HTLV-1-encoded Tax antigen-specific cytotoxic T lymphocyte T cell receptor or its functional fragment

Abstract

To provide T-cell receptors or functional fragments thereof having an excellent anti-tumor effect.SOLUTION: Provided is a T-cell receptor of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1, or a functional fragment thereof, which has an α chain V region and a β chain V region based on an amino acid sequence revealed by cloning the T-cell receptor of a Tax antigen-specific cytotoxic T lymphocyte (CTL), converting a CTL clone to an iPS cell once, and obtaining a CTL, obtained by redifferentiation induction of Tax antigen-specific CTL from the iPS cells, with a stronger antitumor effect than the original CTL clone.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Adult T-cell leukemia-lymphoma (ATL) is caused by infection with a virus called HTLV-1 (human T-lymphotropic virus type-I), which infects CD4+ T cells and develops when the infected T cells turn into cancerous cells (ATL cells) and proliferate indefinitely (Non-Patent Document 1). HTLV-1 contains the gene encoding a viral protein called Tax, which has carcinogenic properties, and CTL therapy targeting this Tax antigen is expected to be an effective treatment. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] National Institute of Infectious Diseases website Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to clone a T cell receptor of a Tax antigen-specific cytotoxic T lymphocyte and to provide a T cell receptor or a functional fragment thereof that has an excellent antitumor effect. [Means for solving the problem]

[0005] Therefore, the inventors induced Tax antigen-specific cytotoxic T lymphocytes from the patient's peripheral blood, performed single-cell cloning, and successfully established Tax antigen-specific cytotoxic T lymphocyte (CTL) clones. The antitumor effects of the resulting clones against autologous tumor cells (ATL cells) were confirmed. However, the proliferation capacity of CTL clones is limited, making it difficult to secure cells for use in actual patient treatment. Therefore, the inventors converted the CTL clones into iPS cells and then induced redifferentiation of Tax antigen-specific CTLs from these iPS cells. As a result, they succeeded in generating CTLs with stronger antitumor effects than the original CTL clones. The amino acid sequence of the T cell receptor (TCR) was elucidated by repertoire analysis of the redifferentiation-induced CTLs, thereby completing the present invention.

[0006] That is, the present invention provides the following inventions [1] to [8]. [1] A T cell receptor or a functional fragment thereof for Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1, having an α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by substituting, deleting or adding 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: 4 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence. [2] A T cell receptor or a functional fragment thereof described in [1], having an alpha chain V region consisting of the amino acid sequence shown in sequence number 1 and a beta chain V region consisting of the amino acid sequence shown in sequence number 4. [3] The T cell receptor or functional fragment thereof according to [1] or [2], further comprising an α chain J region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence obtained by substituting, deleting or adding 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: 5 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence. [4] A T cell receptor or a functional fragment thereof according to [1] or [2], further comprising an alpha chain J region consisting of the amino acid sequence shown in SEQ ID NO: 2 and a beta chain J region consisting of the amino acid sequence shown in SEQ ID NO: 5. [5] A therapeutic composition for adult T-cell leukemia / lymphoma, comprising a Tax antigen-specific cytotoxic T lymphocyte having the T cell receptor or a functional fragment thereof described in any one of [1] to [4]. [6] Use of Tax antigen-specific cytotoxic T lymphocytes having a T cell receptor or a functional fragment thereof described in any one of [1] to [4] for the manufacture of a therapeutic agent for adult T-cell leukemia / lymphoma. [7] A Tax antigen-specific cytotoxic T lymphocyte having a T cell receptor or a functional fragment thereof described in any one of [1] to [4] for treating adult T-cell leukemia / lymphoma. [8] A method for treating adult T-cell leukemia / lymphoma, characterized by administering a Tax antigen-specific cytotoxic T lymphocyte having a T cell receptor or a functional fragment thereof described in any one of [1] to [4]. [Effects of the Invention]

[0007] The Tax antigen-specific cytotoxic T lymphocytes having the T cell receptor of the Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1 of the present invention or a functional fragment thereof are useful as therapeutic agents for adult T-cell leukemia / lymphoma. [Brief explanation of the drawings]

[0008] [Figure 1] The antigen specificity of the obtained T lymphocytes to the Tax antigen is shown. [Figure 2] Figure 1 shows the antigen-specific cytotoxic activity of the original Tax-specific CTL clones and iPSC-derived Tax-specific CTLs. AutoATL shows activity against ATL, and HLA-mismatched LCL shows activity against HLA-mismatched LCL cells as a control. [Figure 3] The results of the obtained TCR repertoire analysis are shown below. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a T cell receptor (TCR) or a functional fragment thereof of a Tax antigen-specific cytotoxic T lymphocyte encoded by HTLV-1, characterized in that it has an α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by substituting, deleting or adding 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: 4 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence.

