JP7725014B2 - T-cell receptor of Tax antigen-specific cytotoxic T lymphocytes encoded by HTLV-1 or functional fragment thereof - Google Patents

Description

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

Adult T-cell leukemia/lymphoma (ATL) is caused by infection with the virus HTLV-1 (human T-lymphotropic virus type-I), which infects CD4+ T cells and develops when the infected T cells become cancerous and grow indefinitely (Non-Patent Document 1). HTLV-1 contains the gene for a viral protein called Tax, which has carcinogenic properties, and CTL therapy targeting this Tax antigen is expected to be an effective treatment.

National Institute of Infectious Diseases website

The objective of the present invention is to clone the T cell receptor of Tax antigen-specific cytotoxic T lymphocytes and provide a T cell receptor or a functional fragment thereof that has excellent antitumor effects.

Therefore, the present inventors induced Tax antigen-specific cytotoxic T lymphocytes from the patient's peripheral blood, performed single-cell cloning, and successfully established a Tax antigen-specific cytotoxic T lymphocyte (CTL) clone. The antitumor effect of the resulting clone against autologous tumor cells (ATL cells) was then confirmed. However, CTL clones have limited proliferation capacity, 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 the iPS cells. They succeeded in generating CTLs with a stronger antitumor effect than the original CTL clones.
The amino acid sequence of the T cell receptor (TCR) was clarified by repertoire analysis of the redifferentiation-induced CTLs, thereby completing the present invention.

That is, the present invention provides the following inventions [1] to [8].
[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 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 SEQ ID NO: 1 and a beta chain V region consisting of the amino acid sequence shown in SEQ ID NO: 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 described in [1] or [2], further having 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 according to any one of [1] to [4].
[6] Use of a Tax antigen-specific cytotoxic T lymphocyte having the T cell receptor or a functional fragment thereof according to 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 the 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, comprising administering a Tax antigen-specific cytotoxic T lymphocyte having the T cell receptor or a functional fragment thereof according to any one of [1] to [4].

Tax antigen-specific cytotoxic T lymphocytes having a T cell receptor for 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.

The antigen specificity of the obtained T lymphocytes to the Tax antigen is shown. Figure 1 shows the antigen-specific cytotoxic activity of the original Tax-specific CTL clones and iPSC-derived Tax-specific CTLs. AutoATL shows the effect on ATL, and HLA-mismatched LCL shows the effect on HLA-mismatched LCL cells as a control. The results of the obtained TCR repertoire analysis are shown below.

The present invention relates to a T cell receptor (TCR) or functional fragment thereof for Tax antigen-specific cytotoxic T lymphocytes 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.

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 such as Tax, Rex, gag, pol, and env. Of these, the full-length Tax protein has approximately 353 amino acid residues and has multiple functions in 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.

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 a TCR α chain protein (TCRα) and a TCR β chain protein (TCRβ), or a heterodimer of γ and δ chains, forms a TCR together with accessory molecules such as the CD3 complex (including γ, δ, ε, and ζ), CD4, or CD8.
TCRα and TCRβ have a variable region (V region + J region) and a constant region (C region). The V region of the variable region contains a complementarity-determining region (CDR). Therefore, TCRs can be characterized by the amino acid sequences of the V region (including the CDR region) or the V and J regions in TCRα and TCRβ.

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.

Preferably, the TCR of the present invention further comprises 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 preferably further comprises 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 preferably has 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.

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.

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, and then performing single cell cloning to establish a Tax antigen-specific cytotoxic T lymphocyte clone.
To induce Tax antigen-specific cytotoxic T lymphocytes from peripheral blood, either healthy individuals or individuals suffering from a viral infection may be used.
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. Examples also include T cells expressing CD3 and CD4, specifically CD4-positive T cells. The antigen specificity of T cells is conferred by an antigen-specific, rearranged TCR gene. From the viewpoint of production efficiency, although not particularly limited thereto, it is preferable to use antigen-specific CD8-positive T cells as the human T cells induced to become T-iPS cells in order 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. Furthermore, T cells that are not antigen-specific are also included as T cells from which T-iPS cells are induced. Specific examples include genetically modified T cells such as CAR T cells or TCR-T cells.

