Method for producing antigen-specific T cells

Abstract

To provide production methods that extremely increase the frequency of appearance of T cells having the same TCR gene rearrangement pattern as the original human T cells, in CD8 single positive cells or CD4 single positive cells obtained by redifferentiation from human T cells having antigen specificity via iPS cells.SOLUTION: Provided is a method for producing human CD8 single positive cells or CD4 single positive cells having antigen specificity, the method comprising the steps of: differentiating iPS cells derived from human T cells into CD4 / CD8 double negative cells; stimulating the T cell receptor of the CD4 / CD8 double negative cells; and differentiating the CD4 / CD8 double negative cells in which the T cell receptor has been stimulated into CD8 single positive cells or CD4 single positive cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing T cells having antigen specificity, more specifically, to a method for producing human CD8 single positive cells and / or CD4 single positive cells having antigen specificity, comprising the steps of differentiating iPS cells induced from human T cells into CD4 / CD8 double negative cells, stimulating the T cell receptors of the CD4 / CD8 double negative cells, and differentiating the CD4 / CD8 double negative cells whose T cell receptors have been stimulated into CD8 single positive cells and / or CD4 single positive cells. The present invention also relates to human CD8 single positive cells or CD4 single positive cells having antigen specificity produced by the method. [Background technology]

[0002] T cells play a central role in the induction of acquired immunity and systemic immunity against pathogens. In particular, cytotoxic T cells (CTLs) recognize antigen peptides derived from viruses, tumors, etc., presented together with class 1 major histocompatibility complexes (MHC class 1, HLA class 1) on antigen-presenting cells via T cell receptors (TCRs) present on the cell surface, and specifically exert cytotoxic activity against cells presenting the antigen peptides, which are foreign substances, and attack them. Furthermore, some CTLs become long-lived memory T cells, which are stored in the host while maintaining their cytotoxicity against foreign substances, and are able to respond when exposed to the same foreign substance again. Therefore, CTLs function as a key element in the immune system that fights against microorganisms, viral infections, and neoplasms (tumors) (Non-Patent Documents 1 and 2).

[0003] CTLs are cells differentiated from CD8 single positive (SP) cells. It is known that CD8SP cells differentiate in the thymus from immature cells that do not express TCR and have neither CD4 nor CD8 (CD4 / CD8 double negative (DN) cells), via cells that express both CD8 and CD4 (CD4 / CD8 double positive (DP) cells).

[0004] Furthermore, when T cells such as CTLs specifically recognize antigen peptides via TCR under HLA restriction, they begin to exert their proliferation and effector functions. Thus, the greatest advantage of T cell immunity is the specific recognition of target cells and their eradication, in addition to long-term immune surveillance by long-lived memory T cells (Non-Patent Documents 3 to 5).

[0005] However, T cell immunity is often hindered by insufficient recognition of antigens associated with latent viral infections or cancer / self-antigens: persistent exposure to latent viral infections or cancer / self-antigens impairs the long-term survival, proliferation and effector functions of T cells, leading to severe T cell exhaustion and ultimately to a loss of the antigen-responsive T cell pool (Non-Patent Documents 6 and 7).

[0006] Adoptive immunotherapy using antigen-specific T cells expressing a specific TCR repertoire has been attracting attention as a promising treatment for various types of cancer and chronic viral infections. However, in vitro expansion of antigen-specific peripheral T cells causes a similar loss of T cell function during the extracorporeal treatment of patient-derived samples, and thus, even when a large amount of ex vivo expanded T cells are used, sufficient therapeutic efficacy has not been demonstrated so far (Non-Patent Document 8).

[0007] In addition, approaches to confer desired antigen specificity to non-specific T cells for such diseases are being developed (Non-Patent Documents 9 and 10). The therapeutic effect of T cells should depend largely on the antigen specificity of the induced and expanded T cells (Non-Patent Documents 11 and 12). In particular, it has been revealed that when TCRs are exogenously introduced to induce hematopoietic stem cells or peripheral mature T cells into antigen-specific T cells, mismatched TCRs usually occur.

[0008] A method for inducing antigen-specific immature T cells from pluripotent stem cells has also been developed (Non-Patent Document 13). However, even when hematopoietic stem / progenitor cells are used, a method for differentiating them into sufficiently functional human T cells has not yet been established, and it is expected that there will be many difficulties in putting this method into practical use.

[0009] Thus, there is a strong demand for the development of a method for producing T cells, particularly CTL (CD8 single positive) cells, that have antigen specificity and high proliferation ability (self-replication ability). Although various trial and error efforts have been carried out, no effective method has yet been provided.

[0010] In view of this situation, the present inventors have explored the possibility of induced pluripotent stem cell (iPS cell) technology that allows unlimited self-replication while maintaining pluripotency, and have succeeded in establishing iPS cells (hereinafter also referred to as "T-iPS cells") from human T cells in which TCR gene rearrangement has been completed and which have antigen specificity. They have also succeeded in inducing differentiation of the T-iPS cells into T cells, and have further demonstrated that this method can be used to obtain T cells with the same gene rearrangement pattern as the original T cells (i.e., T cells that show specificity for the antigen recognized by the original human T cells) (Patent Document 1). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2011 / 096482 [Non-patent literature]

[0012] [Non-Patent Document 1] Greenberg, P.D., Adv Immunol, 1991, 49, 281-355 [Non-Patent Document 2] Zhang, N. & Bevan, M. J. Immunity, 2011, vol. 35, pp. 161-168. [Non-Patent Document 3] Jameson, S.C. & Masopust, D., Immunity, 2009, vol. 31, pp. 859-871. [Non-Patent Document 4] MacLeod, M.K. et al., Immunology, 2010, vol. 130, pp. 10-15 [Non-Patent Document 5] Butler, NS et al., Cell Microbiol, 2011, vol. 13, pp. 925-933 [Non-Patent Document 6] Klebanoff, CA et al., Immunol Rev, 2006, 211, 214-224 [Non-Patent Document 7] Wherry, E. J., Nat Immunol, 2011, 12, 492-499. [Non-Patent Document 8] June, C.H., J Clin Invest, 2007, vol. 117, pp. 1466-1476 [Non-Patent Document 9] Morgan, RA et al., Science, 2006, 314, 126-129 [Non-Patent Document 10] Porter, DL, N Engl J Med, 2011, 365, 725-733 [Non-Patent Document 11] Bendle, GM et al., Nat Med, 2010, vol. 16, pp. 565-570 [Non-Patent Document 12] Topalian, S.L. et al., J Clin Oncol, 2011, vol. 29, pp. 4828-4836 [Non-Patent Document 13] Timmermans, F. et al., J Immunol, 2009, 182, 6879-6888 Summary of the Invention [Problem to be solved by the invention]

[0013] As mentioned above, the present inventors have developed a method for establishing T-iPS cells from human T cells having antigen specificity and then inducing differentiation of the T-iPS cells into T cells. It has also been demonstrated that this method can produce T cells that have the same gene rearrangement pattern as the original T cells (Patent Document 1).

[0014] However, as shown in Comparative Example 1 described below, it was newly discovered that the frequency of T cells having the same gene rearrangement pattern as the original T cells among the obtained T cells was about one-quarter.

[0015] Therefore, the present invention aims to significantly increase the frequency of T cells that have the same TCR gene rearrangement pattern as the original human T cell (i.e., T cells that exhibit specificity for the antigen recognized by the original human T cell) among T cells obtained by redifferentiation from antigen-specific human T cells via T-iPS cells. [Means for solving the problem]

[0016] As mentioned above, it is known that during the maturation process of T cells in the thymus, TCR is not usually expressed in T cells at the most immature stage (CD4 / CD8DN stage) that do not express CD4 or CD8. Therefore, the present inventors first examined the expression of TCR during the process of redifferentiating T-iPS cells into T cells.

[0017] As a result, it was surprisingly found that in the process of redifferentiating T-iPS cells into T cells, TCR was expressed even at the CD4 / CD8DN stage, which is different from the conventional knowledge regarding the maturation process of T cells in the thymus (see Example 1 below).

[0018] In the so-called "positive selection" process in the thymus, TCR signals mediated by antigen peptide-MHC complexes are known to silence RAG gene expression and suppress further assembly of TCR genes. Furthermore, it has been shown that TCR signal-like signals generated by stimulation with anti-CD3 antibodies have a similar effect.

[0019] Therefore, the present inventors next examined whether it is possible to suppress further assembly of TCR genes and increase the frequency of T cells having the same gene rearrangement pattern as the original T cells by stimulating the TCR expressed in CD4 / CD8DN cells redifferentiated from T-iPS cells. Furthermore, it was also examined whether it is possible to differentiate such stimulated CD4 / CD8DN cells into CD8SP cells by applying a molecule that contributes to CD8 lineage selection to the cells (see Example 1 described below).

[0020] As a result, they found that by stimulating the TCR expressed in T-iPS cell-derived CD4 / CD8DN cells with anti-CD3 antibodies, etc., and further treating such stimulated CD4 / CD8DN cells with molecules such as IL-7 that contribute to CD8 lineage selection, it is possible to significantly increase the frequency of appearance of CD8SP cells that have the same TCR gene rearrangement pattern as the original human T cells. Furthermore, they found that the CD8SP cells produced in this way have longer telomeres than the original T cells and have high proliferative potential (replicative ability). They also found that by stimulating the TCR expressed in T-iPS cell-derived CD4 / CD8DN cells with anti-CD3 antibodies, etc., and further treating such stimulated CD4 / CD8DN cells with molecules such as IL-7 that contribute to CD8 lineage selection, it is possible to significantly increase the frequency of appearance of CD8SP cells that have the same TCR gene rearrangement pattern as the original human T cells. It was also possible to obtain CD4SP cells by applying molecules such as IL-7 to given CD4 / CD8DN cells.

[0021] The present invention has been made based on the above findings, and more specifically provides a method for producing human CD8 single positive cells having antigen specificity, comprising the steps of differentiating iPS cells induced from human T cells into CD4 / CD8 double negative cells, stimulating the TCR of the CD4 / CD8 double negative cells, and differentiating the CD4 / CD8 double negative cells whose TCR has been stimulated into single positive T cells, specifically, CD4 single positive cells or CD8 single positive cells.

[0022] The present invention also provides single positive T cells having antigen specificity, specifically CD4 single positive cells and human CD8 single positive cells, produced by the above method.

[0023] That is, the present invention provides the following inventions. (1) A method for producing human single positive T cells having antigen specificity, comprising the steps of: Differentiating iPS cells derived from human T cells into CD4 / CD8 double negative cells; Stimulating the T cell receptors of the CD4 / CD8 double negative cells; and differentiating the CD4 / CD8 double negative cells that have been stimulated via a T cell receptor into single positive T cells. (2) The method according to (1) above, wherein the single positive T cells are CD8 single positive cells. (3) The method according to (1) or (2) above, wherein the human T cells have antigen specificity. (4) A human single positive T cell having antigen specificity, produced by the method according to any one of (1) to (3) above. (5) A pharmaceutical composition comprising the human single positive T cell described in (4) above.