[0010] HTLV-1 causes diseases such as adult T-cell leukemia / lymphoma (ATL), HTLV-1 associated myelopathy (HAM), and HTLV-1 uveitis (HU). HTLV-1 has a proviral gene approximately 9 kb in length. The HTLV-1 proviral gene encodes various proteins, including Tax, Rex, gag, pol, and env. The full-length Tax protein contains approximately 353 amino acid residues and has multiple functions within host cells, including activating host transcription factors such as NF-κB, SRF, and CREB, and suppressing the function of proteins such as p53. Furthermore, most of the cells infected by HTLV-1 are CD4+ T cells.

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

[0012] The TCR of the present invention is characterized by having an α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained by substituting, deleting or adding 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: 4 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence. Here, the amino acid sequence obtained by substituting, deleting, or adding 1 to 3 amino acids to the amino acid sequence may be an amino acid sequence that has 95% or more identity with the amino acid sequence, and more preferably an amino acid sequence that has 97% or more identity with the amino acid sequence. The TCR of the present invention is preferably a TCR having an α chain V region consisting of the amino acid sequence shown in SEQ ID NO:1 and a β chain V region consisting of the amino acid sequence shown in SEQ ID NO:4. The α chain V region consisting of the amino acid sequence shown in SEQ ID NO: 1 contains the CDR3 region shown in SEQ ID NO: 3. The β chain V region consisting of the amino acid sequence shown in SEQ ID NO: 4 contains the CDR3 region shown in SEQ ID NO: 6.

[0013] It is preferable that the TCR of the present invention further has an α chain J region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence obtained by substituting, deleting or adding 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: 5 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence. Here, the amino acid sequence obtained by substituting, deleting, or adding 1 to 3 amino acids to the amino acid sequence may be an amino acid sequence that has 95% or more identity with the amino acid sequence, and more preferably an amino acid sequence that has 97% or more identity with the amino acid sequence. The TCR of the present invention is preferably a TCR further comprising an α chain J region consisting of the amino acid sequence shown in SEQ ID NO:2 and a β chain J region consisting of the amino acid sequence shown in SEQ ID NO:5. Furthermore, the TCR of the present invention is preferably a TCR having an α chain consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence, and a β chain consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence obtained by substituting, deleting or adding 1 to 3 amino acids to said amino acid sequence.

[0014] Functional fragments of the TCR of the present invention include polypeptides having the α chain V region and β chain V region, polypeptides having the α chain V region, α chain J region, β chain V region and β chain J region, and conjugates of these polypeptides.

[0015] The TCR of the present invention can be obtained, for example, by inducing Tax antigen-specific cytotoxic T lymphocytes from the peripheral blood of a patient, followed by single cell cloning to establish a Tax antigen-specific cytotoxic T lymphocyte clone. Tax antigen-specific cytotoxic T lymphocytes can be induced from peripheral blood from either healthy individuals or humans suffering from a viral infection. In the present invention, the T cells induced to become T-iPS cells are preferably T cells with Tax antigen specificity. Examples include T cells expressing CD3 and CD8, specifically CD8-positive CTLs. Other examples include T cells expressing CD3 and CD4, specifically CD4-positive T cells. The antigen specificity of T cells is conferred by antigen-specific rearranged TCR genes. From the viewpoint of production efficiency, although not limited thereto, antigen-specific CD8-positive T cells are preferably used as the human T cells induced to become T-iPS cells to obtain antigen-specific CD8-positive cells. Furthermore, when performing immunotherapy, it is preferable that the human T cells differentiated from iPS cells have the same or substantially the same antigen specificity as the human T cells induced to become iPS cells. T cells from which T-iPS cells are induced also include T cells without antigen specificity. Specific examples include genetically modified T cells such as CART cells or TCR-T cells.