Such T cells can be isolated from, for example, human tissues using 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 diseased tissue. Among these, peripheral blood is preferred due to its low invasiveness and ease of preparation. 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. Desired T cells can also be isolated using cytokine secretion, expression of functional molecules, or signaling molecules such as PD-1 as indicators. Cytotoxic T cells (CTLs) can also be isolated using secretion or production of granzymes and perforins as indicators. Furthermore, when isolating T cells with antigen specificity from human tissues containing them, T cells with 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").

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 following: (a) Oct3/4 gene, (b) c-Myc gene, (c) Sox2 gene, (d) Klf4 gene, (e) NANOG gene, and (f) LIN28 gene.

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 introducing the gene group into T cells in the form of nucleic acid 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.

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 simultaneously. They do not harm cells, can be removed when the introduced gene is not needed, and have stealth properties that prevent cells from recognizing the vector as a foreign body.
Such stealth RNA expression vectors include complexes that do not activate innate immune systems and are composed of a minus single-stranded RNA (A) containing any of 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 constituting 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.

Furthermore, when establishing T-iPS cells, the T cells are preferably activated by stimulation with 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) before the introduction of the group of genes. Alternatively, the T cells may be activated by stimulation with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 antibody, anti-CD28 antibody, and CD3 and CD28 agonists. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody and/or anti-CD28 antibody to a 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, and 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 the surface. Furthermore, the T cells (e.g., human T cells) may be stimulated by adding an antigen peptide recognized by the T cells (e.g., human T cells) to the medium together with feeder cells.

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

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. It is usually 2 to 7 days, but from the viewpoint of gene transfer efficiency, it 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, it is preferable to culture the cells on a culture dish coated with Retronectin.

As a medium for culturing the T cells and adding PHA, IL-2, anti-CD3 antibody, and/or anti-CD28 antibody, etc., there can be used, for example, a known medium suitable for culturing the T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, AIM V ^™ medium, or NS-A2 containing other cytokines and human serum). In addition to PHA, IL-2, anti-CD3 antibody, and/or anti-CD28 antibody, the medium may also contain amino acids necessary for culturing (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin). 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, respectively.

Furthermore, there are no particular limitations on the conditions for or after introducing the four genes into the T cells, but 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 (MEFs), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

Furthermore, during the process of inducing T cells into T-iPS cells, it is preferable to add iPS cell culture medium from the next day. After that, it is preferable to gradually replace the T cell culture medium with iPS culture medium by replacing half of the medium every other day.

It is also preferable to gradually replace the known medium suitable for culturing T cells with a medium suitable for culturing iPS cells as the T cells transition to 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 cells, or Essential 8 Medium is preferable for vitronectin-coated cells. For feeder cells such as MEF cells, Dulbecco's modified Eagle's medium/F12 medium (human iPS cell medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like is preferable.

T-iPS cells can be selected in this way by appropriately selecting a known method. One such known method is to select cells 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 one method is to simply passage all established colonies without selecting each T-iPS cell colony.

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

The time to select and recover these cells is preferably 10 to 40 days, preferably 14 to 28 days, after the introduction of the gene group containing the four genes into the T cells. Unless otherwise specified above, the culture environment is preferably 5% CO ₂ and 35 to 38°C, more preferably 37°C.

Next, Tax antigen-specific CTL cells are induced to differentiate from the established T-iPS cells.
The method for inducing redifferentiation is preferably a method in which T-iPS cells are differentiated into CD8+ single-positive T cells, and more preferably a method in which T-iPS cells are differentiated into CD4/CD8 double-negative T cells and then the CD4/CD8 double-negative T cells are differentiated into CD8+ single-positive T cells.
Furthermore, as described in Patent Document 1, it is preferable to obtain the T-iPS cells by differentiating them 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 cytokines IL-7 and IL-15.

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, and the like.
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, with IMDM medium being preferred because it facilitates the formation of T-iPS sacs (sac-like structures containing hematopoietic progenitor cells). The culture period for these T-iPS cells 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% CO ₂ and 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.

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, 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). From the perspective of inducing differentiation into T lymphocytes via notch signaling, the stromal cells used for this culture are preferably OP9-DL1 cells, OP9-DL4 cells, 10T1/2/DL4 cells, or 10T1/2/DL1 cells that have been treated with radiation or other treatments. 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.

The culture period for the cells contained in the T-iPS sac is preferably the period until the CD4/CD8 double-negative cells obtained by differentiation in this manner begin to express T cell receptors (TCRs) on their cell surfaces, 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.

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.

