 11B, in the intraperitoneal tumor inoculation model, tumor growth was observed up to day 3 in the group (GM2 CAR—T) to which the anti-GM2 CAR-IL—7/CCL19-expressing T cells were administered, but the tumor gradually shrank from the subsequent day. Based on these results, it became clear that the anti-GM2 CAR—IL—7/CCL19—expressing T cells exhibited ent antitumor activity in both the intrathoracic tumor model and the intraperitoneal tumor model.

(Administration of anti—GM2 —7/CCL19—expressing T cells to X—ray non— irradiated mice) Next, an antitumor effect on a model in which an NSG mouse was intrathoracically inoculated with a tumor t atment of X—ray irradiation was examined. The thoracic cavity was inoculated with 1 x 104 luciferase-expressing 1H, and on the next day, 1.6 x 107 anti-GM2 CAR-IL-7/CCL19-expressing T cells (1 x 107 cells for a case of CAR—expressing cells) or the same number of non— transgenic T cells as a control were administered intravenously (the group to which the anti—GM2 CAR—IL—7/CCL19—expressing T cells were administered: 11 = 6, and the group to which the non—transgenic T cells were administered: n = 5). The day of cell administration was considered day 0, and tumor growth was evaluated over time using the IVIS imaging system as described above.

[Results] Results of changes in tumor volume of mice are shown in Fig. 12. As shown in Fig. 12, in the group (non infection) to which the non—transgenic T cells were administered, tumors gradually grew. On the other hand, in the group (GM2 CAR—T) to which the anti—GM2 CAR—lL—7/CCL19—expressing T cells were administered, tumors showed a tendency to grow until day 9, but from the subsequent day, the tumors did not grow except for one mouse and disappeared. It was confirmed that the anti-GM2 CAR- IL—7/CCL19—expressing T cells induce an excellent antitumor effect, as in the above— mentioned model subjected to X—ray irradiation pretreatment.

[Example 7] Therapeutic effect in tumor model (Production of anti—GM2 CAR—expressing T cells) An anti—GM2 CAR—expressing vector was produced with the same configuration as the lL—7/CCL19 expressing—anti—GM2 CAR vector except that no 2A—CCL19 DNA fragment was contained. This anti—GM2 CAR-expressing vector was transduced into T cells in the same manner as in Example 1, and thereby anti—GM2 CAR—expressing T cells were obtained.

(Administration of anti—GM2 CAR—lL—7/CCL19—expressing T cells or anti—GM2 CAR- expressing T cells to mice) NOD/SClD/ILngKO (NSG) mice, which are deficient mice, were inoculated with 1 x 104 luciferase—expressing MSTO211H intrathoracically. After one day, 2.2 x 106 anti—GM2 CAR—IL—7/CCL19—expressing T cells (1 x 106 cells for cells in which CAR expression was confirmed), the same number of anti—GM2 CAR—expressing T cells (1 x 106 cells for cells in which CAR expression was confirmed), or the same number of non—transgenic T cells as a l were stered intravenously to this intrathoracic tumor model group. The day of cell administration was considered day 1, and a tumor volume (2 luminescence intensity due to luciferase ty) was evaluated over time using IVIS imaging system (manufactured by Perkin Elmer).

[Results] Results of changes in tumor volume of mice are shown in Fig. 13. In Fig. 13, "x" indicates that a mouse died. As shown in Fig. 13, in the intrathoracic tumor model, tumor growth was confirmed over time in the group (non infection) to which non— transgenic T cells were administered, and no mice survived at day 57. Even in the group (GM2 CAR—T (-) IL—7/CCL19) to which the anti—GM2 CAR—expressing T cells were administered, tumors grew over time, but suppression in tumor growth was observed as compared to the group to which the non—transgenic T cells were stered. On the other hand, in the group (GMZ CAR—T (+) IL—7/CCL19) to which the anti—GM2 CAR—IL—7/CCL19—expressing T cells were administered, tumor growth was suppressed as compared to the other groups, and mice survived even after day 70.

Fig. 14 is a graph showing changes in luminescence ity in Fig. 13. In the graph of Fig. 14, a l axis indicates the number of days elapsed since intrathoracical inoculation of tumor cells into the mouse, and a vertical axis indicates the luminescence intensity (x 106 photons/sec) from the tumor cells. In the group (non—infection) to which the non—transgenic T cells were administered and the group (GM2 CAR—T (—) IL— 7/CCL19) to which the anti—GM2 CAR—expressing T cells were administered, no mice survived at day 57, and there were no subsequent plots. In Fig. 14, it can be confirmed that tumor growth is slightly suppressed in the group (GM2 CAR—T (—) IL—7/CCL19) to which the anti—GM2 CAR—expressing T cells were administered as compared to the group (non—infection) to which the ansgenic T cells were administered. On the other hand, in the group (GM2 CAR—T (+) IL—7/CCL19) to which the anti—GM2 CAR—IL— 7/CCL19—expressing T cells were administered, tumor growth was suppressed within a ntially constant range. id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"

[0150] In addition, Fig. 15 is a graph which shows transition in a survival ratio of mice.