[0016] Such T cells can be isolated from, for example, human tissues by known techniques. Examples of human tissues include tissues containing T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissue. Among these, peripheral blood is preferred because it is less invasive to humans and easier to prepare. Tumor-infiltrating lymphocytes (TILs) can be isolated from tumor tissue or peripheral blood. Known techniques for isolating human T cells include, for example, magnetic selection using magnetic beads for cell separation, flow cytometry using a cell sorter and antibodies against cell surface markers such as CD4 or CD8, and activated T cell induction methods using anti-CD3 and anti-CD28 antibodies. Furthermore, desired T cells can be isolated using cytokine secretion, expression of functional molecules, or signal molecules such as PD-1 as indicators. Cytotoxic T cells (CTLs) can also be isolated using secretion or production of granzymes or perforins as indicators. Furthermore, when isolating T cells having antigen specificity from human tissues containing such cells, T cells having the desired antigen specificity can be purified from human tissues using a multimer of MHC (major histocompatibility complex) bound to the desired antigen (e.g., "MHC tetramer" or "Pro5 (registered trademark) MHC class I pentamer").

[0017] In the present invention, the genes introduced to convert T cells into iPS cells are preferably a combination of at least four genes selected from the group consisting of (a) the Oct3 / 4 gene, (b) the c-Myc gene, (c) the Sox2 gene, (d) the Klf4 gene, (e) the NANOG gene, and (f) the LIN28 gene.

[0018] In the present invention, the method for introducing the gene group into T cells is not particularly limited, and any known method can be appropriately selected and used. For example, when the gene group is introduced into T cells in the form of nucleic acids encoding the gene group, the nucleic acid (e.g., cDNA, RNA) encoding the gene group can be 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, liposome method, electroporation, calcium phosphate co-precipitation, DEAE-dextran method, microinjection, or electroporation.

[0019] Among these expression vectors, it is more preferable to use a stealth RNA expression vector containing the above-mentioned gene group, in terms of reducing the risk of canceration and introducing efficiency. Stealth RNA expression vectors are designed to avoid chromosomal incorporation and to achieve sustained and stable gene expression in the cytoplasm rather than the nucleus. They can be used to introduce large genes of 13,000 base pairs or more, or even 10 genes at a time, without harming cells. They can be removed when the introduced gene is no longer needed, and their stealth properties mean that cells cannot recognize the vector as a foreign body. Such stealth RNA expression vectors include a complex that does not activate innate immune structures and consists of a minus single-stranded RNA (A) containing the RNA sequences (1) to (8) below, a single-stranded RNA-binding protein (B), and an RNA-dependent RNA polymerase. (1) RNA sequences for the gene group; (2) a human mRNA-derived RNA sequence that constitutes a non-coding region; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA synthetase; (4) a transcription termination signal sequence recognized by the RNA-dependent RNA synthetase; (5) an RNA sequence containing a replication origin recognized by the RNA-dependent RNA synthetase; (6) an RNA sequence encoding the RNA-dependent RNA synthetase; (7) an RNA sequence encoding a protein that regulates the activity of the RNA-dependent RNA synthetase; (8) An RNA sequence encoding the single-stranded RNA-binding protein.

[0020] Furthermore, when establishing T-iPS cells, the T cells are preferably stimulated and activated with anti-CD3 and anti-CD28 antibodies in the presence of interleukin-2 (IL-2) or interleukin-7 (IL-7) and interleukin-15 (IL-15) before the introduction of the gene cluster. Alternatively, the T cells may be stimulated and activated with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 and anti-CD28 antibodies, and CD3 and CD28 agonists. Such stimulation can be carried out, for example, by adding PHA, IL-2, anti-CD3 and / or anti-CD28 antibodies to a medium and culturing the T cells for a certain period of time. Furthermore, the anti-CD3 and anti-CD28 antibodies may be bound to magnetic beads or the like. Furthermore, instead of adding these antibodies to the medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish to which anti-CD3 and anti-CD28 antibodies are bound. Furthermore, the T cells (for example, human T cells) may be stimulated by adding an antigen peptide recognized by the T cells to the medium together with the feeder cells.