[0024] According to the present invention, it is possible to extremely increase the frequency of T cells that have the same TCR gene rearrangement pattern as the original human T cell among T cells obtained by redifferentiating antigen-specific human T cells via T-iPS cells. [Brief description of the drawings]

[0025] [Figure 1] This is a diagram showing an outline of a method for detecting rearrangement of the TCRA gene. That is, 34 Vα primers were designed to amplify all configurations of the Vα segment in the human TCRA locus, which spans more than 1000 kb on chromosome 14q11.2. Furthermore, 12 Jα primers were designed downstream of each sequence of the Jα1, Jα6, Jα10, Jα17, Jα23, Jα29, Jα32, Jα36, Jα41, Jα48, Jα53, or Jα58 segment so as to amplify 3 to 7 different Jα segments. Then, all the Jα primers were mixed in one tube, and the Jα primer mix was used together with each Vα primer to perform 34 PCR reactions for one sample, thereby detecting rearrangement of the TCRA gene. This is a schematic diagram showing this. [Diagram 2] This is a schematic diagram showing the process of generating T cell-derived iPS cells (T-iPS cells) from peripheral blood T cells using retroviral vectors encoding OCT3 / 4, SOX2, or KLF4. In the figure, the tapered region (7 to 11 days after the start of T cell activation) indicates the period during which the medium was gradually replaced with human iPS medium. [Diagram 3] This is a schematic diagram showing the process of generating T-iPS cells from a CTL clone (H25-4T cells) using SeV encoding OCT3 / 4, SOX2, and KLF4 in a polycistronic arrangement, and SeV encoding SV40LTAg. In the figure, the tapered region (9 to 12 days after the start of T cell activation) indicates the period during which the medium was gradually replaced with human iPS medium. [Figure 4]Photographs showing the results of microscopic observation of alkaline phosphatase (AP) activity and expression of pluripotency markers (SSEA-4, Tra-1-60, and Tra-1-81) in T-iPS cells (H254SeVT-3) generated from H25-4T cells. The scale bar in the figure indicates 200 μm, and in b to d, the results of counterstaining the nuclei with DAPI (blue glowing areas in the figure) are shown. [Diagram 5] These are photographs showing the results of microscopic observation of AP activity and expression of pluripotency markers (SSEA-4, Tra-1-60, and Tra-1-81) in iPS cells (TkT3V1-7) generated from peripheral blood T cells. In the figure, the scale bar in (a) indicates 500 μm. In (b) to (e), the scale bar in (b) indicates 200 μm, and the results of counterstaining the nuclei with DAPI (blue glowing areas in the figure) are shown. [Figure 6] This is a photograph showing the results of RT-PCR analysis of the expression of pluripotency genes in TkT3V1-7. In the figure, "Tg" indicates the expression of genes exogenously introduced by retrovirus, "endo" indicates the expression of genes endogenously expressed, and "total" indicates the expression of genes exogenously introduced by retrovirus and genes endogenously expressed. "PB CD3+" indicates the results of peripheral blood T cells from which TkT3V1-7 was derived, "KhES3" indicates the results of ES cells, which are pluripotent stem cells (positive control), and "Water" indicates the results of RT-PCR performed without adding template DNA (negative control). In this RT-PCR, GAPDH was used as an internal standard. [Figure 7]FIG. 1 is a photograph showing the results of RT-PCR detection of remaining SeV RNAs (four cistronic expressed factors (tetracistronic factors: OCT4, SOX2, KLF4, and c-MYC) and T antigen). In the figure, "SeVp(KOSM302L)" and "SeV18+SV40 / TS15ΔF" respectively show the results for SeV used to establish H254SeVT-3, and "Water" shows the results of RT-PCR performed without adding template DNA (negative control). "na" indicates "not assessed." In this RT-PCR, GAPDH was used as an internal standard. [Figure 8] This is a scatter plot showing the results of a comprehensive analysis of gene expression by cDNA microarray. That is, the left panel shows the results of a comprehensive comparison of gene expression between CD4+ T cells (PB CD4+ T-cell) and TkT3V1-7 by cDNA microarray. The right panel shows the results of a comprehensive comparison of gene expression between ES cells (KhES3) and TkT3V1-7 by cDNA microarray. The two parallel lines in the figure indicate a 5-fold difference between the corresponding samples. [Figure 9] This is a graph showing the results of quantitative PCR analysis of the expression of pluripotency genes in H25-4, KhES3, TkT3V1-7, and H254SeVT-3. In each PCR reaction, the expression level of 18SrRNA was used as an internal standard. In the figure, the horizontal axis shows, from the left, the results for H25-4, KhES3, TkT3V1-7, and H254SeVT-3. The vertical axis shows the relative expression level when the expression level of each gene in KhES3 is set to 1.0. In addition, "ND" indicates "not detected." [Figure 10]1 shows the results of bisulfite sequencing of the promoter regions of OCT3 / 4 and NANOG in H25-4 and H254SeVT-3, in which open and closed circles represent unmethylated and methylated CpG dinucleotides, respectively, and "%Me" represents the methylation rate in each region. [Figure 11] Figure 1 shows the results of bisulfite sequencing of the OCT3 / 4 and NANOG promoters of PB CD3+ and TkT3V1-7. In the figure, open and closed circles represent unmethylated and methylated CpG dinucleotides, respectively, and "%Me" represents the methylation rate in each region. [Figure 12] This is a micrograph showing a representative result obtained by observing a section prepared from a TkT3V1-7-derived teratoma formed in the testis of a NOG mouse and staining the section with HE. That is, this is a micrograph showing that the teratoma contains anatomical structures derived from three germ layers. Note that glands / ducts and intestinal-like mucosa are observed as anatomical structures derived from the endoderm, cartilage and striated muscle are observed as anatomical structures derived from the mesoderm, and neural plate and pigment epithelium are observed as anatomical structures derived from the ectoderm. In the figure, the scale bar indicates 100 μm. [Figure 13] This is a micrograph showing a representative result obtained by observing a section prepared from H254SeVT-3 cells formed in the testis of a NOG mouse and staining the section with HE. That is, this is a micrograph showing that H254SeVT-3 cells were differentiated into a cell lineage derived from endoderm (goblet cells in gut-like epithelium), a cell lineage derived from mesoderm (smooth muscle cells in muscle tissue), and a cell lineage derived from ectoderm (retinal cells in pigment epithelium). In the figure, the scale bar indicates 100 μm. [Figure 14] Photographs showing the results of detecting TCRB gene rearrangement in the TkT3V1-7 genome by multiplex PCR analysis, in which tubes A and B show Vβ-(D)Jβ assembly, and tube C shows D-Jβ assembly. [Figure 15]13 is a photograph showing the results of detecting TCRA gene rearrangement (V-Jα assembly) in the TkT3V1-7 genome by multiplex PCR analysis. [Figure 16] Photographs showing the results of detecting TCRB gene rearrangement in the H254SeVT-3 genome by multiplex PCR analysis, in which tubes A and B show Vβ-(D)Jβ assembly, and tube C shows D-Jβ assembly. [Figure 17] 13 shows photographs depicting the results of detection of TCRA gene rearrangement (V-Jα assembly) in the H254SeVT-3 genome by multiplex PCR analysis. [Figure 18] This is a dot plot diagram showing the results of flow cytometry analysis of the developmental progression of TkT3V1-7-derived CD34+ hematopoietic stem / progenitor cells to human T lineage cells. To examine the hematopoietic differentiation potential of T-iPS cells, TkT3V1-7 was cultured on C3H10T1 / 2 feeder cells with VEGF, SCF, and FLT-3L. Then, on the 14th day, the CD34+ hematopoietic stem / progenitor cells (T-iPS sac cells) derived from TkT3V1-7 thus generated were transferred onto OP9 (OP9-DL1) feeder cells expressing delta-like molecule 1. The cells in the process of redifferentiating on OP9-DL1 cells were then collected, and the expression of CD34, CD38, CD7, CD5, CD1a, CD45RA, and CD45 on the cell surface of TkT3V1-7-derived cells was analyzed by flow cytometry every week for 22 to 36 days ("1wks" to "3wks" in the figure) after redifferentiation induction. This is a dot plot diagram showing the results. [Figure 19]This is a dot plot diagram showing the results of flow cytometric analysis of the developmental process from H254SeVT-3-derived T-iPS sac cells to human T lineage cells. That is, to examine the hematopoietic differentiation ability of T-iPS cells, H254SeVT-3 was cultured on C3H10T1 / 2 feeder cells together with VEGF, SCF, and FLT-3L. Then, on the 14th day, the TkT3V1-7-derived T-iPS sac cells thus generated were transferred onto OP9-DL1 feeder cells. Then, the cells undergoing redifferentiation on the OP9-DL1 cells were collected, and the expression of CD34, CD38, CD7, CD5, CD1a, CD45RA, and CD45 on the cell surface of H254SeVT-3-derived cells was analyzed weekly for 15 to 36 days ("0wks" to "3wks" in the diagram) after redifferentiation induction. This is a dot plot diagram showing the results of flow cytometric analysis of the expression of CD34, CD38, CD7, CD5, CD1a, CD45RA, and CD45 on the cell surface of H254SeVT-3-derived cells. [Figure 20] This is a dot plot diagram showing the results of flow cytometric analysis of the presence of CD4 / 8DP cells derived from T-iPS cells established in vitro. In the figure, a shows the results of flow cytometric analysis of the expression of CD45, CD56, CD3, TCRαβ, CD4, and CD8 on the cell surface of TkT3V1-7-derived cells 35 to 42 days after redifferentiation induction. b shows the results of flow cytometric analysis of the expression of CD45, CD56, CD3, TCRαβ, CD4, and CD8 on the cell surface of H254SeVT-3-derived cells 35 to 42 days after redifferentiation induction. The results shown in this figure are representative results of experiments performed at least three times independently. [Figure 21] This is a photograph showing the results of RT-PCR amplification of the TCRA and TCRB genes expressed in the DN cell-derived cDNA library synthesized by the SMART method or the DP cell-derived cDNA library synthesized by the SMART method. In the figure, "Water" indicates the result of RT-PCR performed without adding template DNA (negative control). GAPDH was used as an internal standard for each PCR. [Figure 22]Photographs showing the results of RT-PCR analysis of RAG1 and RAG2 expression in DN and DP cells. In the figure, "Water" indicates the results of RT-PCR performed without adding template DNA (negative control). Samples used as templates for PCR were standardized based on GAPDH expression. [Figure 23] This is a dot plot diagram showing the results of flow cytometry analysis of floating cells collected on OP9-DL1 cells on day 42 after the start of redifferentiation induction. DN cells and DP cells were isolated from cells divided into CD45+, CD56-, CD3+, TCRαβ+, CD2+, and CD5+ by gating in advance on CD1a- and CD1a+, respectively. [Figure 24] 1 is a schematic diagram showing a method for producing human CD8 single positive (SP) cells having antigen specificity of the present invention, i.e., a schematic diagram showing the process of redifferentiation from T-iPS cells to human CD8 SP cells. [Diagram 25] This is a dot plot diagram showing the results of flow cytometry analysis of the phenotype of cells obtained by redifferentiating T-iPS cells by the method of the present invention (cells 50-60 days after the start of redifferentiation induction, as shown in FIG. 24). The diagram shows representative results from at least three independent experiments, in which a and b show the results of analyzing the phenotype as T cells, and c and d show the results of analyzing the phenotype as memory cells. In addition, uniform expression of HLA-A24 was observed in b, indicating that there was no contamination with CD3+ / CD8+ cells from PBMCs. [Figure 26] FIG. 25 is a dot plot diagram showing the results of flow cytometry analysis of cells obtained by redifferentiating T-iPS cells using the method of the present invention (cells 50 to 60 days after the start of redifferentiation induction, as shown in FIG. 24) using A24 / Nef-138-8(wt) tetramer (upper panel), and the results of culturing tetramer-positive cells sorted by FACS or magnetic selection for 14 days or more and reanalyzing T cells amplified by this culture using the tetramer. [Figure 27]This is a photograph showing the results of PCR detection of TCR mRNA from cDNA libraries of CD8SP cells (reT-1, reT-2.1, and reT-3) that react with A24 / Nef-138-8 (wt) tetramer, which were synthesized by the SMART method. In the figure, "Water" indicates the results of RT-PCR performed without adding template DNA (negative control). GAPDH was used as an internal standard for each PCR. [Figure 28] This is a graph showing the results of comparing the expression levels of major cell surface molecules between CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells using quantitative PCR. In each PCR reaction, the expression level of 18SrRNA was used as an internal standard. In the figure, the vertical axis shows the expression ratio when the expression level of each gene in CD8+ cells is set to 1.0. The horizontal axis shows the results for CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells, from the left. [Figure 29] This is a graph showing the results of comparing the expression levels of major cytolytic molecules between CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells using quantitative PCR. In each PCR reaction, the expression level of 18SrRNA was used as an internal standard. In the figure, the vertical axis shows the expression ratio when the expression level of each gene in CD8+ cells is set to 1.0. The horizontal axis shows the results for CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells, from the left. [Diagram 30] This is a graph showing the results of comparing the expression levels of major transcription factors and signaling molecules between CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells using quantitative PCR. In each PCR reaction, the expression level of 18SrRNA was used as an internal standard. In the figure, the vertical axis shows the expression ratio when the expression level of each gene in CD8+ cells is set to 1.0. The horizontal axis shows the results for CD4+ cells, CD8+ cells, reT-2.1 cells, and H25-4 cells, from the left. [Diagram 31] This is a heat map showing correlation coefficients between samples obtained by comprehensively analyzing gene expression using cDNA microarrays. [Diagram 32]This is a heat map showing genes with a 3-fold or greater difference in expression level compared to NK cells, obtained by comprehensive gene expression analysis using cDNA microarrays. In the figure, the areas shown in red and green indicate increased and decreased expression, respectively. [Diagram 33] 1 is a graph showing the expansion rate of each cell type two weeks after stimulation of reT-1, reT-2.2, and reT-3 with PHA, IL-7, and IL-15. The vertical axis shows the expansion ratio when the number of each cell type upon stimulation is set to 100. [Diagram 34] This is a dot plot diagram showing the results of flow cytometry analysis of the expression of CD45RA, CCR7, CD27, and CD28 on the cell surface of cells (reT-2.1 and reT-3) purified and expanded with A24 / Nef-138-8(wt) tetramer. In this figure, the data of reT-2.1 are shown as representative results. [Diagram 35] FIG. 1 is a graph showing relative telomere length (RTL) determined by flow-FISH. [Diagram 36]1 is a histogram showing the results of analyzing the secretion of cytolytic molecules in reT-2.1 cells stimulated with α-CD3 / CD28 beads. The left panel is a histogram showing the amount of intracellular production of Granzyme B. The white histogram area surrounded by a gray line shows the reactivity of unstimulated cells with anti-Granzyme B antibody, the white histogram area surrounded by a black line shows the reactivity of the stimulated cells with anti-Granzyme B antibody, and the filled histogram area shows the reactivity of the stimulated cells with an antibody used as a negative control (an antibody of the same isotype as the anti-Granzyme B antibody). The right panel is a histogram showing the results of a CD107a mobilization assay in reT-2.1 cells stimulated with α-CD3 / CD28 beads. The white histogram area surrounded by gray lines shows the reactivity of unstimulated cells with anti-CD107a antibody, the white histogram area surrounded by black lines shows the reactivity of the stimulated cells with anti-CD107a antibody, and the filled histogram area shows the reactivity of the stimulated cells with an antibody used as a negative control (an antibody of the same isotype as the anti-CD107a antibody). [Figure 37] 1 is a graph showing the results of ELISPOT analysis of IFN-γ produced in response to stimulation with Nef-138-8 (wt). The graph shows a representative result of at least three independent experiments. The vertical axis in the graph shows the number of spots formed in 500 cells. [Figure 38] Graph showing the results of 51Cr release assay performed using various concentrations of epitope peptides such as Nef-138-8 (wt). The 51Cr release assay was performed by reacting effector cells with target cells loaded with 51Cr at a ratio of 5:1. The vertical axis shows the specific release rate (%) of 51Cr. [Figure 39] FIG. 1 is a schematic diagram of a method for producing human CD4SP cells with antigen specificity from T-iPS cells. [Diagram 40]This is a dot plot diagram showing the results of flow cytometric analysis of the phenotype of cells obtained by redifferentiating T-iPS cells, specifically, the cells 50 to 60 days after the start of redifferentiation induction in the schematic diagram of Figure 39. Figure 40 shows the results of analysis of the phenotype of the obtained T cells using various cell surface markers and flow cytometric analysis using the A24 / Nef-138-8(wt) tetramer, and is a representative result of at least three independent experimental results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] <Method for producing human CD8SP cells or CD4SP cells having antigen specificity> The present invention provides a method for producing human CD8SP cells and / or CD4SP cells having antigen specificity, comprising the steps of differentiating iPS cells induced from human T cells (hereinafter also referred to as "T-iPS cells") into CD4 / CD8 double negative (DN) cells, stimulating the T cell receptors of the CD4 / CD8DN cells, and differentiating the CD4 / CD8DN cells whose T cell receptors have been stimulated into single positive T cells, specifically CD8SP cells and / or CD4SP cells.