In a method for producing antigen-specific human CD8 single-positive cells, further TCRA gene rearrangement can be suppressed by stimulating T-iPS cell-derived CD4/CD8 double-negative cells via the TCR expressed on their cell surface. This in turn significantly increases the frequency of T cells with the same TCR gene rearrangement pattern as the original human T cells among the CD8 single-positive cells obtained by redifferentiation.

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 a complex with HLA that restricts the T cell receptor, and an MHC multimer to which the antigenic peptide is bound. From the perspective of providing physiological stimulation, a more preferred method is to contact the cells with specific peptide/HLA complex-expressing cells. Furthermore, from the perspective of emphasizing uniformity of stimulation, a more preferred method is to contact the cells with an antibody or reagent.

The contact can be achieved, 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 also 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 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.

To stimulate the TCR of CD4/CD8 double-negative cells, the concentration of PHA added to the culture medium is preferably 1 to 100 μg/ml. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the culture 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 the 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.

The culture period for the cells contained in the T-iPS sac preferably includes the period required for the expression of T cell receptors (TCR) 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.

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, and examples thereof include 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.

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. Furthermore, from the perspective 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.

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% CO ₂ and 35 to 38°C, more preferably 37°C.

Confirmation that the CD8 single-positive cells induced to differentiate in this way are derived from T-iPS cells and from the T cells that gave rise to those T-iPS cells can be made, for example, by detecting the state of TCR gene rearrangement using genomic PCR.

The CD8 single-positive cells obtained in this manner can be isolated using an appropriate known method. Examples of such known methods 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, purification methods using an affinity column immobilized with an antigen recognized by the T cells from which the CD8 single-positive cells were derived, or purification methods using an MHC multimer (e.g., MHC tetramer) bound to the antigen can also be employed.

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. Furthermore, their telomeres are longer than those of the original T cells, and they have a high self-renewal capacity. 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 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.

To maintain the CD8 single-positive cells obtained in this manner, the cells may be stimulated every one to two 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 CD8 SP 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.

To confirm whether the obtained iPSC-derived T lymphocytes possess Tax antigen-specific cytotoxic activity, MHC pentamers, MHC tetramers, etc. were used to confirm that they retained the same antigen specificity as the original peripheral blood-derived CTLs. Furthermore, TCR sequence analysis was performed to identify TCRαβ.

Alternatively, Tax antigen-specific cytotoxic T lymphocytes can 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 having 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 may be selected by confirming the Tax antigen-specific cytotoxic activity described above, or by detecting the ability to produce cytokines such as IFN-γ.

The resulting 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.

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.

The present invention will now be described in more detail using examples, but the present invention is not limited to these examples.

Example 1
Establishment of T-iPS cells from Tax-specific CTL clones using Sendai virus vectors.
1) Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy individuals, and dendritic cells were then induced for antigen presentation. Seven days later, Tax antigen peptide (Tax11-19, A0201) was added to the induced dendritic cells, and co-culture with peripheral blood mononuclear cells was initiated. Approximately 8 to 10 days later, to detect Tax-specific CTLs, the 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 was performed followed by limiting dilution.

2) After approximately 3 to 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) below. The transfected CTLs were transferred to a 6-well plate coated with iMatrix, and culture was initiated in a _CO2 incubator using CTL medium.

A) SeV4 vector + SV40 large T antigen

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, colonies of T-iPS cells were observed, which were then picked and expanded. The iPSC-derived rejuvenated Tax-CTL (Tax-rejT) were then induced to differentiate.
5) When a cytotoxicity test was performed, it was found that the cells exhibited stronger antigen-specific cytotoxic activity against tumor cells than the original peripheral blood CTLs.

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

Example 2
(Cytotoxicity test)
1) To compare the cytotoxic activity of Tax-rejT and peripheral blood-derived Tax-CTL against ATL tumor cells, a 51Cr release assay was performed. Tax-rejT or peripheral blood-derived Tax-CTL were co-cultured for 6 hours with chromium-labeled patient-derived (autologous) ATL cells as effectors and an HLA-mismatched EB virus-infected tumor cell line (LCL) as control targets at effector:target ratios of 20:1, 10:1, 5:1, and 2.5:1.
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 (<10%) against control HLA-mismatched tumor cell lines. Peripheral blood-derived Tax-CTL showed cytotoxicity of approximately 15-20% against ATL cells. They also showed no cytotoxicity (<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).

Example 3
The results of the analysis of the TCR sequence are shown in FIG.

Claims (3)

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. 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. 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.