T cell receptor of tax antigen-specific cytotoxic t lymphocyte coded by HTLV-1 or functional fragment thereof

Abstract

To clone a T cell receptor of a Tax antigen-specific cytotoxic T lymphocyte, thereby providing a T cell receptor or a functional fragment thereof having a superior antitumor effect.SOLUTION: The present invention provides a T cell receptor of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1, or a functional fragment thereof, having: an α-chain CDR3 region consisting of an amino acid sequence represented by Sequence ID No. 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added in the amino acid sequence; and a β-chain CDR3 region consisting of an amino acid sequence represented by Sequence ID No. 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added in the amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a T cell receptor of Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1, or a functional fragment thereof, and use thereof.

Background Art

[0002] Adult T-cell leukemia / lymphoma (ATL) is caused by infection with the virus HTLV-1 (human T-lymphotropic virus type-I), infects CD4+ T cells, and develops by the uncontrolled proliferation of cancerous cells (ATL cells) derived from the infected T cells (Non-Patent Document 1). HTLV-1 has a gene for the viral protein Tax that has carcinogenic activity, and CTL therapy targeting this Tax antigen is expected to be an effective treatment method.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to clone a T cell receptor of Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1 and provide a T cell receptor or a functional fragment thereof having an excellent antitumor effect.

Means for Solving the Problems

[0005] Therefore, the present inventors induced HLA-A2402-restricted Tax antigen-specific cytotoxic T lymphocytes, which are common in Japanese people, from the peripheral blood of patients, and then performed single cell cloning, successfully establishing clones of Tax antigen-specific cytotoxic T lymphocytes (CTLs). Subsequently, the antitumor effect of the obtained clones against autologous tumor cells (ATL cells) was confirmed. However, CTL clones have limited proliferative capacity, and it was considered difficult to secure cells for use in the treatment of actual patients. Therefore, once the CTL clones were converted into iPS cells, and Tax antigen-specific CTLs were induced to redifferentiate from the iPS cells, successful production of CTLs with a more potent antitumor effect than the original CTL clones was achieved. By performing repertoire analysis on the redifferentiated CTLs, the amino acid sequences of the T cell receptors (TCRs) were clarified, completing the present invention.

[0006] That is, the present invention provides the following inventions [1] to

[20] . [1] An α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence, and a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence, and a T cell receptor of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1 or a functional fragment thereof. [2] The T cell receptor or a functional fragment thereof according to [1], having an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8. [3] Further, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence, and a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence, and the T cell receptor or a functional fragment thereof according to [1] or [2]. [4] Furthermore, the T cell receptor or a functional fragment thereof according to [1] or [2], which has an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6. [5] Furthermore, the T cell receptor or a functional fragment thereof according to any one of [1] to [4], which has an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. [6] Furthermore, the T cell receptor or a functional fragment thereof according to any one of [1] to [4], which has an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2 and a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7. [7] Furthermore, the T cell receptor or a functional fragment thereof according to any one of [1] to [6], which has an α-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. [8] Furthermore, the T cell receptor or a functional fragment thereof according to any one of [1] to [6], which has an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 4 and a β-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 9. [9] A cell having the T cell receptor or a functional fragment thereof according to any one of [1] to [8].

[10] The cell according to [9], which is a Tax antigen-specific cytotoxic T cell.

[11] An iPS cell having the T cell receptor or a functional fragment thereof according to any one of [1] to [8].

[12] The iPS cell according to

[11] , which is a Tax antigen-specific cytotoxic T-iPS cell.

[13] A vector containing the DNA of the T cell receptor or a functional fragment thereof according to any one of [1] to [8]. A pharmaceutical composition containing a Tax antigen-specific cytotoxic T cell or T-iPS cell having the T cell receptor according to any one of [1] to [8] or a functional fragment thereof. The pharmaceutical composition according to

[14] , which is a pharmaceutical composition for treating adult T cell leukemia / lymphoma. A cellular pharmaceutical product containing a Tax antigen-specific cytotoxic T cell having the T cell receptor according to any one of [1] to [8] or a functional fragment thereof. The cellular pharmaceutical product according to

[16] , which is a cellular pharmaceutical product for treating adult T cell leukemia / lymphoma. A Tax antigen-specific cytotoxic T cell or T-iPS cell, characterized by containing a T cell whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+. A pharmaceutical composition containing a Tax antigen-specific cytotoxic T cell or T-iPS cell, characterized by containing a T cell whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+. A cellular pharmaceutical product containing a Tax antigen-specific cytotoxic T cell or T-iPS cell, characterized by containing a T cell whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+.