[0027] In the present invention, the "human" from which T cells are isolated is not particularly limited, and may be a healthy individual, or a person with a weakened immune function, or a person suffering from a malignant tumor, an infectious disease such as a chronic infectious disease, an autoimmune disease, etc. When human CD8SP cells or CD4SP cells obtained by the present invention are used in immune cell therapy, from the viewpoint of preventing rejection reactions, it is preferable that the human from which T cells are isolated has the same HLA type as the human to which the T cells obtained by the present invention are administered, and it is more preferable that the human is the same person to which the T cells obtained by the present invention are administered.

[0028] In the present invention, "T cells" refers to cells expressing an antigen receptor called T cell receptor (TCR) on the surface, and their precursor cells (pro-T cells not expressing TCR (TCRαβ), pre-T cells in which TCRβ and pre-TCRα are associated, etc.). Furthermore, "CD4 / CD8 double negative (DN) cells" refers to T cells expressing neither CD4 nor CD8, "CD4 / CD8 double positive (DP) cells" refers to T cells expressing both CD4 and CD8, and "CD8 single positive (SP) cells" refers to T cells expressing only CD8 out of CD4 and CD8. Furthermore, "CD4 single positive (SP) cells" refers to T cells expressing only CD4 out of CD4 and CD8.

[0029] In the present invention, the "human T cells induced to iPS cells" are T cells expressing CD3 and CD8, for example, cytotoxic T cells that are CD8 positive cells. The "human T cells induced to iPS cells" also include T cells expressing CD3 and CD4, for example, T cells that are CD4 positive cells. Specific examples of such human T cells include helper / regulatory T cells that are CD4 positive cells, cytotoxic T cells (CTLs) that are CD8 positive cells, naive T cells (CD45RA+CD62L+ cells), central memory T cells (CD45RA-CD62L+ cells), effector memory T cells (CD45RA-CD62L- cells), and terminal effector T cells (CD45RA+CD62L- cells). The antigen specificity of T cells is provided by antigen-specific, rearranged TCR genes. From the viewpoint of production efficiency, although not particularly limited thereto, in order to obtain antigen-specific CD8SP cells, it is preferable to use antigen-specific CD8+ T cells as the human T cells induced into iPS cells, and in order to obtain antigen-specific CD4SP cells, it is preferable to use antigen-specific CD4+ T cells as the human T cells induced into iPS cells. Furthermore, when performing the immunotherapy described below, 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 into iPS cells.

[0030] In the present invention, "human T cells to be induced into iPS cells" can be isolated from human tissues by known techniques. There are no particular limitations on the human tissues as long as they contain the T cells, and examples of such tissues include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and diseased tissues. Among these, peripheral blood and umbilical cord blood are preferred from the viewpoint of low invasiveness to humans and easy preparation. Known methods for isolating human T cells include, for example, flow cytometry using antibodies against cell surface markers such as CD4 or CD8 and a cell sorter, as shown in the Examples below. In addition, it is also possible to isolate desired T cells using the secretion of cytokines and the expression of functional molecules as indicators. In such cases, for example, T cells secrete different cytokines depending on whether they are Th1 type or Th2 type, so that T cells having a desired Th type can be isolated by selecting using such cytokines as an indicator. In addition, it is possible to isolate cytotoxic T cells (CTLs) using the secretion or production of granzymes, perforins, etc. as an indicator. Furthermore, when isolating from human tissues containing "T cells having desired antigen specificity", a method of purification using an affinity column or the like on which a desired antigen is immobilized can be adopted. Furthermore, "T cells having a desired antigen specificity" can be purified from human tissues using a polymerized MHC (major histocompatibility complex) bound to a desired antigen (e.g., "MHC tetramer" or "Pro5 (registered trademark) MHC class I pentamer").

[0031] In the present invention, the term "iPS cells" refers to cells also called induced pluripotent stem cells or induced pluripotent stem cells, and can be induced by introducing a cellular reprogramming factor into the T cells. The "cellular reprogramming factor" is not particularly limited as long as it is a factor that can confer pluripotency to the somatic cells alone or in cooperation with other pluripotency factors by being introduced into the T cells, but is preferably at least one protein selected from the group consisting of Oct3 / 4, c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, ERas, ECAT7, ECAT8, Gdf3, Sox15, ECAT15-1, ECAT15-2, Fthl17, Sal14, Rex1, Utf1, Tcl1, Stella, β-catenin, Stat3, and Grb2. Furthermore, among these proteins, from the viewpoint of efficiently establishing iPS cells with fewer factors, it is more preferable to introduce Oct3 / 4, c-Myc, Sox2, and Klf4 (four factors) into the T cells. As shown in International Publication No. 2011 / 096482, from the viewpoint of further increasing the efficiency of inducing iPS cells from human CD8-positive T cells or CD4-positive T cells, it is more preferable to introduce OCT4, SOX2, KLF4, C-MYC, and NANOG into human CD8-positive T cells or CD4-positive T cells, and it is particularly preferable to introduce OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28 into human CD8-positive T cells or CD4-positive T cells. Furthermore, from the viewpoint of reducing the risk of canceration of the obtained pluripotent stem cells, it is more preferable to introduce Oct3 / 4, Sox2, and Klf4 (three factors) excluding c-Myc into the T cells.

[0032] In the present invention, the method for establishing iPS cells induced from human T cells (i.e., the method for introducing a cell reprogramming factor into T cells) is not particularly limited, and a known method can be appropriately selected and used. For example, when the cell reprogramming factor is introduced into the T cells in the form of a protein, the method using a protein introduction reagent, the method using a protein introduction domain (PTD) fusion protein, the electroporation method, and the microinjection method can be mentioned. In addition, when the cell reprogramming factor is introduced into the T cells in the form of a nucleic acid encoding the cell reprogramming factor, the nucleic acid (e.g., cDNA) encoding the cell reprogramming factor can be inserted into an appropriate expression vector containing a promoter that functions in T cells, and the expression vector can be introduced into the cells by infection, lipofection, liposome, electroporation, calcium phosphate coprecipitation, DEAE dextran, microinjection, or electroporation.

[0033] Examples of such expression vectors include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus. Examples of suitable plasmids include a plasmid for expressing a cellular reprogramming factor or an animal cell expression plasmid. However, from the viewpoints that insertional mutagenesis is unlikely to occur, gene transfer efficiency is high, and the number of copies of the transferred gene is large, it is preferable to use a Sendai virus to transfer a nucleic acid encoding the cellular reprogramming factor into the T cells.

[0034] Examples of promoters used in such expression vectors include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, etc. Furthermore, such promoters may be capable of controlling the expression of a gene inserted downstream of the promoter depending on the presence or absence of a drug (e.g., tetracycline). The expression vector may further contain, in addition to the promoter, an enhancer, a polyA addition signal, a selection marker gene (e.g., neomycin resistance gene), an SV40 replication origin, etc.

[0035] In addition, when establishing iPS cells induced from human T cells, as shown in the Examples below, the T cells are preferably stimulated and activated with anti-CD3 antibody and anti-CD28 antibody in the presence of interleukin-2 (IL-2) before the introduction of the cell reprogramming factor, and may be stimulated and activated with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), allogeneic antigen-expressing cells, anti-CD3 antibody, and anti-CD28 antibody. Such stimulation can be performed, for example, as shown in the Examples below, 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. In addition, the anti-CD3 antibody and anti-CD28 antibody may be bound to magnetic beads, and further, 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 antibody and anti-CD28 antibody are bound on the surface. 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.

[0036] In order to provide such a stimulus to the T cells, the concentration of PHA added to the medium is not particularly limited, but is preferably 1 to 100 μg / ml. In addition, the concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / ml. Furthermore, 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. In order to provide such a stimulus 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 concentration at the time of coating 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.

[0037] 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 allows the T cells to proliferate to the number of cells required for the introduction of the cellular reprogramming factor, but is usually 1 to 14 days, may be 1 to 6 days, and is preferably 2 to 7 days, more preferably 2 to 4 days from the viewpoint of gene transfer efficiency. Furthermore, from the viewpoint of increasing gene transfer efficiency, it is preferable to culture the cells on a culture dish coated with Retronectin.

[0038] As a medium for culturing the T cells and adding PHA, IL-2, anti-CD3 antibody, and / or anti-CD28 antibody, etc., a known medium suitable for culturing the T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, minimum essential medium (α-MEM), Dulbecco's modified Eagle medium (DMEM), F12 medium, etc., containing other cytokines and human serum) can be used. In addition to PHA, IL-2, anti-CD3 antibody, and / or anti-CD28 antibody, the medium may also contain amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin, penicillin) necessary for culturing. In addition, apoE may be added to the medium. From the viewpoint of suppressing ptosis, it is preferable to add IL-7 and IL-15 to the medium. There are no particular limitations on the concentrations of IL-7 and IL-15 added, but it is preferable that each be added at 1 to 100 ng / ml.

[0039] In addition, although there are no particular limitations on the conditions when or after introducing a cellular reprogramming factor into the T cells, it is preferable to culture the T cells into which the cellular reprogramming factor has been introduced on a feeder cell layer. There are no particular limitations on such feeder cells, but examples thereof include mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

[0040] Furthermore, from the viewpoint of suppressing cell differentiation during the process of inducing the T cells into iPS cells, it is preferable to add basic fibroblast growth factor (bFGF) to the culture medium when introducing the cell reprogramming factor into the T cells or thereafter.