[0021] To provide such stimulation to the T cells, the concentration of PHA added to the medium is not particularly limited, but is preferably 1 to 100 μg / mL. The concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / mL. The concentrations of anti-CD3 antibody and anti-CD28 antibody added to the medium are not particularly limited, but are preferably 1 to 10 times the culture volume of the T cells. To provide such stimulation to the T cells, the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of the culture dish are not particularly limited, but the concentrations at the time of coating are preferably 0.1 to 100 μg / mL, preferably 1 to 100 μg / mL, for anti-CD3 antibody and 0.1 to 10 μg / mL for anti-CD28 antibody.

[0022] The culture period for such stimulation is not particularly limited, as long as it is a period sufficient to provide such stimulation to the T cells and to allow the T cells to proliferate to the number of cells required for the introduction of the four genes, but is usually 2 to 7 days, and from the viewpoint of gene transfer efficiency, is preferably 3 to 5 days. Infection is preferably carried out by mixing the T cells with the vector in a 15 mL tube, or, from the viewpoint of increasing gene transfer efficiency, culture is preferably carried out on a culture dish coated with Retronectin.

[0023] Examples of the medium to culture the T cells and to which PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, etc. are added include known media suitable for culturing the T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, AIM V medium, etc., which contain other cytokines and human serum). TM The medium may contain, in addition to PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin) necessary for culture. It is also preferable to add IL-7 and IL-15 to the medium instead of IL-2. The concentrations of IL-7 and IL-15 added are not particularly limited, but are preferably 1 to 100 ng / mL each.

[0024] Furthermore, there are no particular limitations on the conditions for or after introducing the four genes into the T cells. However, it is preferable to culture the T cells into which the four genes have been introduced under feeder-free conditions. Examples include wells coated with iMatrix-511 solution, which is a laminin 511E8 fragment, or vitronectin. T cells can also be established by culturing under feeder cell conditions. Examples of feeder cells include mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

[0025] Furthermore, during the process of inducing T cells to T-iPS cells, it is preferable to add iPS cell medium from the next day, and then replace the medium by half every other day, gradually replacing the T cell medium with iPS medium.

[0026] Furthermore, it is preferable to culture the iPS cells while gradually replacing a known medium suitable for culturing the T cells with a medium suitable for culturing iPS cells as the T cells transition from the iPS cells to the iPS cells. A known medium can be appropriately selected and used as the medium suitable for culturing iPS cells. For example, StemFit AK03N is preferable for iMatrix-coated iPS cells, or Essential 8 Medium is preferable for vitronectin-coated iPS cells. For feeder cells such as MEF cells, Dulbecco's modified Eagle's medium / F12 medium (human iPS cell culture medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like is preferable.

[0027] In this way, T-iPS cells can be selected by appropriately selecting known techniques. Examples of such known techniques include selection by observing the morphology of ES cell / iPS cell-like colonies under a microscope. On the other hand, in the case of T-iPS cells established from single-cell CTL clones, their properties are often similar, so an alternative method is to simply passage all established colonies without selecting each T-iPS cell colony.

[0028] The identity of the cells selected in this manner as T-iPS cells can be confirmed by, for example, detecting the expression of undifferentiated cell-specific markers (ALP, SSEA-4, Tra-1-60, Tra-1-81, etc.) in the selected cells by immunostaining, RT-PCR, or by transplanting the selected cells into mice and observing the formation of teratomas. Furthermore, the identity of the cells selected in this manner as T cells can be confirmed by detecting the state of TCR gene rearrangement by genomic PCR.

[0029] The time to select and recover these cells is preferably 10 to 40 days, preferably 14 to 28 days, after the gene group containing the four genes is introduced into the T cells. Unless otherwise specified above, the culture environment is preferably 5% CO2, 35 to 38°C, more preferably 37°C.

[0030] Next, Tax antigen-specific CTL cells are induced to differentiate from the established T-iPS cells. The preferred method for inducing redifferentiation is to differentiate T-iPS cells into CD8+ single-positive T cells, and more preferably to differentiate T-iPS cells into CD4 / CD8 double-negative T cells and then differentiate the CD4 / CD8 double-negative T cells into CD8+ single-positive T cells. Furthermore, as described in Patent Document 1, it is preferable to obtain these cells by differentiating T-iPS cells into CD4 / CD8 double-negative cells, stimulating the CD4 / CD8 double-negative cells by adding a substance that stimulates the T cell receptor, and then differentiating the CD4 / CD8 double-negative cells whose T cell receptors have been stimulated into CD8 single-positive T cells in the presence of the cytokines IL-7 and IL-15.