Advantages of the Invention

[0007] The Tax antigen-specific cytotoxic T lymphocyte having the T cell receptor of the Tax antigen-specific cytotoxic T lymphocyte of the present invention or a functional fragment thereof is useful as a therapeutic agent for adult T cell leukemia / lymphoma.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0009] One aspect of the present invention is a T cell receptor of Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1 or a functional fragment thereof, which comprises an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Another aspect of the present invention is a T cell receptor of Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1 or a functional fragment thereof, which comprises an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Another aspect of the present invention is a T cell receptor or a functional fragment thereof of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1, which consists of an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Another aspect of the present invention is a T cell receptor or a functional fragment thereof of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1, which comprises an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence.

[0010] HTLV-1 causes diseases such as Adult T-cell leukemia (ATL), HTLV-1 associated myelopathy (HAM), and HTLV-1 uveitis (HU). HTLV-1 has a proviral gene with a length of approximately 9 kb. The proviral gene of HTLV-1 encodes various proteins such as Tax, Rex, gag, pol, and env. Among these, the Tax protein has approximately 353 amino acid residues as the full length and has multiple functions such as activation of host transcription factors such as NF-κB, SRF, and CREB, and suppression of the functions of proteins such as p53 in host cells. Moreover, most of the target cells infected with HTLV-1 are CD4+ T cells.

[0011] The T cell receptor (hereinafter referred to as TCR) is responsible for the antigen recognition function of T cells and is composed of proteins such as α chain, β chain, γ chain, and δ chain. Among these, a heterodimer of TCRα chain protein (TCRα) and TCRβ chain protein (TCRβ), or a heterodimer of γ and δ chains, together with a CD3 complex (including γ, δ, ε, ζ), CD4, or CD8 as auxiliary molecules, forms a TCR. TCRα and TCRβ have a variable region (V region + J region) and a constant region (C region). In addition, there are complementarity-determining regions (CDRs) in the V region of the variable region. Therefore, TCR can be characterized by the amino acid sequences of the V regions (including the CDR regions) in TCRα and TCRβ, or the V region and J region.

[0012] As described above, the TCR of the present invention comprises an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Here, the amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence may be an amino acid sequence having 97% or more identity with the amino acid sequence, and more preferably an amino acid sequence having 99% or more identity with the amino acid sequence. Preferably, the TCR of the present invention has an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0013] In addition to the α-chain CDR3 region and the β-chain CDR3 region, the TCR of the present invention preferably further has an α-chain L region consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain L region consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Here, the amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence may be an amino acid sequence having 95% or more identity with the amino acid sequence, more preferably an amino acid sequence having 97% or more identity with the amino acid sequence, and even more preferably an amino acid sequence having 99% or more identity with the amino acid sequence. As the TCR of the present invention, it is more preferable to have an α-chain CDR3 region consisting of the amino acid sequence represented by SEQ ID NO: 3, an α-chain L region consisting of the amino acid sequence represented by SEQ ID NO: 1, a β-chain CDR3 region consisting of the amino acid sequence represented by SEQ ID NO: 8, and a β-chain L region consisting of the amino acid sequence represented by SEQ ID NO: 6.

[0014] In addition to the α-chain CDR3 region, the α-chain L region, the β-chain CDR3 region, and the β-chain L region, the TCR of the present invention more preferably has an α-chain V region consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence, and a β-chain V region consisting of the amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence. Here, the amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added to the amino acid sequence may be an amino acid sequence having 95% or more identity with the amino acid sequence, more preferably an amino acid sequence having 97% or more identity with the amino acid sequence, and even more preferably an amino acid sequence having 99% or more identity with the amino acid sequence. The TCR of the present invention preferably has an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1, an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2, a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8, a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0015] In addition to the α-chain CDR3 region, the α-chain L region, the β-chain CDR3 region, the α-chain V region, the β-chain L region, and the β-chain V region described above, the TCR of the present invention preferably has an α-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence, and a β-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence. Here, the amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or added to the amino acid sequence may be an amino acid sequence having 95% or more identity with the amino acid sequence, more preferably an amino acid sequence having 97% or more identity with the amino acid sequence, and even more preferably an amino acid sequence having 99% or more identity with the amino acid sequence. The TCR of the present invention preferably has an α-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3, an α-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1, an α-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2, an α-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 4, a β-chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8, a β-chain L region consisting of the amino acid sequence shown in SEQ ID NO: 6, a β-chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a β-chain J region consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0016] Examples of the functional fragments of the TCR of the present invention include polypeptides having the α-chain CDR3 region and the β-chain CDR3 region, polypeptides having the α-chain CDR3 region, the α-chain L region, the β-chain CDR3 region, and the β-chain L region, polypeptides having the α-chain CDR3 region, the α-chain L region, the α-chain V region, the β-chain CDR3 region, the β-chain L region, and the β-chain V region, polypeptides having the α-chain CDR3 region, the α-chain L region, the α-chain V region, the α-chain J region, the β-chain CDR3 region, the β-chain L region, the β-chain V region, and the β-chain J region, conjugates of these polypeptides, and the like.