[0041] Furthermore, in order to further increase the efficiency of establishing iPS cells, it is preferable to add a histone deacetylase (HDAC) inhibitor (small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, M344, etc., siRNA against HDAC, etc.), a G9a histone methyltransferase inhibitor (small molecule inhibitors such as BIX-01294, etc., siRNA against G9a, etc.), or a p53 inhibitor (small molecule inhibitors such as pifithrin-α (PFT-α), siRNA against p53, etc.) to the culture medium when introducing the cellular reprogramming factor into the T cells or thereafter.

[0042] Furthermore, when a viral vector is used to introduce a cellular reprogramming factor into T cells, it is preferable to add protamine sulfate to the culture medium, since this makes it easier for the vector to bind to the T cells.

[0043] As shown in the Examples below, it is preferable to gradually replace a 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 such iPS cells, and examples thereof include 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, etc.

[0044] As shown in WO 2011 / 096482, when human CD8-positive T cells or CD4-positive T cells are induced to iPS cells, from the viewpoint of further increasing the efficiency of induction into iPS cells, it is preferable to culture the cells after transfer onto the feeder cells under low oxygen concentration conditions (oxygen concentration: for example, 5%) in a medium further containing 1 to 1000 μM ROCK inhibitor, and it is also preferable to add 1 to 1000 μM MEK inhibitor (for example, PD0325901) and 1 to 1000 μM GSK3 inhibitor (for example, CHIR99021) to the medium until colony formation described below.

[0045] The iPS cells induced from the T cells in this way (T-iPS cells) can be selected by appropriately selecting a known method, such as a method of selecting by observing the morphology of ES cell / iPS cell-like colonies under a microscope as shown in the Examples below, or a method of selecting using drug resistance or reporter activity as an index, using recombinant T cells in which a drug resistance gene or a reporter gene (such as the GFP gene) is targeted to the locus of a gene known to be specifically expressed in iPS cells (for example, the cellular reprogramming factor).

[0046] It can be confirmed that the cells selected in this manner are iPS cells (i.e., T-iPS cells) 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, etc., as shown in the Examples below, or by transplanting the selected cells into mice and observing the formation of teratomas. It can also be confirmed that the cells selected in this manner are derived from the T cells by, for example, detecting the state of TCR gene rearrangement by genomic PCR, as shown in the Examples below.

[0047] The timing of selecting and recovering these cells can be appropriately determined while observing the growth state of the colonies, and is generally 10 to 40 days, preferably 14 to 28 days, after the cell reprogramming factor is introduced into the T cells. The culture environment is preferably 5% CO2, 35 to 38°C, more preferably 37°C, unless otherwise specified above.

[0048] -The process of differentiating T-iPS cells into CD4 / CD8DN cells- In order to differentiate iPS cells induced from human T cells (i.e., T-iPS cells) into CD4 / CD8DN cells, from the viewpoint of facilitating induction of differentiation into mesodermal lineage, T-iPS cells are first cultured on feeder cells (preferably stromal cells, more preferably human stromal cells) in a medium that does not necessarily contain cytokines, but preferably contains cytokines, serum (e.g., fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine, α-monothioglycerol, ascorbic acid, etc. From the viewpoint of facilitating induction of differentiation into hematopoietic lineage, the stromal cells used are preferably OP9 cells or 10T1 / 2 cells (C3H10T1 / 2 cells) that have been treated with irradiation or the like. From the viewpoint of efficiently inducing T-iPS cells into CD34 hematopoietic progenitor cells, the cytokine added to the medium is preferably at least one cytokine selected from the group consisting of VEGF, SCF, TPO, SCF, 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, and from the viewpoint of high efficiency in forming a sac-like structure (also called "T-iPS sac" or "ES sac") containing hematopoietic progenitor cells, IMDM medium is preferred. The culture period of the T-iPS cells is preferably the period until the T-iPS sac is formed, and is preferably 8 to 14 days, more preferably 10 to 14 days, from the start of the culture of the T-iPS cells. The culture environment is not particularly limited, but is preferably 5% CO2, 35 to 38°C, and more preferably 37°C. Furthermore, from the viewpoints of high efficiency of T-iPS sac formation and a large number of blood cells contained in the T-iPS sac, it is more preferable to culture under low oxygen concentration conditions (oxygen concentration: for example, 5 to 20%).

[0049] In order to differentiate the T-iPS cells into CD4 / CD8DN cells, the cells contained in the T-iPS sac obtained above (hematopoietic progenitor cells, CD34 positive cells, blood cells, etc.) are then cultured in a medium containing cytokines, serum (e.g., FBS), etc., on feeder cells. It is preferable to culture the T-iPS sac on a sac (preferably stromal cells, more preferably human stromal cells). The cells present inside the T-iPS sac can be separated by physical means, for example, by passing through a sterilized sieve-like device (e.g., a cell strainer, etc.). From the viewpoint of inducing differentiation into T lymphocytes via the notch signal, the stromal cells used in this culture are preferably OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2 / DL4 cells, or 10T1 / 2 / DL1 cells that have been treated with irradiation or the like. Examples of cytokines to be added to the medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15. Although not particularly limited, any of SCF, TPO, FLT-3L, and IL-7 is preferable for differentiation into CD4SP cells. One or more of these, or all of them can be added in combination. Among these, IL-7 and FLT3L are preferred from the viewpoint of helping the differentiation of early T cells. From the viewpoint of suppressing the differentiation of T-iPS cells into NKT cells, it is preferred that SCF is not included in the medium. The concentration of IL-7 added to the medium is preferably 0.1 to 9.5 ng / ml, more preferably 1 to 4 ng / ml, from the viewpoint of facilitating the production of CD3-positive CD56-negative T lineage cells and facilitating the induction of differentiation into CD8SP cells or CD4SP cells. The concentration of FLT3L added to the medium is preferably 0.1 to 100 ng / ml from the viewpoint of enhancing the proliferation of blood cells. Examples of the medium include α-MEM medium, DMEM medium, and IMDM medium, and α-MEM medium is preferred from the viewpoint of facilitating the maintenance of stromal cells. In addition to IL-7 and FLT3L, the medium may contain amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin) necessary for the culture.

[0050] The culture period of the cells contained in the T-iPS sac is preferably the period until the T cell receptor (TCR) is expressed on the cell surface of the CD4 / CD8DN cells thus differentiated, and is preferably 14 to 28 days from the start of the culture of the cells contained in the T-iPS sac. There are no particular limitations on the culture environment, but the conditions are preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0051] In addition, whether or not T cell receptors (TCR) are expressed on the cell surface of CD4 / CD8DN cells can be evaluated by flow cytometry using anti-TCRαβ antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD8 antibodies, as shown in Comparative Example 1 described below (see Figure 20).

[0052] -A process for stimulating the T cell receptors of CD4 / CD8DN cells- As shown in Comparative Example 1 described later, the present inventors have revealed for the first time that in T cells redifferentiated from T-iPS cells, the expression of RAG1 and RAG2 is stronger at the CD4 / CD8DP stage than at the CD4 / CD8DN stage. Furthermore, the present inventors have revealed for the first time that in the TCRA mRNA of T cells redifferentiated from T-iPS cells, the DP stage has a higher frequency of having a rearrangement pattern of TCR genes different from that of the original human T cells than the DN stage, strongly suggesting that further assembly of TCRA genes (receptor modification) occurs by RAG1 and RAG2 in the process of redifferentiating T-iPS cells into T cells, particularly in the process from the CD4 / CD8DN stage to the CD4 / CD8DP stage.

[0053] In addition, as shown in Example 1 below, the present inventors have revealed for the first time that TCR is expressed even at the DN stage in T cells obtained by redifferentiating T-iPS cells. Normally, T cells do not express TCR at the DN stage in vivo. Therefore, it was surprising that TCR was expressed in DN cells obtained by differentiating T-iPS cells.

[0054] Furthermore, it has been known that in the process of positive selection, TCR signals mediated by peptide-MHC complexes stop the expression of RAG genes and suppress further assembly of TCR genes. It has also been revealed that TCR signal-like signals by anti-CD3 antibodies have a similar effect (i.e., the effect of suppressing further assembly of TCR genes). The present inventors have succeeded in suppressing further rearrangement of TCR genes by stimulating the TCR expressed in DN cells induced from T-iPS cells, thereby succeeding in increasing the frequency of appearance of T cells having the same TCR gene rearrangement pattern as the original human T cells.

[0055] Therefore, in the method of the present invention for producing antigen-specific human CD8SP cells and / or CD4SP cells, further rearrangement of the TCR gene can be suppressed by stimulating T-iPS cell-derived CD4 / CD8DN cells via the TCR expressed on the cell surface, thereby making it possible to extremely increase the frequency of T cells having the same TCR gene rearrangement pattern as the original human T cells in the CD8SP cells and / or CD4SP cells obtained by redifferentiation.

[0056] As a method for stimulating the T cell receptor of CD4 / CD8DN cells derived from T-iPS cells, a method of contacting the T-iPS cell-derived CD4 / CD8DN cells with at least one substance selected from the group consisting of PHA, anti-CD3 antibody, anti-CD28 antibody, an antigen peptide that specifically binds to the human T cells that are the source of the T-iPS cells, cells expressing a complex with HLA that shows restriction to the T cell receptor, an MHC multimer to which the antigen peptide is bound, PMA, and ionomycin is preferred, and from the viewpoint of providing a physiological stimulus, a method of contacting with cells expressing a specific peptide / HLA complex is more preferred. Also, from the viewpoint of emphasizing uniformity of stimulation, a method of contacting with an antibody or a reagent is more preferred.

[0057] The contact can be carried out, for example, by adding PHA or the like to a medium and culturing the T cells for a certain period of time, as shown in the Examples below. The anti-CD3 antibody and the 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 having anti-CD3 antibody and anti-CD28 antibody bound to the surface. Furthermore, the antigen peptide may be stimulated by adding the antigen peptide to the medium together with feeder cells.

[0058] The concentration of PHA added to the medium to stimulate the TCR of CD4 / CD8DN cells is not particularly limited, but is preferably 1 to 100 μg / ml. The concentrations of anti-CD3 and anti-CD28 antibodies added to the medium are not particularly limited, but are preferably 1 to 10 times the culture volume of the T cells. The concentrations of anti-CD3 and anti-CD28 antibodies bound to the surface of the culture dish to stimulate the TCR of CD4 / CD8DN cells are not particularly limited, but the concentrations at the time of coating are preferably 0.1 to 100 μg / ml for anti-CD3 antibodies and 0.1 to 10 μg / ml for anti-CD28 antibodies.

[0059] The culture period of the cells contained in the T-iPS sac preferably includes the period during which T cell receptors (TCR) are expressed on the cell surface of the CD4 / CD8DN cells thus differentiated, and is preferably 7 to 29 days from the start of culture of the cells contained in the T-iPS sac. There are no particular limitations on the culture environment, but the conditions are preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0060] During this culture period, CD8 T cells with self-renewal ability and pluripotency capable of differentiating into central memory T cells, effector memory T cells, and effector T cells were also cultured. + Memory stem cells (stem cell-like memory T cells (T SCMFrom the viewpoint of enhancing the production of T, an inhibitor against glycogen synthase kinase-3β (GSK3β) may be added to the medium. Such an inhibitor against GSK3β may be any one that can activate the Wnt signaling pathway by suppressing phosphorylation of β-catenin by GSK3β, and an example of such an inhibitor is a 4,6-disubstituted pyrrolopyrimidine compound (TWS119). SCM For more information, see Luca Gattinoni et al., Nature Medicine, 2011, vol. 17, pp. 1290-1298. SCM Regarding the relationship with the enhancement of the production of β-lactamase, see "Luca Gattinoni et al., Nature Medicine, 2009, Vol. 15, pp. 808-813" See “Ji.”

[0061] -A step of differentiating CD4 / CD8DN cells into CD8SP cells or CD4SP cells- As shown in the Examples below, it was revealed that by stimulating T-iPS cell-derived CD4 / CD8DN cells via the TCR expressed on the cell surface in the above process, further rearrangement (receptor modification) of the TCRA gene that may occur as the T-iPS cells differentiate from the DN stage to the DP stage and then to the SP stage (e.g., the CD8SP stage or CD4SP stage) can be suppressed.