[0031] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, it is preferable to culture T-iPS cells on feeder cells (preferably mouse stromal cells) in a medium containing cytokines, serum (e.g., fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine, α-monothioglycerol, ascorbic acid, etc. The stromal cells used are preferably OP9 cells or 10T1 / 2 cells (C3H10T1 / 2 cells) that have been treated with radiation, etc. 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 culture media include X-VIVO medium, Iscove's Modified Dulbecco's Medium (IMDM medium), α-MEM, and DMEM. IMDM medium is preferred because it facilitates the formation of T-iPS sacs (sac-like structures containing hematopoietic progenitor cells). The T-iPS cell culture period is preferably 8 to 14 days, more preferably 10 to 14 days, from the start of T-iPS cell culture. The culture environment is not particularly limited, but is preferably 5% CO2 at 35 to 38°C, more preferably 37°C. It is more preferable to culture the cells under low oxygen concentration conditions (oxygen concentration: e.g., 5 to 20%) for about a week.

[0032] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, the cells contained in the T-iPS sac are preferably cultured on feeder cells (preferably stromal cells, more preferably human stromal cells) in a medium containing cytokines and serum (e.g., FBS), etc., using cytokine-coated wells under feeder-free conditions. Cells present inside the T-iPS sac can be separated, for example, by passing them through a sterilized sieve-like device (e.g., a cell strainer). The stromal cells used for this culture are preferably irradiated OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2 / DL4 cells, or 10T1 / 2 / DL1 cells, from the viewpoint of inducing differentiation into T lymphocytes via Notch signaling. Examples of cytokines added to the medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15. Examples of media include α-MEM, DMEM, and IMDM, with α-MEM being preferred. In addition to IL-7 and FLT3L, the media may also contain amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin) necessary for culture.

[0033] The culture period for the cells contained in the T-iPS sac is preferably the period until the CD4 / CD8 double-negative cells differentiated in this manner begin to express T cell receptors (TCRs) on their surface, and is preferably 14 to 28 days from the start of culture of the cells contained in the T-iPS sac. The culture environment is not particularly limited, but is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0034] Whether or not T cell receptors (TCRs) are expressed on the cell surface of CD4 / CD8 double-negative cells can be evaluated by flow cytometry using anti-TCRαβ antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.

[0035] In the method for producing antigen-specific human CD8 single-positive cells, further rearrangement of the TCR gene can be suppressed by stimulating T-iPS cell-derived CD4 / CD8 double-negative cells via the TCR expressed on the cell surface, thereby extremely increasing the frequency of T cells with the same TCR gene rearrangement pattern as the original human T cell among the CD8 single-positive cells obtained by redifferentiation.

[0036] A preferred method for stimulating the T cell receptors of T-iPS cell-derived CD4 / CD8 double-negative cells is to contact the T-iPS cell-derived CD4 / CD8 double-negative cells with at least one substance selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, an antigenic peptide that specifically binds to the human T cells from which the T-iPS cells were derived, cells expressing an HLA complex that restricts the T cell receptor, and an MHC multimer to which the antigenic peptide is bound.From the perspective of providing physiological stimulation, contact with cells expressing a specific peptide / HLA complex is more preferred.Furthermore, from the perspective of emphasizing uniformity of stimulation, contact with an antibody or reagent is more preferred.

[0037] The contact can be carried out, for example, by adding PHA or the like to the medium and culturing the T cells for a certain period of time. The anti-CD3 antibody and anti-CD28 antibody may be bound to magnetic beads or the like. Instead of adding these antibodies to the medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish with anti-CD3 antibody and anti-CD28 antibody bound to its surface. Furthermore, stimulation may also be achieved by adding the antigen peptide to the medium together with feeder cells.

[0038] 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. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the medium are preferably 1 to 10 times the culture volume of the T cells. Furthermore, to stimulate the TCR of CD4 / CD8 double-negative cells, the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of a culture dish at the time of coating are preferably 0.1 to 100 μg / ml for anti-CD3 antibody and 0.1 to 10 μg / ml for anti-CD28 antibody.