[0017] The TCR of the present invention can be obtained, for example, by inducing HLA-A2402-restricted Tax antigen-specific cytotoxic T lymphocytes, which are common in Japanese people, from a patient's peripheral blood and then performing single cell cloning to establish Tax antigen-specific cytotoxic T lymphocyte clones. To induce HLA-A2402-restricted Tax antigen-specific cytotoxic T lymphocytes from peripheral blood, it may be a healthy person or a human suffering from a viral infectious disease. In the present invention, the T cells induced into T-iPS cells are preferably T cells having Tax antigen specificity. For example, T cells expressing CD3 and CD8, specifically, CTLs that are CD8-positive cells. Also, for example, T cells expressing CD3 and CD4, specifically, T cells that are CD4-positive cells. Note that the antigen specificity in T cells is brought about by an antigen-specific, reconstituted TCR gene. From the perspective of production efficiency, although not particularly limited thereto, in order to obtain antigen-specific CD8-positive cells, as human T cells induced into T-iPS cells, it is preferable to use antigen-specific CD8-positive T cells. Also, when performing immunotherapy, the human T cells differentiated from iPS cells are preferably the same or substantially the same in antigen specificity as the human T cells induced into iPS cells. Also, T cells without antigen specificity are included as T cells for inducing T-iPS cells. Specifically, gene-modified T cells such as CART cells or TCR-T cells can be mentioned.

[0018] Such T cells can be isolated, for example, from human tissues by known methods. Examples of human tissues include tissues containing the T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, cord blood, and diseased tissue. Among these, peripheral blood is preferred from the viewpoints of low invasiveness to humans and ease of preparation. When isolating tumor-infiltrating lymphocytes (TIL), it can be isolated from tumor tissue or peripheral blood. Known methods for isolating human T cells include, for example, magnetic selection using magnetic beads for cell separation, flow cytometry using an antibody against a cell surface marker such as CD4 or CD8 and a cell sorter, an activated T cell induction method using an anti-CD3 antibody and an anti-CD28 antibody, and the like. In addition, desired T cells can be isolated using the secretion of cytokines, the expression of functional molecules, or signal molecules such as PD-1 as an index. Also, cytotoxic T cells (CTL) can be isolated using the secretion or production of granzymes, perforin, etc. as an index. Furthermore, when isolating from a human tissue containing T cells having antigen specificity, a multimerized MHC (major histocompatibility complex) bound to a desired antigen (for example, "MHC tetramer", "Pro5 (registered trademark) MHC class I pentamer") can be used to purify T cells having the desired antigen specificity from human tissue.

[0019] In the present invention, the gene introduced to convert T cells into iPS cells is preferably a combination of at least 4 types of genes among (a) Oct3 / 4 gene, (b) c-Myc gene, (c) Sox2 gene, (d) Klf4 gene, (e) NANOG gene, and (f) LIN28 gene, and a combination of these 6 types of genes is more preferred.

[0020] In the present invention, the method for introducing the gene group into T cells is not particularly limited, and known methods can be appropriately selected and used. For example, when introducing the gene group into T cells in the form of nucleic acid encoding the gene group, the nucleic acid encoding the gene group (for example, cDNA, RNA) is inserted into an appropriate expression vector containing a promoter that functions in T cells, and the expression vector can be introduced into cells by infection, lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, electroporation method.

[0021] Among such expression vectors, it is more preferable to use a stealth type RNA expression vector containing the gene group in terms of reducing the risk of carcinogenesis and introduction efficiency. A stealth type RNA expression vector is a vector designed to avoid the vector from entering the chromosome and to continuously and stably express genes in the cytoplasm rather than in the nucleus. It can introduce a large gene of 13,000 base pairs or more and simultaneously introduce 10 genes, does not damage cells, can be removed when the introduced gene is unnecessary, and has the stealth property that cells cannot recognize the vector as a foreign substance. Such a stealth type RNA expression vector includes a minus single-stranded RNA (A) containing the following RNA sequences (1) to (8), a single-stranded RNA binding protein (B), and an RNA-dependent RNA synthase, and a complex that does not activate the innate immune structure. (1) An RNA sequence for the gene group, (2) An RNA sequence derived from human mRNA constituting a non-coding region, (3) A transcription start signal sequence recognized by the RNA-dependent RNA synthase, (4) A transcription termination signal sequence recognized by the RNA-dependent RNA synthase, (5) An RNA sequence containing an origin of replication recognized by the RNA-dependent RNA synthase, (6) An RNA sequence encoding the RNA-dependent RNA synthase, (7) An RNA sequence encoding a protein that regulates the activity of the RNA-dependent RNA synthase (8) An RNA sequence encoding the single-stranded RNA-binding protein.