In the graph of Fig. 15, a lateral axis indicates the number of days elapsed since intrathoracical inoculation of tumor cells into the mouse, and a vertical axis tes a survival ratio (%) of mice. As shown in Fig. 15, it was confirmed that, in group (GM2 CAR—T (—) IL—7/CCL19) to which the anti—GM2 CAR—expressing T cells were stered, a survival period tended to slightly extend as compared to the group (non infection) to which the non—transgenic T cells were stered. On the other hand, it was confirmed that, in the group (GM2 CAR—T (+) IL—7/CCL19) to which the anti—GM2 CAR—IL—7/CCL19-expressing T cells were administered, the survival ratio was improved (an effect of extending a survival ) as compared to the group to which the non— transgenic T cells were administered and the group to which the anti-GM2 CAR— expressing T cells were administered.

Based on these results, it became clear that the anti—GM2 CAR—IL—7/CCL19— expressing T cells had excellent mor activity.

[Industrial Applicability] ing to the present ion, a novel CAR that targets a solid tumor antigen as a target antigen, and a CAR—T cell that is effective against solid tumors are provided. The CAR—T cells of the present invention can be applied to treatment or prevention of solid tumors expressing GM2, such as lung cancer, neuroblastoma, glioma, melanoma, malignant elioma, and myeloma.

The present application is based on Japanese Patent Application No. 2017—61461 filed on March 27, 2017, the content of which is incorporated in the present specification by reference in its entirety. [

Claims (16)

CLAIMS ]

1. [Claim 1] An isolated cell which expresses a chimeric antigen receptor, IL-7 and CCL19, the ic antigen receptor comprising: 5 a target n-binding region; a embrane region; and a T cell activation signal transduction region, wherein the target antigen-binding region comprises a heavy-chain le region and a light-chain variable region of an anti-ganglioside GM2 antibody, wherein the anti-ganglioside GM2 antibody is an antibody selected from the 10 group consisting of the following (a) to (c): (a) an antibody that comprises a heavy-chain variable region comprising CDR1, CDR2, and CDR3 of a heavy-chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain variable region sing CDR1, CDR2, and CDR3 of a light-chain variable region consisting of the amino acid sequence 15 set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein CDR1s, CDR2s, and CDR3s of the heavy-chain variable region and the light-chain variable region are determined by the definition of Kabat, Chothia, AbM or contact; (b) an antibody that comprises a chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid 20 sequence set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of a mutation in the chain variable region and the light-chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the 25 definition of Kabat, Chothia, AbM or contact; and (c) an antibody that comprises a heavy-chain le region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and a light-chain le region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in 5 SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of a mutation in the heavy-chain le region and the light-chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, Chothia, AbM or contact.

2. [Claim 2] 10 The isolated cell according to Claim 1, wherein CDR1, CDR2 and CDR3 of the chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 63, the amino acid sequence set forth in SEQ ID NO: 64 and the amino acid sequence set forth in SEQ ID NO: 65, respectively; and 15 CDR1, CDR2 and CDR3 of the light-chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 66, the amino acid sequence set forth in SEQ ID NO: 67 and the amino acid sequence set forth in SEQ ID NO: 68, respectively.

3. [Claim 3] The isolated cell according to Claim 1, wherein the chain variable region 20 comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light-chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4.

4. [Claim 4] The isolated cell according to any one of Claims 1 to 3, wherein the antiganglioside GM2 antibody is a single-stranded dy (scFv).