[0039] The culture period for the cells contained in the T-iPS sac preferably includes the period required for the expression of T cell receptors (TCRs) on the cell surface of the CD4 / CD8 double-negative cells obtained by differentiation in this manner, and is preferably 7 to 29 days from the start of culture of the cells contained in the T-iPS sac. The culture environment is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0040] In the present invention, in order to differentiate CD4 / CD8 double-negative cells whose T cell receptors have been stimulated into CD8 single-positive cells, the CD4 / CD8 double-negative cells are preferably cultured in a medium containing cytokines, serum (e.g., human serum), and the like. The cytokines added to the medium may be any cytokine capable of differentiating CD4 / CD8 double-negative cells into CD8 single-positive cells, such as IL-7 and IL-15. Among these, adding IL-7 and IL-15 in combination is preferred, from the viewpoint of selecting the CD8 lineage and facilitating the generation of memory CD8 T cells during differentiation into CD8 single-positive cells. The concentrations of IL-7 and IL-15 added are preferably 1 to 20 ng / ml. Examples of media include RPMI-1640 medium, X-VIVO medium, DMEM medium, and α-MEM medium, with RPMI-1640 medium or X-VIVO medium being preferred. In addition to IL-7, IL-15, etc., the medium may also contain amino acids necessary for culture (e.g., L-glutamine), antibiotics (e.g., streptomycin, penicillin), and cytokines other than IL-7, IL-15, etc.

[0041] 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 (PBMCs). These PBMCs are preferably allogeneic to the CD4 / CD8 double-negative cells. From the viewpoint of continuously stimulating TCR and continuously suppressing further TCR rearrangement, it is more preferable to use peripheral blood mononuclear cells that present antigen peptides that specifically bind to the human T cells that are the source of the CD4 / CD8 double-negative cells.

[0042] The culture period for differentiating these CD4 / CD8 double-negative cells into CD8 single-positive cells is preferably 2 to 4 weeks, and the culture environment is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0043] The CD8 single-positive cells induced to differentiate in this manner can be confirmed to be derived from T-iPS cells and the T cells from which the T-iPS cells were derived, for example, by detecting the state of TCR gene rearrangement by genomic PCR.

[0044] Furthermore, the CD8 single-positive cells thus obtained can be isolated by appropriately selecting a known technique. Examples of such known techniques include flow cytometry using an antibody against the CD8 cell surface marker and a cell sorter. For example, in the case of CD8 single-positive cells, a purification method using an affinity column immobilized with an antigen recognized by the T cells from which the CD8 single-positive cells were derived, or a purification method using an MHC multimer (e.g., MHC tetramer) bound to the antigen can also be employed.

[0045] Furthermore, the CD8 single-positive cells obtained according to the present invention do not express PD-1, but express CCR7 along with CD27 and CD28, which are representative of the central memory T cell phenotype; their telomeres are also longer than those of the original T cells, and they have high self-renewal ability. Therefore, according to the present invention, it is possible to produce CD8 single-positive T cells that have the same TCR gene rearrangement pattern as the original T cells, but which do not express PD-1 and express CD27, CD28, and CCR7. Furthermore, T cells collected from humans differ from the obtained T cells in that they express PD-1 and have a low proportion of the immature memory phenotype.

[0046] To maintain the CD8 single-positive cells obtained in this manner, the cells may be stimulated every 1 to 2 weeks. Such stimulation may involve 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 to which the antigen is bound, feeder cells in an allogeneic relationship with the CD8 single-positive cells, and feeder cells in an autogeneic relationship with the CD8 single-positive cells.

[0047] To confirm whether the iPSC-derived T lymphocytes possessed Tax antigen-specific cytotoxic activity, we confirmed that they retained the same antigen specificity as the original peripheral blood-derived CTLs using MHC pentamers, MHC tetramers, etc. Furthermore, TCR sequence analysis identified TCRαβ.

[0048] Tax antigen-specific cytotoxic T lymphocytes can also be produced by genetic recombination using a gene encoding the TCR of the present invention, for example, by introducing a gene encoding the TCR of the present invention into host T cells and selecting cells with Tax antigen-specific cytotoxicity. To introduce a gene encoding a TCR into host T cells, it is preferable to incorporate the gene into various viral vectors. Cells having Tax antigen-specific cytotoxicity can be selected by confirming the Tax antigen-specific cytotoxic activity described above, or by detecting the ability to produce cytokines such as IFN-γ.