[0022] Also, when establishing T-iPS cells, before introduction of the gene group, the T cells are preferably stimulated and activated by an anti-CD3 antibody and an anti-CD28 antibody in the presence of interleukin-2 (IL-2) or interleukin-7 (IL-7) and interleukin-15 (IL-15), and may be stimulated and activated by at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), allogeneic antigen-expressing cells, anti-CD3 antibody, anti-CD28 antibody, CD3, and CD28 agonists. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody, and / or anti-CD28 antibody, etc. to the medium and culturing the T cells for a certain period. Also, the anti-CD3 antibody and anti-CD28 antibody may be those to which magnetic beads or the like are bound. Further, instead of adding these antibodies to the medium, stimulation may be given by culturing the T cells on a culture dish having the anti-CD3 antibody and anti-CD28 antibody bound to its surface for a certain period. Furthermore, stimulation may also be given by adding an antigen peptide recognized by the T cells (for example, human T cells) to the medium together with feeder cells.

[0023] In order to apply such a stimulus to the T cells, the concentration of PHA added to the medium is not particularly limited, but it is preferably 1 to 100 μg / mL. Also, the concentration of IL-2 added to the medium is not particularly limited, but it is preferably 1 to 200 ng / mL. Further, the concentration of the anti-CD3 antibody and anti-CD28 antibody added to the medium is not particularly limited, but it is preferably 1 to 10 times the amount of the cultured T cells. Also, in order to apply such a stimulus to the T cells, the concentration of the anti-CD3 antibody and anti-CD28 antibody bound on the surface of the culture dish is not particularly limited, but the concentration during coating is preferably 0.1 to 100 μg / mL for the anti-CD3 antibody, preferably 1 to 100 μg / mL, and 0.1 to 10 μg / mL for the anti-CD28 antibody.

[0024] Also, the culture period for applying such a stimulus is a period sufficient to apply such a stimulus to the T cells and is a period capable of growing the T cells to the number of cells required for the introduction of the four genes, and there is no particular limitation, but it is usually 2 to 7 days, and from the viewpoint of gene introduction efficiency, it is preferably 3 to 5 days. From the viewpoint of infecting by mixing the T cells and the vector in a 15 mL tube or increasing the gene introduction efficiency, it is preferable to culture on a culture dish coated with Retronectin.

[0025] As the medium for culturing the T cells and adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, etc., for example, a known medium suitable for culturing the T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium containing other cytokines and human serum, AIM V TM medium, NS-A2 can be used. In addition to PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, amino acids necessary for culture (for example, L-glutamine), antibiotics (for example, streptomycin, penicillin) may be added to the medium. Also, it is preferable to add IL-7 and IL-15 to the medium instead of IL-2. The added concentrations of IL-7 and IL-15 are not particularly limited, but are preferably 1 to 100 ng / mL each.

[0026] In addition, when introducing the four genes into the T cells, or as subsequent conditions, there are no particular restrictions. However, the T cells into which the four genes have been introduced are preferably cultured under feeder-free conditions. For example, wells coated with an iMatrix-511 solution, which is a laminin 511E8 fragment, or vitronectin can be mentioned. Culturing under feeder cell conditions is also possible. Examples of feeder cells include mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation or treatment with antibiotics.

[0027] Furthermore, in the process of inducing T-iPS cells from the T cells, it is preferable to add the iPS cell medium from the next day. After that, it is preferable to change the medium by half every other day and gradually replace it from the T cell medium to the iPS medium.

[0028] In addition, in accordance with the transition from the T cells to iPS cells, it is preferable to culture while gradually replacing the medium suitable for culturing the T cells with a medium suitable for culturing iPS cells. As a medium suitable for culturing such iPS cells, a known medium can be appropriately selected and used. For example, when coated with iMatrix, StemFit AK03N, or when coated with vitronectin, Essential 8 Medium. On feeder cells such as MEF cells, Dulbecco's modified Eagle medium / F12 medium (human iPS cell medium) containing knockout serum replacement, L-glutamine, non-essential amino acids, 2-mercaptoethanol, and b-FGF, etc. is desirable.

[0029] In this way, the selection of T-iPS cells can be carried out by appropriately selecting known methods. Such known methods include, for example, a method of observing and selecting the morphology of ES cell / iPS cell-like colonies under a microscope. On the other hand, in the case of T-iPS established from CTL clones that are single cells, since their properties are often similar, there is also a method of subculturing all the established colonies as they are without selecting each colony of T-iPS cells.