[0062] Therefore, in the method of the present invention for producing human CD8SP cells or CD4SP cells having antigen specificity, T-iPS cell-derived CD4 / CD8DN cells are stimulated via the TCR expressed on the cell surface, and then the cells are differentiated into single positive T cells, specifically CD8SP cells or CD4SP cells, which makes it possible to extremely increase the frequency of T cells having the same TCR gene rearrangement pattern as the original human T cells among the CD8SP cells or CD4SP cells obtained by redifferentiation.

[0063] In the "step of stimulating the T cell receptor of CD4 / CD8DN cells", some T-iPS cell-derived T lineage cells differentiate not only into CD4 / CD8DN cells but also into CD4 / CD8DP cells. Therefore, the "step of differentiating CD4 / CD8DN cells whose T cell receptors have been stimulated into single-positive T cells" includes not only the step of differentiating CD4 / CD8DN cells whose T cell receptors have been stimulated into CD8SP cells or CD4SP cells, but also the differentiation of CD4 / CD8DP cells whose TCRs have been stimulated at the DN stage into CD8SP cells or CD4SP cells.

[0064] In the present invention, in order to differentiate CD4 / CD8DN cells into CD8SP cells or CD4SP cells after stimulating the T cell receptor, the CD4 / CD8DN 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 / CD8DN cells into CD8SP cells or CD4SP cells, and examples of such cytokines include IL-7, IL-15, and IL-2. Among these, the cytokines that are preferred for selecting the CD8 lineage and for differentiation into CD8SP cells are those that are preferred for selection of memory-type CD8 cells. + From the viewpoint of facilitating T cell generation, it is preferable to add IL-7 and IL-15 in combination. There is no particular limitation on the concentration of IL-7 and IL-15 added, but it is preferable to add IL-7 and IL-15 at a concentration of 1 to 20 ng / ml. Examples of the medium include RPMI-1640 medium, X-VIVO medium, DMEM medium, and α-MEM medium, and RPMI-1640 medium or X-VIVO medium is preferable from the viewpoint of being more suitable for the growth of blood cells. In addition to IL-7, IL-15, etc., the medium may contain amino acids (e.g., L-glutamine), antibiotics (e.g., streptomycin, penicillin), and cytokines other than IL-7, IL-15, etc., necessary for culture.

[0065] In such a culture, the CD4 / CD8DN cells may be co-cultured with feeder cells. There is no particular limitation on the feeder cells, but from the viewpoint of further promoting differentiation and proliferation into CD8SP cells or CD4SP cells through cell contact, etc., peripheral blood mononuclear cells (PBMCs) are preferable. From the viewpoint of effective stimulation of TCR, such PBMCs are preferably in an allogeneic (allogeneic) relationship with CD4 / CD8DN cells. Moreover, from the viewpoint of supporting survival while preventing excessive TCR stimulation, it is preferable that the PBMCs are in an auto (derived from the same person) relationship with CD4 / CD8DN cells. Moreover, from the viewpoint of continuing to stimulate TCR and continuing to suppress further TCR rearrangement, it is more preferable to use peripheral blood mononuclear cells that present an antigen peptide that is specifically bound by human T cells that are the origin of CD4 / CD8DN cells.

[0066] The culture period for differentiating the CD4 / CD8DN cells into CD8SP cells or CD4SP cells is preferably 2 to 4 weeks. The culture environment is not particularly limited, but is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0067] It has been pointed out in "Serwold, T. et al., Proc Natl Acad Sci USA, 107, 18939-18943. 2010, vol. 107, pp. 18939-18943" that abnormally early TCR signal transduction in the mouse thymus may cause lymphoma development. Therefore, in the method of producing human CD8SP cells or CD4SP cells of the present invention, a system using a suicide gene may be incorporated from the viewpoint of avoiding tumor development due to early (CD4 / CD8DN stage) TCR signal activation. Examples of such "systems using suicide genes" include a system that uses a gene encoding inducible caspase 9 (iCasp9) consisting of human caspase 9 and modified FK-binding protein, as described in "Antonio Di Stasi et al., N Engl J Med, 2011, vol. 365, pp. 1673-1683" and a system that uses a thymidine kinase (TK) gene as described in "Kaneko, S. et al., BLOOD, 2009, vol. 113, pp. 1006-1015," "Fabio Ciceri et al., THE LANCET, 2009, vol. 10, pp. 489-500," and "Attilio Bondanza et al., Blood, 2011, vol. 24, pp. 6469-6478," etc. Can be obtained.

[0068] It can be confirmed that the CD8SP cells or CD4SP cells induced to differentiate in this manner are derived from T-iPS cells and from the T cells that were the source of the T-iPS cells by, for example, detecting the state of TCR gene rearrangement by genomic PCR, as shown in the Examples described later.

[0069] The CD8SP cells or CD4SP cells thus obtained can be isolated by appropriately selecting a known method. Examples of such known methods include flow cytometry using an antibody against a cell surface marker such as CD8 or CD4 and a cell sorter, as shown in the Examples below. When isolating "T cells having a desired antigen specificity" from a human, a purification method using an affinity column or the like on which a desired antigen (for example, in the case of CD8SP cells, an antigen recognized by the T cells that became the origin of the CD8SP cells, and in the case of CD4SP cells, an antigen recognized by the T cells that became the origin of the CD4SP cells) is immobilized, or a method of purifying "T cells having a desired antigen specificity" using an MHC multimer (for example, an MHC tetramer) to which the antigen is bound can also be adopted.

[0070] Furthermore, the CD8SP cells obtained by the present invention do not express PD-1, but express CCR7 along with CD27 and CD28, which are representative of the phenotype of central memory T cells, and also have longer telomeres than the original T cells, and have high self-replication ability. Therefore, according to the present invention, it is possible to produce T cells (e.g., CD8SP cells) that have the same TCR gene rearrangement pattern as the original T cells, do not express PD-1, and express CD27, CD28, and CCR7. The T cells collected from humans are different from the obtained T cells in that they express PD-1 and do not express CD27, CD28, and CCR7. The same is thought to be true for CD4SP cells.

[0071] To maintain the CD8SP cells or CD4SP cells thus obtained, the cells may be stimulated every 1 to 2 weeks. Such stimulation may include, for CD8SP cells, 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, a feeder cell in an allo-relationship with the CD8SP cells, and a feeder cell in an auto-relationship with the CD8SP cells. Similarly, for CD4SP cells, such stimulation may be 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 CD4SP cells, an MHC multimer having the antigen bound thereto, a feeder cell in an allo-relationship with the CD4SP cells, and a feeder cell in an auto-relationship with the CD4SP cells.

[0072] <Human CD8SP cells or CD4SP cells, pharmaceutical compositions, and immune cell therapy methods> The human CD8SP cells or CD4SP cells produced by the method of the present invention have antigen-specific immune functions, as shown in the Examples below. Furthermore, in particular, CD8SP cells do not express PD-1, which is one of the markers of exhausted T cells (i.e., T cells with significantly weakened long-term survival, self-renewal and / or effector functions), but express CCR7 along with CD27 and CD28, which are representative of the phenotype of central memory T cells, and also have longer telomeres than the original T cells, giving them a high self-renewal ability. This is thought to be the same for CD4SP cells. Therefore, the human T cells produced by the method of the present invention are useful in treating or preventing diseases such as tumors, infectious diseases (e.g., chronic infectious diseases), and autoimmune disorders.

[0073] Thus, the present invention provides human T cells produced by the method of the present invention, a pharmaceutical composition comprising the human T cells, and a method of immune cell therapy (immune cell therapy or immunotherapy) using the human T cells. In particular, the present invention provides human CD8SP cells and / or human CD4SP cells produced by the method of the present invention, a pharmaceutical composition comprising the human CD8SP cells and / or human CD4SP cells, and a method of immune cell therapy using the human CD8SP cells and / or human CD4SP cells.

[0074] The immune cell therapy method of the present invention can be carried out, for example, as follows. First, T cells are collected from a human, preferably a human with a matching HLA type, more preferably from a subject to be treated. Next, T-iPS cells are produced from the T cells, and then the T-iPS cells are differentiated into T cells having the same TCR gene rearrangement pattern as the original T cells, preferably CD4SP cells or CD8SP cells. The obtained T cells express CCR7 together with CD27 and CD28, which are representative of the phenotype of central memory T cells, do not express PD-1, and have longer telomeres than the original T cells, and have high self-replication ability. Therefore, if desired, the expression of CD27, CD28, CCR7 and / or PD-1 may be confirmed for the obtained cells. The T cells thus obtained can be administered to a subject to be treated.

[0075] In the immune cell therapy method of the present invention, the administration of T cells to a subject is not particularly limited, but is preferably parenteral, for example, intravenous, intraperitoneal, subcutaneous or intramuscular, more preferably intravenous, or may be administered locally to the affected area.

[0076] The pharmaceutical composition of the present invention can be prepared by formulating the human CD8SP cells or CD4SP cells produced by the method of the present invention using a known pharmaceutical method, and can be administered primarily parenterally, for example, in the form of capsules, liquids, film coatings, suspensions, emulsions, injections (intravenous injections, drip injections, etc.).

[0077] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, solubilizing agent, or other additives. In addition, the composition may be used in combination with known pharmaceutical compositions or immunostimulants that are used for the treatment or prevention of the above-mentioned diseases.

[0078] When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected depending on the age, weight, symptoms, health condition, type of composition (drug, food or drink, etc.) of the subject, and the like.

[0079] The product (pharmaceutical product) of the composition of the present invention or its instructions may be labeled to the effect that it is used to treat or prevent a decrease in immune function. Here, "labeling on the product or instructions" means that the product itself, container, packaging, etc., is labeled, or that the instructions, package insert, advertising materials, other printed matter, etc., disclosing information about the product are labeled.

[0080] The immune cell therapy method of the present invention comprises the steps of isolating T cells having a desired antigen specificity from a human, inducing iPS cells from the T cells having the desired antigen specificity, differentiating the iPS cells into CD4 / CD8 double negative cells, stimulating the T cell receptors of the CD4 / CD8 double negative cells, differentiating the CD4 / CD8 double negative cells whose T cell receptors have been stimulated into CD8 single positive cells and / or CD4 single positive cells, and administering the obtained CD8 single positive cells and / or CD4 single positive cells into the human body.

[0081] When carrying out the immune cell therapy method of the present invention, from the viewpoint of preventing rejection, it is preferable that the human from whom the T cells are isolated has the same HLA type as the human to whom the CD8SP cells and / or CD4SP cells obtained by the present invention are administered, and it is more preferable that the human is the same person to whom the CD8SP cells and / or CD4SP cells obtained by the present invention are administered. The human CD8SP cells and / or CD4SP cells to be administered may be human T cells produced by the method of the present invention as they are, or may be administered in the form of a formulated pharmaceutical composition as described above. EXAMPLES

[0082] Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples. In the following examples and comparative examples, the following experimental methods were used unless otherwise specified.

[0083] <Preparation of CTL clones> A Nef138-8(wt)-specific CTL strain was established from PBMCs of an HLA-A24 type HIV-1-infected patient according to the description in "Kawana-Tachikawa, A. et al., J Virol, 2002, Vol. 76, pp. 11982-11988".

[0084] <Preparation of T-iPS cells> Human iPS cells were established from peripheral blood T cells or CTL clones under optimized T cell culture conditions as described in "Takayama, N. et al., J Exp Med, 2010, Vol. 207, pp. 2817-2830".

[0085] That is, first, peripheral blood T cells were stimulated and activated with beads (manufactured by Miltenyi Biotec) coated with α-CD3 / CD28 antibodies. On the other hand, CTL clones were stimulated and activated with PHA (manufactured by Sigma-Aldrich). In addition, CTL clones were also activated by stimulation with PBMCs (allogeneic antigen-expressing cells) derived from someone other than the irradiated HIV-1-infected patient. When stimulated with PHA and when stimulated with allogeneic antigen-expressing cells, It has been confirmed that there is no significant difference in the properties of the obtained T-iPS cells.

[0086] Reprogramming factors were introduced into the activated cells via a retroviral vector (pMXs retroviral vector) or a Sendai virus vector (SeV vector), and in RH10 medium, 10 ng / ml (200 U) IL-2, 5-10 ng / ml The cells were cultured together with IL-7 and 5 to 10 ng / ml IL-15 (Peprotech), and the medium was gradually replaced with human iPS medium (Wako) containing bFGF and other ingredients.

[0087] The viral vector was introduced into the cells by centrifugation on a RetroNectin-coated plate (Takara Co., Ltd.) The composition of RH10 medium is as follows. RPMI-1640 supplemented with 10% human AB serum, 2 mM L-glutamine, 100 U / ml penicillin and 100 ng / ml streptomycin. The composition of the human iPS culture medium is as follows: DMEM / F12FAM supplemented with 20% KSR, 2 mM L-glutamine, 1% non-essential amino acids, 10 μM 2-mercaptoethanol and 5 ng / ml b-FGF.