5. [Claim 5] The isolated cell according to Claim 4, wherein the scFv is a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide that comprises the amino acid sequence selected from the 5 group ting of SEQ ID NOs: 10, 12, 14, and 16; (b) a polypeptide that ts of the amino acid sequence having 90% or more sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a binding ability to ganglioside GM2, wherein a location of mutation in the amino acid sequence selected from the group consisting of 10 SEQ ID NOs: 10, 12, 14, and 16 is a region other than CDR1s , CDR2s and CDR3s of the heavy-chain variable region and the light-chain variable region ined by the definition of Kabat, Chothia, AbM or t; and (c) a ptide that consists of the amino acid sequence in which one or 2 to 20 amino acids are mutated in the amino acid sequence selected from the group 15 consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a g ability to ganglioside GM2, wherein a location of mutation in the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16 is a region other than CDR1s , CDR2s and CDR3s of the heavy-chain variable region and the light-chain variable region determined by the definition of Kabat, Chothia, AbM or contact. 20 [

6. Claim 6] The isolated cell according to any one of Claims 1 to 4, wherein the cell is an immune cell.

7. [Claim 7] A polynucleotide comprising a base sequence that encodes the chimeric antigen 25 or, a base sequence that encodes IL7, and a base sequence that encodes CCL19, the chimeric n receptor comprising: a target n-binding region; a transmembrane region; and a T cell activation signal transduction region, wherein the target antigen-binding region comprises a heavy-chain viable region 5 and a light-chain variable region of an anti-ganglioside GM2 antibody, wherein the anti-ganglioside GM2 antibody is an antibody selected from the group consisting of the following (a) to (c): (a) an antibody that comprises a heavy-chain variable region comprising CDR1, CDR2, and CDR3 of a heavy-chain variable region ting of the amino acid 10 sequence set forth in SEQ ID NO: 2, and a chain variable region comprising CDR1, DR2, and CDR3 of a light-chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein CDR1s, CDR2s and CDR3s of the heavy-chain variable region and the light-chain le region are determined by the definition of Kabat, a, AbM or contact; 15 (b) an antibody that comprises a heavy-chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid ce set forth in SEQ ID NO: 2, and a chain variable region consisting of an amino acid sequence in which one or 2 to 10 amino acids are mutated in the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, 20 wherein a location of mutation in the heavy-chain variable region and the light-chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, Chothia, AbM or contact; and (c) an antibody that comprises a heavy-chain variable region consisting of an amino acid sequence having 90% or more ce identity to the amino acid sequence 25 set forth in SEQ ID NO: 2, and a light-chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and that has a binding ability to ganglioside GM2, wherein a location of mutation in the heavy-chain variable region and the chain variable region is a region other than CDR1s , CDR2s and CDR3s determined by the definition of Kabat, Chothia, 5 AbM or contact.

8. [Claim 8] The cleotide according to Claim 7, wherein CDR1, CDR2 and CDR3 of the heavy-chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 63, the amino acid sequence set forth 10 in SEQ ID NO: 64 and the amino acid sequence set forth in SEQ ID NO: 65, respectively; and CDR1, CDR2 and CDR3 of the light-chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 66, the amino acid sequence set forth in SEQ ID NO: 67 and the amino acid sequence set forth in SEQ ID NO: 68, respectively. 15 [

9. Claim 9] The polynucleotide according to Claim 8, wherein the heavy-chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light-chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4.

10. [Claim 10] 20 The polynucleotide ing to any one of Claims 7 to 9, wherein the antiganglioside GM2 antibody is a single-stranded antibody (scFv).

11. [Claim 11] The polynucleotide ing to Claim 10, wherein the scFv is a polypeptide selected from the group consisting of the ing (a) to (c): 25 (a) a polypeptide that comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16; (b) a polypeptide that comprises the amino acid sequence having 90% or more sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a binding y to ganglioside GM2, wherein a 5 location of mutation in the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16 is a region other than CDR1s , CDR2s and CDR3s of the heavy-chain variable region and the light-chain variable region determined by the definition of Kabat, a, AbM or contact; and (c) a ptide that comprises the amino acid sequence in which one or 2 to 20 10 amino acids are mutated in the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16, and that has a g ability to ganglioside GM2, wherein a location of mutation in the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16 is a region other than CDR1s , CDR2s and CDR3s of the heavy-chain variable region and the light-chain variable region determined 15 by the definition of Kabat, Chothia, AbM or contact.

12. [Claim 12] A vector sing the polynucleotide according to any one of Claims 7 to 11.

13. [Claim 13] A method for producing an isolated cell expressing a chimeric antigen receptor, 20 comprising introducing the polynucleotide according to any one of Claims 7 to 11 or the vector ing to Claim 12 into a cell.

14. [Claim 14] A pharmaceutical composition comprising the isolated cell according to any one of Claims 1 to 6.

15. [Claim 15] Use of the pharmaceutical composition according to Claim 14 in the manufacture of a medicament for ng or preventing a tumor.

16. [Claim 16] 5 The isolated cell according to Claim 1, ntially as herein described with reference to any one of the examples and/or