[0030] Confirmation that the cells selected in this way are T-iPS cells can be carried out, for example, by detecting the expression of undifferentiated cell-specific markers (such as ALP, SSEA-4, Tra-1-60, and Tra-1-81) in the selected cells by immunostaining, RT-PCR, etc., or by transplanting the selected cells into a mouse and observing teratoma formation. Also, confirmation that the cells selected in this way are derived from the above T cells can be carried out by detecting the state of TCR gene rearrangement by genomic PCR.

[0031] When selecting and collecting these cells, it is preferable to collect them while observing the growth state of the colonies. Generally, it is 10 to 40 days, preferably 14 to 28 days, after introducing the gene group containing the reprogramming factor into the T cells. As the culture environment, unless otherwise specified above, it is preferably under the conditions of 5% CO2, 35 to 38 °C, more preferably 37 °C.

[0032] Next, Tax antigen-specific CTL cells are induced to differentiate from the established T-iPS cells. As this redifferentiation induction method, a method of differentiating T-iPS cells into CD8+ single-positive T cells is preferable, and a method of differentiating T-iPS cells into CD4 / CD8 double-negative T cells and then differentiating the CD4 / CD8 double-negative T cells into CD8+ single-positive T cells is more preferable. Furthermore, as described in Patent Document 1, it is preferable to differentiate T-iPS cells into CD4 / CD8 double-negative cells, add a substance that stimulates the T cell receptor to stimulate the CD4 / CD8 double-negative cells, and then differentiate the CD4 / CD8 double-negative cells stimulated by the T cell receptor into CD8 single-positive T cells in the presence of cytokines IL-7 and IL-15.

[0033] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, the T-iPS cells are cultured on feeder cells (preferably mouse stromal cells) in a medium containing cytokines, serum ( For example, fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine, α-monothioglycerol, ascorbic acid, etc. It is preferable to culture in the medium. As the stromal cells to be used, it is preferable to use OP9 cells or 10T1 / 2 cells (C3H10T1 / 2 cells) that have been treated such as by radiation. The cytokine added to the medium is preferably at least one cytokine selected from the group consisting of VEGF, SCF, TPO, and FLT3L, and more preferably VEGF, SCF, and TPO, or VEGF, SCF, and FLT3L. Examples of the medium include X-VIVO medium, Iscove's modified Dulbecco's medium (IMDM medium), α-MEM, and DMEM. From the viewpoint of facilitating the formation of T-iPS sacs (bag-like structures containing hematopoietic progenitor cells), IMDM medium is preferable. The culture period of these T-iPS cells is preferably 8 to 14 days, more preferably 10 to 14 days, after starting the culture of T-iPS cells. The culture environment is not particularly limited, but is preferably under the conditions of 5% CO2, 35 to 38 °C, more preferably 37 °C. Furthermore, it is more preferable to culture for about one week under low oxygen concentration conditions (oxygen concentration: for example, 5 to 20%).

[0034] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, the cells contained in the above-mentioned T-iPS sac are preferably cultured on feeder cells (preferably stromal cells, more preferably human stromal cells) in a medium containing cytokines, serum (e.g., FBS), etc. However, a cytokine-coated well is used under feeder-free conditions. The cells present inside the T-iPS sac can be separated, for example, by passing them through a sterilized sieve-like instrument (e.g., a cell strainer). As the stromal cells used for this culture, from the viewpoint of inducing differentiation into T lymphocytes via the notch signal, OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2 / DL4 cells, and 10T1 / 2 / DL1 cells treated with radiation or the like are preferable. Examples of cytokines added to the medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15. Examples of the medium include α-MEM medium, DMEM medium, and IMDM medium, but α-MEM medium is preferable. In addition to IL-7 and FLT3L, amino acids necessary for culture (e.g., L-glutamine) and antibiotics (e.g., streptomycin, penicillin) may be added to the medium.

[0035] The culture period of the cells contained in this T-iPS sac is preferably the period until a T cell receptor (TCR) is expressed on the cell surface of the CD4 / CD8 double-negative cells thus differentiated, and is preferably 14 to 28 days after starting the culture of the cells contained in the T-iPS sac. The culture environment is not particularly limited, but is preferably under the conditions of 5% CO2, 35 to 38 °C, more preferably 37 °C.

[0036] Whether a T cell receptor (TCR) is expressed on the cell surface of CD4 / CD8 double-negative cells can be evaluated by flow cytometry using anti-TCRαβ antibody, anti-CD3 antibody, anti-CD4 antibody, and anti-CD8 antibody.

[0037] In the method for producing human CD8 single-positive cells having antigen specificity, by stimulating CD4 / CD8 double-negative cells derived from T-iPS cells via the TCR expressed on the cell surface, for example, by stimulating CD4 / CD8 double-negative cells derived from T-iPS cells via the TCR complex expressed on the cell surface, further rearrangement of the TCRA gene can be suppressed, and as a result, in the CD8 single-positive cells obtained by redifferentiation, the frequency of appearance of T cells having the same TCR gene rearrangement pattern as the original human T cells can be extremely increased. The rearrangement of the TCR is carried out by RAG, and similarly, by knocking out RAG1 and / or RAG2, the frequency of appearance of T cells having the same TCR gene rearrangement pattern as the original human T cells can be extremely increased.