[0088] Furthermore, to remove SeVs from the cytoplasm, siRNA L527 (see Nishimura, K. et al., J. Biol. Chem., 2011, vol. 286, pp. 4760-4771) was introduced into the established iPS clones using Lipofectamine RNAi Max (Invitrogen). <Alkaline phosphatase staining and immunocytochemistry> Alkaline phosphatase activity was evaluated using Alkaline Phosphatase Substrate Kit II (Vector Laboratories) according to the manufacturer's instructions.

[0089] Immunocytochemical staining was performed using the following antibodies according to the description in "Takayama, N. et al., J Exp Med, 2010, Vol. 207, pp. 2817-2830." The numbers in parentheses indicate the dilution rate of each antibody. SSEA-4 (1:50, FAB1435P, R&D Systems) Tra-1-60 (1:100, MAB4360, Millipore) Tra-1-81 (1:100, MAB4381, Millipore) HLA - A24 (1:100, manufactured by BIH0964, Veritas) Microscopic photographs were taken using an Axio Observer Z1 fluorescence microscope (manufactured by Carl Zeiss).

[0090] <Teratoma formation> Through teratoma formation in a system using NOD - Scid mice, the pluripotency of human iPS cells was evaluated in accordance with the description in "Masaki, H. et al., Stem Cell Res, 2007, Volume 1, Pages 105 - 115".

[0091] <Bisulfite sequencing> Genomic DNA was processed using the MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (manufactured by Human Genetic Signatures) according to the instruction manual.

[0092] The promoter regions of the human Oct3 / 4 and Nanog genes were amplified by PCR using EpiTaq HS (manufactured by Takara). The obtained PCR products were inserted into the pGEM - T - Easy vector (manufactured by Promega) and cloned for sequencing and subjected to singly. The sequences of the primers used for PCR (SEQ ID NOs: 5 - 8) are shown in Table 1.

[0093]

Table 1

[0094] <RT - PCR and quantitative PCR> Total RNA was extracted from ES cells, iPS cells, and T cells using an RNeasy micro kit (Qiagen). Then, using the total RNA as a template, a reverse transcription reaction was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random 6-base primers. RT-PCR was performed as described in "Takayama, N. et al., J Exp Med, 2010, vol. 207, pp. 2817-2830". The sequences of the target genes and PCR primers (SEQ ID NOs: 9-43) used in the analysis are shown in Table 2.

[0095] [Table 2]

[0096] Quantitative PCR was performed using TaqMan Array Human Stem Cell Pluripotency Card and Customized Card (Applied Biosystems).

[0097] <Analysis of TCR gene rearrangements in genomic DNA> Genomic DNA was prepared using the QIAamp DNA kit (Qiagen) according to the manufacturer's instructions, at approximately 5 x 10 6 extracted from the cells.

[0098] To analyze the rearrangement of the TCRB gene, multiplex PCR analysis was performed according to the BIOMED-2 protocol (see van Dongen, JJ et al., Leukemia, 2003, vol. 17, pp. 2257-2317) with some modifications. The primers (SEQ ID NOs: 44-81) used in PCR to analyze the rearrangement of the TCRB gene are shown in Table 3.

[0099] To analyze the rearrangement of the TCRA gene, PCR was performed using the primers shown in Fig. 1 and Table 4 (SEQ ID NOs: 82 to 127) and LATaq HS (manufactured by Takara). The PCR program consisted of 3 cycles of an amplification step at 95°C for 30 seconds, 68°C for 45 seconds, and 72°C for 6 minutes, 15 cycles of an amplification step at 95°C for 30 seconds, 62°C for 45 seconds, and 72°C for 6 minutes, and 12 cycles of an amplification step at 95°C for 15 seconds, 62°C for 30 seconds, and 72°C for 6 minutes. The main band within the expected molecular weight range was purified using the QIAquick Gel Extraction Kit (manufactured by Qiagen) and subjected to sequencing. The use of segments for V, D, and J was identified by using an online tool (IMGT / V-Quest) and comparing it with the ImMunoGeneTics (IMGT) database (http: / / www.cines.fr / ) (see "Lefranc, M.P., Leukemia, 2003, Vol. 17, 2003, pp. 260 - 266"). The naming of gene fragments (segments) followed the IMGT nomenclature.

[0100]

Table 3

[0101]

Table 4

[0102] <Detection of TCR gene rearrangement in mRNA> the 5' end of the reverse transcription product (switch mechanism at Double-stranded cDNA was synthesized using a Super SMART(TM) cDNA synthesis kit (manufactured by Clontech Laboratories) according to the manufacturer's instructions, using a method based on the 5'-end of the reverse transcriptase (SMART method; see Du, G. et al., J Immunol Methods, 2006, vol. 308, pp. 19-35 (2006)). The synthesized double-stranded cDNA was amplified using an Advantage 2 PCR kit (manufactured by BD Clontech), and TCRA-specific amplification or TCRB-specific amplification was performed from the amplified cDNA using the primers shown in Table 5 (SEQ ID NOs: 128-130). The PCR products were inserted into pGEM-T-Easy vector (Promega), cloned, and subjected to sequencing.

[0103] [Table 5]

[0104] <Intracellular staining> To stain for intracellular granzyme B, T cells were incubated with α-CD3 / 28 beads and 10 μg / ml brefeldin A (BFA, Invitrogen).

[0105] The cells were then harvested, fixed with Fixation / Permeabilization solution (BD Pharmingen), and intracellular staining was carried out using FITC-conjugated anti-granzyme B antibody (BD Bioscience) according to the manufacturer's instructions.

[0106] To capture CD107a transiently expressed on the cell surface, T cells were incubated with α-CD3 / 28 beads and cultured with FITC-conjugated anti-CD107a antibody (BioLegend).

[0107] Then, the cells prepared in this way were sorted using a FACS AriaII device (manufactured by BD Bioscience) and analyzed using Flowjo software (manufactured by Treestar).

[0108] <Microarray analysis> Total RNA was extracted from human ES cells, T-iPS cells, redifferentiated T cells, peripheral blood T cells, and peripheral blood NK cells using an RNeasy micro kit (manufactured by Qiagen).

[0109] The fluorescently labeled complementary RNA was hybridized with a whole human genome microarray 4×44K (G4112F, manufactured by Agilent Technologies) or SurePrint G3 human gene expression 8×60K (G4851A, manufactured by Agilent Technologies) using a one-color protocol. Then, the obtained signal data was analyzed using GeneSpring GX software (manufactured by Agilent Technologies).

[0110] <Measurement of telomere length by Flow-FISH> As described in "Neuber, K. et al., Immunology, 2003, Vol. 109, pp. 24 - 31", the telomere length was measured using a DAKO telomere PNA kit / FITC (manufactured by DAKO).

[0111] <ELISPOT assay and 51 Cr release assay> As antigen-expressing cells, HLA-A24-expressing autologous B-LCLs were used, and according to the descriptions in "Kawana -Tachikawa, A. et al., J Virol, 2002, Vol. 76, pp. 11982 - 11988" and "Tsunetsugu-Yokota, Y. et al., J Virol, 2003, Vol. 77, pp. 10250 - 10259", an ELISPOT assay targeting IFN-γ and a standard 51Antigen-specific responsiveness of T cells was measured by performing a Cr release assay.

[0112] <Statistical analysis> All data in this example are expressed as mean ± standard deviation (mean ± SD). In all statistical analyses in this example, unpaired two-tailed Student's t-tests were performed using Excel (Microsoft) and Prism (Graphpad Software), and a value of P<0.05 was considered significant.

[0113] (Preparation Example 1) <Reprogramming of antigen-specific cytotoxic T cell clones into pluripotent cells> To establish T cell-derived iPS cells, the inventors isolated CD3 + T cells were magnetically isolated. The isolated CD3 + T cells were incubated with the above CD3 in the presence of 10 ng / ml IL-2. + The T cells were stimulated with microbeads coated with anti-human CD3 antibody and anti-human CD28 antibody (α-CD3 / CD28 beads) in an amount three times the amount of the T cells. + T cells were transduced with retroviruses encoding OCT4, SOX2, KLF4, and c-MYC, respectively (Figure 2 reference).

[0114] Similarly, PBMCs from a chronically HIV-infected individual (HLA type: A24) were isolated and CD8 specific for an antigen peptide derived from the HIV-1 Nef protein (Nef-138-8 (wt); RYPLTFGW, SEQ ID NO: 1, see Miyazaki, E. et al., AIDS, 2009, Vol. 23, pp. 651-660). +CTL clones were established. One of the clones, designated H25-4, was stimulated with 5 μg / ml PHA. The thus-activated H25-4 was then transfected with two types of Sendai virus (SeV) vectors: a SeV vector encoding four cistronic factors (OCT4, SOX2, KLF4, and c-MYC) and a miR-302 target sequence (SeVp[KOSM302L], see Nishimura, K. et al., J Biol Chem, 2011, vol. 286, pp. 4760-4771), and a SeV vector encoding SV40 large T antigen (SeV18+SV40 / TS15ΔF, see Fusaki, N. et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009, vol. 85, pp. 348-362) was introduced into H25-4 cells activated with PHA. By introducing these two types of SeV vectors into H25-4 cells activated with PHA, the appearance of a sufficient number of human ES cell-like colonies was confirmed after 40 days of culture (see Figures 3 and 4).

[0115] As a result, CD3 + The T cell-derived ES cell-like colonies and H25-4-derived ES cell-like colonies (also referred to as "TkT3V1-7" and "H254SeVT-3", respectively) had alkaline phosphatase (AP) activity and expressed pluripotent cell markers (SSEA-4, Tra-1-60, and Tra-1-81) (see Figures 4 and 5). It was also confirmed that human ES cell-associated genes were expressed even after the expression of exogenous reprogramming factors from integrated provirus (TkT3V1-7) or cytoplasmic SeVRNAs (H254SeVT-3) was terminated (see Figures 6 and 7).

[0116] Comparison of gene expression profiles revealed that the expression patterns of all genes in these ES-like cells were similar to those in human ES cells, but not to those in peripheral blood T cells. were significantly different (see Figures 6, 8 and 9).

[0117] Furthermore, it was confirmed by bisulfite PCR assay that slight methylation occurred in the OCT4 and NANOG promoter regions. Therefore, in light of the description in "Freberg, C.T. et al., Mol Biol Cell, 2007, Vol. 18, pp. 1543-1553", it became clear that reprogramming had occurred in these ES-like cells (see Figures 10 and 11).

[0118] Furthermore, in light of the description in "Brivanlou, A.H. et al., Science, 2003, Vol. 300, pp. 913-916", when these ES-like cells were injected into NOD-Scid mice, the formation of teratomas containing characteristic tissues derived from each of the three germ layers was observed, and it became clear that these cells had pluripotency (see Figures 12 and 13).

[0119] <TCR gene rearrangement in T-iPS cells> It is known that the rearrangement of the TCRαβ gene is involved in the normal developmental process of αβ T cells in the thymus. Therefore, next, it was retrospectively confirmed based on such rearrangement whether the T-iPS cells established above were derived from αβ T cells. That is, multiplex PCR primers for analyzing TCRB gene assembly were designed by the BIOMED-2 consortium (see "van Dongen, J.J. et al., Leukemia, 2003, Vol. 17, pp. 2257-2317"). In addition, primers for detecting TCRA gene assembly were designed independently (see Figure 1). Then, by performing PCR using such primers, the rearrangement of the TCRαβ gene in the T-iPS cells was detected. The results obtained are shown in Figures 14 to 17.

[0120] As shown in Figures 14 to 17, the assembly of the TCRB gene and the TCRA gene was identified as a single band in each of the alleles of TkT3V1-7 and H254SeVT-3.

[0121] The structure of the antigen recognition site of TCR is composed of three complementarity determining regions (CDR1, CDR2 and CDR3). Furthermore, among these three regions, CDR3 has the most diverse sequence because it spans the V(D)J joining region where various random nucleotides (N-nucleotides or P-nucleotides) are inserted (see Alt, FW et al., Proc Natl Acad Sci USA, 1982, vol. 79, pp. 4118-4122 and Lafaille, JJ et al., Cell, 1989, vol. 59, pp. 859-870).

[0122] Therefore, the sequences (SEQ ID NOs: 131 to 133 and 145 to 148) of the CDR3 of the TCRA and TCRB genes assembled in the T-iPS cells (TkT3V1-7 and H254SeVT-3) were determined. The results are shown in Tables 6 to 9.