[0049] The obtained Tax antigen-specific cytotoxic T lymphocytes have excellent Tax antigen-specific cytotoxic activity and are therefore useful as therapeutic agents for diseases such as adult T-cell leukemia / lymphoma, HTLV-1-associated myelopathy, and HTLV-1 uveitis (HTLV-1 uveitis: HU), particularly as therapeutic compositions for adult T-cell leukemia / lymphoma.

[0050] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the T lymphocytes of the present invention. Examples of such carriers include physiological saline and Ringer's solution. Furthermore, the pharmaceutical composition of the present invention may contain known pharmaceutically acceptable additives such as preservatives and coloring agents, as necessary. The pharmaceutical composition of the present invention is preferably in the form of an injection, and is preferably an injection for T cell infusion therapy. [Example]

[0051] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0052] Example 1 T-iPS cells were established from Tax-specific CTL clones using Sendai virus vectors. 1) Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors, and dendritic cells were induced for antigen presentation. After 7 days, Tax antigen peptides (Tax11-19, A0201) were added to the induced dendritic cells, and co-culture with peripheral blood mononuclear cells was initiated. Approximately 8-10 days later, to detect Tax-specific CTLs, CTLs were stained with MHC tetramers and the tetramer positivity rate was confirmed by flow cytometry. After confirming Tax-specific CTLs, single cell sorting or tetramer / PE bead selection followed by limiting dilution was performed.

[0053] 2) After approximately 3-6 weeks, colonies that had formed were stained with tetramers, and the establishment of CTL clones was confirmed by flow cytometry. After confirming establishment, the CTL clones were stimulated with CD3 / 28 and then transfected with the two vectors listed in A). The transfected CTLs were transferred to a 6-well plate coated with iMatrix, and culture was initiated in a CO2 incubator using CTL medium.

[0054] A) SeV4 vector + SV40 large T antigen

[0055] 3) The day after SeV gene transfection, an equal volume of iPS medium (StemFitAK03N) was added, and thereafter half the volume was replaced with StemFitAK03N every other day. 4) After 7 days, T-iPS cell colonies were observed, which were then picked and expanded. The iPSC-derived rejuvenated Tax-CTLs (Tax-rejT) were then induced to differentiate. 5) Cytotoxicity tests showed that the CTLs exhibited stronger antigen-specific cytotoxic activity against tumor cells than the original peripheral blood CTLs.

[0056] Figure 1 shows the results of tetramer staining of Tax-CTLs induced from peripheral blood, the established Tax-specific CTL clones, and iPSC-derived Tax-CTLs (Tax-rejT).

[0057] Example 2 (Cytotoxicity test) 1) To compare the cytotoxicity of Tax-rejT and peripheral blood-derived Tax-CTL against ATL tumor cells, we performed a 51Cr release assay. Tax-rejT or peripheral blood-derived Tax-CTLs were co-cultured with chromium-labeled patient-derived (autologous) ATL cells as effectors and HLA-mismatched EB virus-infected small cell lung cancer (LCL) as control targets at effector:target ratios of 20:1, 10:1, 5:1, and 2.5:1 for 6 hours. 2) After co-cultivation, the culture supernatant was transferred to another counter plate, dried, and then measured using a plate reader. 3) Tax-rejT showed strong antigen-specific cytotoxicity against ATL cells (60-70%), but showed no cytotoxicity (less than 10%) against control HLA-mismatched tumor cell lines. Peripheral blood-derived Tax-CTL showed cytotoxicity (approximately 15-20%) against ATL cells. They also showed no cytotoxicity (less than 10%) against control HLA-mismatched tumor cell lines. The Tax antigen-specific cytotoxic activity of Tax-rejT was stronger than that of Tax-CTL (Figure 2).

[0058] Example 3 The results of the analysis of the TCR sequence are shown in Figure 3.

Claims

1. A T cell receptor or antigen-binding fragment thereof for Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1, having an alpha chain V region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a beta chain V region consisting of the amino acid sequence shown in SEQ ID NO:

4.

2. The T cell receptor or antigen-binding fragment thereof according to claim 1, further comprising an α chain J region consisting of the amino acid sequence shown in SEQ ID NO: 2, and a β chain J region consisting of the amino acid sequence shown in SEQ ID NO:

5.

3. A therapeutic composition for adult T-cell leukemia / lymphoma, comprising Tax antigen-specific cytotoxic T lymphocytes having the T cell receptor or antigen-binding fragment thereof according to claim 1 or 2.

Images