[0038] As a method for stimulating the T cell receptor of CD4 / CD8 double-negative cells derived from T-iPS cells, at least one substance selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, an antigen peptide to which the human T cells from which the T-iPS cells were originally derived specifically bind, a cell expressing a complex with HLA that shows binding affinity for the T cell receptor, and an MHC multimer to which the antigen peptide is bound is preferably contacted with the CD4 / CD8 double-negative cells derived from T-iPS cells. From the viewpoint of giving a physiological stimulus, a method of contacting specific peptide / HLA complex-expressing cells is more preferable. Also, from the viewpoint of emphasizing the uniformity of stimulation, a method of contacting an antibody or a reagent is more preferable.

[0039] The contacting method can be carried out, for example, by culturing the T cells for a certain period by adding PHA or the like to the medium. The anti-CD3 antibody and the anti-CD28 antibody may be those to which magnetic beads or the like are bound. Further, instead of adding these antibodies to the medium, the T cells may be stimulated by culturing them for a certain period on a culture dish having the anti-CD3 antibody and the anti-CD28 antibody bound to its surface. Furthermore, stimulation may also be given by adding the antigen peptide together with feeder cells to the medium.

[0040] To stimulate the TCR of CD4 / CD8 double-negative cells, the concentration of PHA added to the medium is preferably 1 to 100 μg / ml. Further, the concentration of the anti-CD3 antibody and the anti-CD28 antibody added to the medium is preferably 1 to 10 times the amount of the cultured T cells. Further, to stimulate the TCR of CD4 / CD8 double-negative cells, the concentration of the anti-CD3 antibody and the anti-CD28 antibody bound on the surface of the culture dish is preferably 0.1 to 100 μg / ml for the anti-CD3 antibody and 0.1 to 10 μg / ml for the anti-CD28 antibody at the time of coating.

[0041] The culture period of the cells contained in this T-iPS sac preferably includes the period required for the expression of the T cell receptor (TCR) on the cell surface of the CD4 / CD8 double-negative cells thus differentiated, and is preferably 7 to 29 days after the start of the culture of the cells contained in the T-iPS sac. The culture environment is preferably conditions of 5% CO2, 35 to 38 °C, more preferably 37 °C.

[0042] In the present invention, in order to differentiate CD4 / CD8 double-negative cells stimulated by a T cell receptor into CD8 single-positive cells, the CD4 / CD8 double-negative cells are preferably cultured in a medium containing cytokines, serum (such as human serum), and the like. As the cytokine to be added to the medium, any cytokine can be used as long as it can differentiate CD4 / CD8 double-negative cells into CD8 single-positive cells. Examples thereof include IL-7 and IL-15. Among these, from the viewpoint of selecting the CD8 lineage and facilitating the generation of memory-type CD8+ T cells during differentiation into CD8 single-positive cells, it is preferable to add IL-7 and IL-15 in combination. The addition concentration of IL-7 and IL-15 is preferably 1 to 20 ng / ml. Examples of the medium include RPMI-1640 medium, X-VIVO medium, DMEM medium, and α-MEM medium, with RPMI-1640 medium or X-VIVO medium being preferable. In addition to IL-7, IL-15, etc., amino acids necessary for culture (such as L-glutamine), antibiotics (such as streptomycin and penicillin), and cytokines other than IL-7, IL-15, etc. may be added to the medium.

[0043] In such culture, the CD4 / CD8 double-negative cells may be co-cultured with feeder cells. The feeder cells are preferably peripheral blood mononuclear cells (PBMC). Such PBMC is preferably in an allogeneic relationship with the CD4 / CD8 double-negative cells. Further, from the viewpoint of continuously stimulating the TCR and suppressing further reconstitution of the TCR, it is more preferable to use peripheral blood mononuclear cells that present an antigen peptide specifically bound by the human T cells from which the CD4 / CD8 double-negative cells originated.

[0044] The culture period for differentiating these CD4 / CD8 double-negative cells into CD8 single-positive cells is preferably 2 to 4 weeks. The culture environment is preferably under the conditions of 5% CO2, 35 to 38 °C, and more preferably 37 °C.

[0045] To confirm that the thus induced CD8 single-positive cells are derived from T-iPS cells and also from the T cells from which the T-iPS cells were originally derived, for example, the state of TCR gene rearrangement can be detected by genomic PCR.