[0123] [Table 6]

[0124] [Table 7]

[0125] [Table 8]

[0126] [Table 9]

[0127] As shown in Tables 6 to 9, in TkT3V1-7 and H254SeVT-3, one set each of the assembly of the effective TCRA and TCRB genes, for example, those that are ligated in-frame and have no stop codon, was identified. Furthermore, it was also revealed that the CDR3 sequence of H254SeVT-3 and that of H25-4 are completely identical in the TCRA and TCRB gene regions. From these results, it became clear that iPS cells were established from a single T cell, and that the antigen specificity inscribed in genomic DNA is conserved even in the process of reprogramming.

[0128] (Comparative Example 1) <Redifferentiation of T-iPS cells into T cells> Next, in order to examine the hematopoietic differentiation ability of the T-iPS cells, according to a specific in vitro differentiation protocol, the T-iPS cells were redifferentiated into cells of the mesoderm-derived type, particularly hematopoietic stem / progenitor cells (see International Publication No. 2011 / 096482, "Vodyanik, M. A. et al., Blood, 2005, Vol. 105, pp. 617-626" and "Takayama, N. et al., Blood, 2008, Vol. 111, pp. 5298-5306").

[0129] That is, small pieces (<100 cells or less) of the T-iPS cells were transferred onto irradiated C3H10T1 / 2 cells and co-cultured in EB medium in the presence of 20 ng / mL VEGF, 50 ng / mL SCF, and 50 ng / mL FLT-3L (manufactured by Peprotech). The composition of the EB medium is as follows. IMDM supplemented with 15% fetal bovine serum (FBS), a cocktail consisting of 10 μg / mL human insulin, 5.5 μg / mL human transferrin, and 5 ng / mL sodium selenite, 2 mM L-glutamine, 0.45 mM α-monothioglycerol, and 50 μg / mL ascorbic acid.

[0130] Then, on the 14th day after transfer onto the C3H10T1 / 2 feeder cells, the hematopoietic cells (CD34 +Hematopoietic stem / progenitor cells (HPCs) were harvested and transferred onto irradiated OP9-DL1 cells. OP9-DL1 cells were provided by the Riken Bioresource Center through the National Bioresource Project of the Ministry of Education, Culture, Sports, Science and Technology (Japan) (see Watarai, H. et al., Blood, 2010, vol. 115, pp. 230-237). Hematopoietic cells were differentiated into T lineage cells in OP9 medium in the presence of 10 ng / mL FLT-3L and 1 ng / mL IL-7 (see Ikawa, T. et al., Science, 2010, vol. 329, pp. 93-96). The composition of OP9 medium is as follows: αMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 ng / ml streptomycin.

[0131] Then, 21 to 28 days after starting culture on OP9-DL1 feeder cells, CD45 + ,CD38 + ,CD7 + ,CD45RA + ,CD3 + and TCRαβ + The differentiation of these cells into T lineage cells was confirmed (see Figures 18 and 19).

[0132] As shown in FIG. 20, in one population of these T lineage cells, there was also differentiation into a small number of SP T cells that could not be characterized in detail by the inventors, but there was also the presence of cells at the CD4 / CD8 double positive (DP) stage, as well as populations of cells that differentiated into more mature CD4 single positive (SP) or CD8 single positive (SP) stage cells.

[0133] In the maturation process of T cells in the thymus, the CD4 / CD8DN stage or the CD4 / CD8DP stage corresponds to the β-chain assembly stage or the α-chain assembly stage, respectively (see Von Boehmer, H. Advances in Immunology, 2004, vol. 84, pp. 201-238). In addition, negative feedback control of gene assembly and inhibition of further rearrangement at the TCRB locus are extremely strict (see Khor, B. et al., Current Opinion in Immunology, 2002, vol. 14, pp. 230-234). However, there is also a relatively loose negative feedback control system and further gene assembly of preassembled genes, which tends to occur at the TCRA locus and is known as "receptor revision" (see Huang, C. et al., J Immunol, 2001, vol. 166, pp. 2597-2601; and Krangel, MS, Curr Opin Immunol, 2009, vol. 21, pp. 133-139).

[0134] Furthermore, in experiments using TCRα transgenic mice, reactivation of genes involved in the rearrangement mechanism (e.g., Rag1 and Rag2) occurred at the DP stage, and gene assembly of the endogenous Tcra gene was also observed (see Petrie, HT et al., J Exp Med, 1993, vol. 178, pp. 615-622 and Padovan, E. et al., Science, 1993, vol. 262, pp. 422-424).

[0135] To clarify whether such receptor correction occurs during the redifferentiation process of T-iPS cells into T lineage cells, we investigated the CD45 + , CD3 + , TCRαβ + and CD5 + From T lineage cells, CD1a - DN and CD1a +Cells at the DP stage were collected and analyzed for TCR mRNA expression and sequence (SEQ ID NOs: 131 to 149). The results are shown in FIG. 21 and Tables 10 to 13.

[0136] For TkT3V1-7, CD1a - DN and CD1a + CD8 cells that are more differentiated than the DP stage + The cells at the SP stage were collected and analyzed for TCR mRNA expression. The results are shown in Tables 14 and 15.

[0137] [Table 10]

[0138] [Table 11]

[0139] [Table 12]

[0140] [Table 13]

[0141] [Table 14]

[0142] [Table 15]

[0143] As shown in Figure 21, at the DN and DP stages, the base sequences of TCRB mRNA in T lineage cells were identical to those of T-iPS cells.

[0144] On the other hand, as shown in Tables 10 and 12, TCRA mRNA contained both identical and different sequences at the DN and DP stages. Different sequences were confirmed more frequently at the DP stage than at the DN stage, and in particular, the frequency of T cells having the same gene rearrangement pattern as the original T cells was about one-quarter in redifferentiated DP cells derived from H254SeVT-3.

[0145] Furthermore, as shown in Tables 10, 12 and 14, TkT3V1-7 derived T lineage cells In the TCRA gene, the DN stage, DP stage, CD8 + As the SP stage and differentiation progressed, the frequency of T cells with the same gene rearrangement pattern as the original T cell decreased to 100%, 89%, and 75%.

[0146] Furthermore, as shown in FIG. 22, the expression of RAG1 and RAG2 was observed at both the DN and DP stages, but stronger expression was confirmed at the DP stage than at the DN stage.

[0147] Example 1 <Redifferentiation of T-iPS cells into CD8 single positive cells by the method of the present invention> As shown in Comparative Example 1, it was revealed that the TCRA mRNA of T cells redifferentiated from T-iPS cells had a higher frequency of TCR gene rearrangement patterns different from those of the original human T cells at the DP stage than at the DN stage. It was also revealed that the expression of RAG1 and RAG2 was stronger at the DP stage than at the DN stage in T cells redifferentiated from T-iPS cells.

[0148] On the other hand, as shown in Figure 23, it was also revealed for the first time that TCR (TCRαβ) is expressed on the cell surface of T cells redifferentiated from T-iPS cells, even at the DN stage, where it is known that TCR is not normally expressed in the thymus.

[0149] As described in "Turka, LA et al., Science, 1991, Vol. 253, pp. 778-781," it is known that in the process of "positive selection," TCR signals mediated by peptide-MHC complexes stop the expression of RAG genes and suppress further assembly of TCR genes. Turka et al. have also confirmed that TCR signal-like signals induced by anti-CD3 antibodies have a similar effect.

[0150] Based on the new finding that TCR (TCRαβ) is expressed on the cell surface of T cells redifferentiated from T-iPS cells, as well as previous reports that activating TCR signals can stop the expression of RAG genes and thus suppress further assembly of TCR genes, we attempted to stimulate the TCR of redifferentiated T lineage cells before the transition from the DN stage to the DP stage was completely completed in order to produce mature CD8SP cells from T-iPS cell-derived T lineage cells without the receptor correction shown in Comparative Example 1.

[0151] That is, as shown in Figure 24, small clumps (<100 cells) of T-iPS cells (H254SeVT-3) obtained in Preparation Example 1 were transferred onto irradiated C3H10T1 / 2 cells and co-cultured in EB medium (Peprotech) in the presence of 20 ng / mL VEGF, 50 ng / mL SCF, and 50 ng / mL FLT-3L. On the 14th day of culture, hematopoietic cells contained in the iPS sac were collected and transferred onto irradiated OP9-DL1 cells, and the hematopoietic cells were differentiated into T lineage cells in OP9 medium in the presence of 10 ng / mL FLT-3L and 1 ng / mL IL-7.

[0152] Then, on day 35 of culture, the cells were stimulated by adding α-CD3 / CD28 beads or 5 μg / ml PHA in an amount three times the amount of the hematopoietic cells to the OP9 medium, and the cells committed to the T lineage were continued to be cultured on OP9-DL1 (stimulation with α-CD3 / CD28 beads or PHA was considered the first stimulation).

[0153] Then, on day 45 of the culture, the T lineage cells were harvested and treated with 10 ng / mL IL-7 and 10 The cells were cultured in RH10 medium together with irradiated HLA-A24-PMBCs in the presence of ng / mL IL-15.

[0154] It has been reported that IL-7 signaling contributes to CD8 lineage selection (see Chong, MM et al., Immunity, 2003, vol. 18, pp. 475-487; Singer, A. et al., Nat Rev Immunol, 2008, vol. 8, pp. 788-801; and Park, JH et al., Nat Immunol, 2010, vol. 11, pp. 257-264). Furthermore, IL-7 and IL-15 are involved in the selection of memory CD8 lineage. + It has also been reported that it is necessary for T cell generation (see Becker, TC et al., J Exp Med, 2002, vol. 195, pp. 1541-1548; Tan, JT et al., J Exp Med, 2002, vol. 195, pp. 1523-1532; Prlic, M. et al., J Exp Med, 2002, vol. 195, pp. F49-52; and Kaneko, S. et al., Blood, 2009, vol. 113, pp. 1006-1015).

[0155] As shown in Figure 25, on day 60 of culture, CD8SP cells appeared, and these cells were confirmed to be derivatives of H254SeV-3 by expression of HLA-A24. + Expression of CD56, which is expressed on T cells, was observed in these CD8SP cells (for CD56, see Lu, PH et al., J. Immunol, 1994, vol. 153, pp. 1687-1696). Furthermore, CD7 was also expressed in those CD8SP cells, and expression of CD2 was observed in some cells. Furthermore, PD-1, one of the markers of exhausted T cells, was not expressed in most of those CD8SP cells. On the other hand, expression of CCR7 was observed in some of those CD8SP cells along with CD27 and CD28, which represent the phenotype of central memory T cells. Note that FIG. 25, FIGS. 26-39 described later, and Tables 8 and 9 are diagrams showing the results when the T lineage cells were stimulated with PHA as the first stimulation. Although not shown in the figures, it has been confirmed that the same results as those obtained when the T lineage cells were stimulated with α-CD3 / CD28 beads as the first stimulation were obtained.

[0156] Example 2 <Antigen specificity of the CD8SP cells of the present invention> The redifferentiated CD8SP cells obtained in Example 1 had the same rearrangement pattern as the TCR gene of the original H25-4, and therefore it was examined whether they could recognize the antigen peptide for which H25-4 shows specificity. That is, all the redifferentiated T cells obtained in Example 1 were mixed with A24 / Nef-138-8(wt) tetramer, and flow cytometry analysis was performed according to the description in "Kawana-Tachikawa, A. et al., J Virol, 2002, Vol. 76, pp. 11982-11988". The "A24 / Nef-138-8(wt) tetramer" is a tetramer of the antigen (Nef-138-8(wt)) and HLA (A24) recognized by H25-4. The obtained results are shown in FIG. 26.

[0157] As is clear from the results shown in Figure 26, most of the CD8SP cells derived from T-iPS cells were stained with the A24 / Nef-138-8 (wt) tetramer. Furthermore, the ratio of CD8SP positive cells (antigen-specific CD8SP cells) among the CD8SP cells derived from T-iPS cells (CD8SP tetramer positive cells and CD8SP tetramer negative cells) was 77%. However, although not shown in the figure, the T-iPS cell-derived CD8SP cells were not stained by the HLA-A24 tetramer presenting a peptide derived from the HIV-1 envelope (RYLRDQQLL, SEQ ID NO: 2) used as a control. Thus, it is possible that the TCR of redifferentiated T lineage cells is stimulated before the transition from the DN stage to the DP stage is completely completed. It was revealed that by stimulating the T cells, the frequency (one-fourth) of T cells having the same gene rearrangement pattern as the original T cells confirmed in Comparative Example 1 was significantly improved.