[0046] In addition, the CD8 single-positive cells thus obtained can be isolated by appropriately selecting known methods. Examples of such known methods include flow cytometry using an antibody against the cell surface marker of CD8 and a cell sorter. For example, in the case of CD8 single-positive cells, a method of purification using an affinity column or the like on which the antigen recognized by the T cells from which the CD8 single-positive cells were originally derived is immobilized, or a method of purification using an MHC multimer (for example, MHC tetramer) to which the antigen is bound can also be employed.

[0047] In addition, the CD8 single-positive cells obtained by the present invention do not express PD-1, but express CCR7 together with CD27 and CD28, which represent the phenotype of central memory T cells, and also have longer telomeres than the original T cells and have a high self-renewal ability. Therefore, according to the present invention, it is possible to produce CD8 single-positive T cells having the same TCR gene rearrangement pattern as the original T cells, which do not express PD-1 and express CD27, CD28 and CCR7. And the T cells collected from humans are different from the obtained T cells in that they express PD-1 and have a small proportion of a naive memory phenotype.

[0048] In order to maintain the CD8 single-positive cells thus obtained, the cells may be stimulated every 1 to 2 weeks. Such stimulation may include contact with at least one substance selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, IL-2, IL-7, IL-15, an antigen recognized by the CD8SP cells, an MHC multimer conjugated with the antigen, feeder cells in an allogeneic relationship with the CD8 single-positive cells, and feeder cells in an autologous relationship with the CD8 single-positive cells.

[0049] To confirm whether the obtained iPSC-derived T lymphocytes have Tax antigen-specific cytotoxic activity, it is confirmed that they retain the same antigen specificity as the original peripheral blood-derived CTL using MHC pentamers, MHC tetramers, etc. Furthermore, TCR sequence analysis was performed to identify TCRαβ.

[0050] In addition, Tax antigen-specific cytotoxic T lymphocytes can also be produced by genetic recombination using the gene encoding the TCR of the present invention. That is, for example, the gene encoding the TCR of the present invention may be introduced into host T cells, and cells having Tax antigen-specific cytotoxicity may be selected. To introduce the gene encoding the TCR into host T cells, it is preferable to incorporate the gene into various viral vectors. The selection of cells having Tax antigen-specific cytotoxicity may be the confirmation of the above-described Tax antigen-specific cytotoxic activity or the detection of the ability to produce cytokines such as IFN-γ.

[0051] Since the obtained Tax antigen-specific cytotoxic T lymphocytes have excellent Tax antigen-specific cytotoxic activity, they are useful as therapeutic agents for diseases such as adult T-cell leukemia / lymphoma, HTLV-1-associated myelopathy, and HTLV-1 uveitis (HU), particularly as a therapeutic agent composition for adult T-cell leukemia / lymphoma.

[0052] In addition to the T lymphocytes of the present invention, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Examples of such carriers include physiological saline, Ringer's solution, and the like. Furthermore, the pharmaceutical composition of the present invention can contain known pharmaceutically acceptable additives such as preservatives and coloring agents, if necessary. The form of the pharmaceutical composition of the present invention is preferably an injection, and more preferably an injection for T cell infusion therapy.

Examples

[0053] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0054] Example 1 Establishment of T-iPS cells using a Sendai virus vector from Tax-specific CTL clones. 1) Peripheral blood mononuclear cells were isolated from HLA-A2402-restricted healthy human peripheral blood, and dendritic cells were induced for antigen presentation purposes. After 7 days, the Tax antigen peptide (Tax301-309, A2402) was added to the induced dendritic cells, and co-culture with peripheral blood mononuclear cells was started. Approximately 8 to 10 days later, for the detection of Tax-specific CTL, CTL was stained with MHC tetramer, and the tetramer-positive rate was confirmed by flow cytometry. After confirming Tax-specific CTL, single cell sorting or tetramer / PE bead selection was performed followed by limiting dilution.

[0055] 2) Tetramer staining of colonies that emerged after about 3 to 6 weeks was performed, and the establishment of Tax antigen-specific CTL clones was confirmed by flow cytometry.

[0056] The established Tax antigen-specific CTL clones were stimulated with CD3 / 28 and infected with a Sendai virus (SeV) vector (SeVp[KOSM302L]) containing nuclear reprogramming factors (Oct4, Sox2, Klf4, c-Myc) and a SeV vector containing a nucleic acid encoding SV40 large T antigen for gene transfer. CTLs after gene transfer were transferred to a 6-well plate coated with iMatrix, and culture was started in a CO2 incubator using a T cell medium supplemented with IL-2 (medium composition: RPMI, 10% human AB serum).

[0057] 3) On the day after SeV gene transfer, an equal volume of iPS medium (StemFit AK03N) was added, and thereafter it was replaced with Stem Fit AK03N in half amounts every other day. 4) After 7 days, colonies of T-iPS cells were observed, and thereafter the colonies were picked up and subjected to expansion culture. Thereafter, iPSC-derived rejuvenated Tax-CTLs (Tax-rejT) were induced to differentiate. 5) When a cytotoxicity test was performed, it was able to show antigen-specific cytotoxic activity against tumor cells more strongly than the original peripheral blood CTLs.

[0058] Figure 1 shows the tetramer staining results of Tax-CTLs induced from peripheral blood, established Tax-specific CTL clones, and iPSC-derived Tax-CTLs (Tax-rejT).

[0059] Example 2 (Cytotoxicity test) 1) To compare the cytotoxic activity of Tax-rejT against ATL tumor cells and the cytotoxicity of peripheral blood-derived Tax-CTLs, a 51-chromium release test was performed. As effectors, Tax-rejT or peripheral blood-derived Tax-CTLs, patient-derived ATL cells labeled with chromium as targets, and HLA-mismatched Epstein-Barr virus-infected tumor cell lines (LCLs) as control targets were co-cultured for 6 hours at effector:target ratios of 20:1, 10:1, 5:1, and 2.5:1. 2) After co-culture, the culture supernatant was transferred to another plate for counting, dried, and measured with a plate reader. 3) Tax-rejT showed strong antigen-specific cytotoxicity against ATL cells (90 - 100%), but showed cytotoxicity of 10% or less against control HLA-mismatched tumor cell lines. Peripheral blood-derived Tax-CTL showed cytotoxicity of about 20 - 50% against ATL cells. Against control HLA-mismatched tumor cell lines, it showed cytotoxicity of 20% or less. The Tax antigen-specific cytotoxic activity of Tax-rejT was stronger than that of Tax-CTL (Figure 2).

[0060] Example 3 The analysis results of the TCR sequences are shown in Figures 3 and 4.

Claims

1. A T cell receptor for Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1, or a functional fragment thereof, having an α chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence, and a β chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence.

2. The T cell receptor or its functional fragment according to claim 1, having an α chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 3, and a β chain CDR3 region consisting of the amino acid sequence shown in SEQ ID NO:

8.

3. The T cell receptor or a functional fragment thereof according to claim 1, further comprising an α chain L region consisting of the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence, and a β chain L region consisting of the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence.

4. The T cell receptor or its functional fragment according to claim 1, further comprising an alpha chain L region consisting of the amino acid sequence shown in SEQ ID NO: 1, and a beta chain L region consisting of the amino acid sequence shown in SEQ ID NO:

6.

5. The T cell receptor or a functional fragment thereof according to claim 1, further comprising an α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence, and a β chain V region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence.

6. The T cell receptor or its functional fragment according to claim 1, further comprising an alpha chain V region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a beta chain V region consisting of the amino acid sequence shown in SEQ ID NO:

7.

7. The T cell receptor or a functional fragment thereof according to claim 1, further comprising an α chain J region consisting of the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence, and a β chain J region consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence resulting from the substitution, deletion or addition of 1 to 3 amino acids to said amino acid sequence.

8. The T cell receptor or its functional fragment according to claim 1, further comprising an α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 4, and a β chain J region consisting of the amino acid sequence shown in SEQ ID NO:

9.

9. A cell having a T cell receptor or a functional fragment thereof according to any one of claims 1 to 8.

10. The cell according to claim 9, which is a Tax antigen-specific cytotoxic T cell.

11. An iPS cell having the T cell receptor or a functional fragment thereof according to any one of claims 1 to 8.

12. The iPS cell according to claim 11, which is a Tax antigen-specific cytotoxic T-iPS cell.

13. A vector comprising the DNA of the T cell receptor or a functional fragment thereof according to any one of claims 1 to 8.

14. A pharmaceutical composition comprising a Tax antigen-specific cytotoxic T cell or T-iPS cell having the T cell receptor or a functional fragment thereof according to any one of claims 1 to 8.

15. The pharmaceutical composition according to claim 14, which is a pharmaceutical composition for treating adult T-cell leukemia / lymphoma.

16. A cellular pharmaceutical comprising Tax antigen-specific cytotoxic T cells having the T cell receptor or a functional fragment thereof according to any one of claims 1 to 8.

17. The cellular pharmaceutical product according to claim 16, which is a cellular pharmaceutical product for treating adult T-cell leukemia / lymphoma.

18. A Tax antigen-specific cytotoxic T cell or T-iPS cell, characterized in that it comprises a T cell whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+.

19. A pharmaceutical composition comprising Tax antigen-specific cytotoxic T cells or T-iPS cells, characterized in that the composition comprises T cells whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+.

20. A cellular pharmaceutical comprising Tax antigen-specific cytotoxic T cells or T-iPS cells, characterized in that the pharmaceutical comprises T cells whose surface antigen is CD4-, CD8+, PD-1-, CD27+, CD28+ or CCR7+.

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