[0158] Next, as shown in FIG. 26, CD8 cells reacting with A24 / Nef-138-8(wt) tetramer were + The cells were collected, expanded, and stimulated again with PHA (this stimulation with PHA was designated the second stimulation). By performing such redifferentiation experiments several times independently, we finally obtained CD8SP cells (reT-1, reT-2.1, reT-2.2, and reT-3) that reacted with A24 / Nef-138-8(wt) tetramer.

[0159] Analysis of the TCRA and TCRB mRNA sequences of these redifferentiated CD8SP cells revealed that the rearrangement patterns of the TCR genes were consistent with those of the original CD8T cell clone H25-4, as shown in FIG. 27 and Tables 8 and 9. Furthermore, although the amino acid sequences (especially the amino acid sequences of CDR3) often differ even between antibodies that recognize such MHC tetramers, as shown in Tables 8 and 9, the rearrangement patterns of the TCR genes of CD8SP cells (reT-1, reT-2.1, reT-2.2, and reT-3) that react with the A24 / Nef-138-8(wt) tetramer and the original CD8T cell clone H25-4 are completely consistent. This data also supports the idea that the frequency (one-fourth) of T cells having the same gene rearrangement pattern as the original T cells confirmed in Comparative Example 1 can be significantly improved by stimulating the TCR of redifferentiated T lineage cells before the transition from the DN stage to the DP stage is completely completed.

[0160] To investigate whether these redifferentiated CD8SP cells are of T cell lineage, we analyzed gene expression profiles in reT-2.1 and peripheral blood (PB) CD4 + T cells, PB CD8 + The results were compared between those in T cells and H25-4 by quantitative PCR, and the results are shown in Figures 28 to 30.

[0161] As is evident from the results shown in Figure 28, PB CD4 + Except for T cells, the expression patterns of CD3, CD4, and CD8 were similar to those of PB CD8 + This was the same among T cells, redifferentiated CD8SP cells, and the original T cell clone H25-4 (hereinafter also referred to as "H25-4 original T cell clone").

[0162] In addition, as shown in FIG. 29, genes characteristic of cytotoxicity, such as granzyme B (GZMB), perforin (PFR1), IFN-γ (IFNG), and FAS ligand (FASLG), were upregulated in PB CD8 +It was also revealed that it was expressed in T cells, and was also expressed at relatively high levels in CD8SP cells and the H25-4 original T cell clone, which are T cells that have already been stimulated with an antigen.

[0163] Furthermore, as shown in FIG. 30, the expression patterns of several factors in transcription or signal transduction and cell surface molecules were significantly different in PB CD8 + This was the same among T cells, redifferentiated CD8SP cells and the H25-4 original T cell clone.

[0164] To exclude the possibility that the redifferentiated CD8SP cells acquired NK-like properties during coculture with OP9-DL1 or PBMCs, the global gene expression profiles of the redifferentiated CD8 cells, the H25-4 original T cell clone, and the peripheral blood NK cells were analyzed by cDNA microarray. The results are shown in Figures 31 and 32. As is clear from the results shown in Figures 31 and 32, the gene expression profile of the redifferentiated CD8SP cells was similar to that of the original H25-4 T cell clone. However, correlation coefficient and cluster analysis did not reveal any correlation between the redifferentiated CD8SP cells and NK cells. Therefore, T-iPS cells are CD8 cells that show the same antigen specificity as the original T cells. + It was shown that they could redifferentiate into T cells.

[0165] Example 3 <High proliferation of the CD8SP cells of the present invention> In Example 1, the number of T lineage cells obtained from the coculture of T-iPS cells and OP9-DL1 on a 6 cm dish was 10 5 However, although not shown in the figure, the first stimulation increased the number of 8The number of cells could be expanded to more than 1000 cells. Therefore, in order to examine the proliferation of the CD8SP cells obtained by the method of the present invention, reT-1, reT-2.2 and reT-3 were stimulated with 5 μg / ml PHA, 10 ng / mL IL-7 and 10 ng / mL IL-15, and the proliferation rate (expansion rate) of each cell was measured two weeks later. The obtained results are shown in FIG.

[0166] As is clear from the results shown in FIG. 33, the H25-4 original T cell clone expanded about 20-fold two weeks after the stimulation, whereas the CD8 + The cells were amplified 100-1000-fold.

[0167] Furthermore, as shown in Figure 34, even after 100- to 1000-fold expansion, expression of central memory T cell markers, such as CCR7, CD27, and CD28, was observed in some populations (for central memory T cell markers, see Romero, P. et al., J Immunol, 2007, Vol. 178, pp. 4112-4119).

[0168] Regarding the high proliferation ability (replicative ability) of the CD8SP cells of the present invention shown above, it is assumed that the high replicative ability could be imparted to the redifferentiated T cells by re-elongating the telomeres that had become short in the H25-4 original T cell clone through the state of iPS cells with extremely high telomerase activity (see "Takahashi, K. et al., Cell, 2007, vol. 131, pp. 861-872", "Marion, RM et al., Cell Stem Cell, 2009, vol. 4, pp. 141-154", "Monteiro, J. et al., J Immunol, 1996, vol. 156, pp. 3587-3590" and "Weng, NP et al., Immunity, 1998, vol. 9, pp. 151-157"). Therefore, the telomere length of the CD8SP cells obtained by the method of the present invention was measured. The obtained results are shown in FIG. 35.

[0169] As is clear from the results shown in FIG. 35, the telomere length was indeed longer in the redifferentiated T cells than in the original T cell clone.

[0170] Therefore, it was revealed that the CD8SP cells (cloned cytotoxic T cells) obtained by the method of the present invention can be rejuvenated into central memory-like T cells that exhibit excellent proliferation and survival capabilities by passing through the T-iPS cell state.

[0171] Although not shown in the figures, neither autonomous cell proliferation nor abnormal cell survival was observed in the absence of cytokines throughout this Example.

[0172] Example 4 <Antigen-specific functions of the CD8SP cells of the present invention> One of the main mechanisms of cytotoxicity of CTLs is the secretion of cytolytic molecules that occurs with TCR signaling. Therefore, we investigated by intracellular staining whether cytolytic molecules are secreted by stimulating TCR with α-CD3 / CD28 beads in an amount three times that of the CD8SP cells obtained by the method of the present invention. The results are shown in Figure 36.

[0173] As shown in FIG. 36, after stimulation with α-CD3 / 28 beads, the cytolytic molecule granzyme B was produced and accumulated in the granules of redifferentiated CD8 T cells (see the left panel of FIG. 36).

[0174] In addition, CD107a, known as lysosomal membrane protein 1 (LAMP1), is a granulocyte membrane protein that is transiently expressed in conjunction with degranulation, which secretes cytolytic molecules, when CTLs are stimulated, and is known to return to the cytoplasm after secretion (Rubio, V. et al., Nat Med, 2003, vol. 9, pp. 1377-1382). Therefore, the redifferentiated CD8 T cells of the present invention were cultured with or without stimulation by α-CD3 / 28 beads, and the CD107a molecules on the cell surface were captured by an antibody bound to a fluorescent dye. The results obtained are shown in FIG. 36.

[0175] As shown in FIG. 36, antibody uptake was detected only when the redifferentiated T cells of the present invention were stimulated with α-CD3 / 28 beads (see the right panel of FIG. 36).

[0176] Next, to evaluate whether the redifferentiated CD8 T cells of the present invention can exert cytotoxicity by recognizing a specific peptide in the MHC complex, functional assays (ELISPOT (Enzyme-Linked ImmunoSpot) and 51 Cr release assay) was performed. The results are shown in Figures 37 and 38.

[0177] In addition, Gag-28-9(wt) (KYKLKHIVW, SEQ ID NO: 3) used in these evaluations is an antigenic peptide of HIV-1 Gag protein (amino acid residues: 28 to 36), and Nef-138-8(2F) (RFPLTFGW, SEQ ID NO: 4) is a single residue mutant of Nef-138-8(wt) in which tyrosine is replaced with phenylalanine, and both are present on HLA-A24.

[0178] As shown in Figure 37, the cytokine production ability per cell was evaluated by ELISPOT, and the redifferentiated CD8 T cells of the present invention were found to significantly produce IFN-γ in response to stimulation with the specific antigen Nef-138-8 (wt).

[0179] Also, as shown in Figure 38, 51 The cytolytic ability was examined by Cr release assay, and the redifferentiated CD8 T cells of the present invention were found to be able to lyse only in the presence of Nef-138-8 (wt). 51 It was found that Cr dissolved the incorporated B-LCL.

[0180] Therefore, it has been demonstrated that the CD8SP cells obtained by the method of the present invention are functional antigen-specific T cells because they release cytotoxic molecules and specifically kill target cells expressing the antigen.

[0181] Example 5 <Redifferentiation of T-iPS cells into CD4 single positive cells by the method of the present invention> In this example, we attempted to produce mature CD4SP cells from T-iPS cell-derived T lineage cells by using a technique to stimulate the TCR of redifferentiated T lineage cells before the transition from the DN stage to the DP stage was completely completed.

[0182] That is, as shown in Figure 39, small clumps (<100 cells) of T-iPS cells (H254SeVT-3) obtained in Preparation Example 1 were transferred onto irradiated C3H10T1 / 2 cells and co-cultured in EB medium containing 20 ng / mL VEGF. On the 14th day of culture, hematopoietic cells contained in the iPS sacs were collected and co-cultured with irradiated OP9-DL 1 cells and co-cultured in EB medium containing 20 ng / mL SCF, 10 ng / mL TPO, 10 ng / mL FLT-3L, and 10 ng / mL IL-7. The hematopoietic cells were transferred onto irradiated OP9-DL1 cells, and differentiated into T lineage cells in OP9 medium in the presence of 5 ng / mL FLT-3L and 1 ng / mL IL-7. On the 35th day of culture, the hematopoietic cells were stimulated by adding α-CD3 / CD28 beads or 5 μg / ml PHA to the OP9 medium in an amount three times the amount of the hematopoietic cells, and the cells committed to the T lineage were continued to be cultured on OP9-DL1 (stimulation with α-CD3 / CD28 beads or PHA was considered as the first stimulation). On the 45th day of culture, the T lineage cells were collected and cultured in RH10 medium together with irradiated HLA-A24-PMBCs in the presence of 10 ng / mL IL-7 and 10 ng / mL IL-15.

[0183] As a result, as shown in Figure 40, when stimulation with PHA was given as the first stimulation, CD4SP cells appeared together with CD8SP cells on culture day 60. In addition, although not shown in the figure, it was confirmed that similar results were obtained when stimulation with α-CD3 was given to the T lineage cells as the first stimulation.

[0184] As described above, according to the present invention, in CD8SP cells obtained by redifferentiating human T cells having antigen specificity into iPS cells, the frequency of T cells having the same TCR gene rearrangement pattern as the original human T cells can be extremely increased. Furthermore, the human CD8SP cells thus obtained have antigen-specific immune functions. This is also considered to be true for human CD4SP cells. Furthermore, while PD-1, one of the markers of exhausted T cells, is not expressed, CCR7 is expressed together with CD27 and CD28, which represent the phenotype of central memory T cells, and the telomeres are longer than those of the original T cells, and the cells have a high self-replication ability.

[0185] Therefore, for example, the production method of the present invention and the CD8 SP cells or CD4 SP cells obtained by said method are useful in treating or preventing diseases such as tumors, infectious diseases (chronic infectious diseases), and autoimmune disorders. [Sequence List Free Text]

[0186] SEQ ID NO:1 <223> Nef-138-8 (wild type) SEQ ID NO:2 <223> HIV-1 envelope-derived peptides SEQ ID NO:3 <223> Gag-28-9 (wild type) SEQ ID NO:4 <223> Artificially synthesized polypeptide sequence (Nef-138-8(2F)) SEQ ID NO:5-130 <223> Artificially synthesized primer sequence SEQ ID NO:131 <223> TRAV8-3*01 TRAJ10*01 SEQ ID NO:132 <223> TRAV13-1*01 TRAJ29*01 SEQ ID NO:133 <223> TRAV38-2 / DV8*01 TRAJ31*01 SEQ ID NO:134 <223> TRAV12-1*01 TRAJ7*01 SEQ ID NO:135 <223> TRAV21*02 TRAJ16*01 SEQ ID NO:136 <223> TRAV1-2*01 TRAJ6*01 SEQ ID NO:137 <223> TRAV1-2*01 TRAJ7*01 SEQ ID NO:138 <223> TRAV1-2*01 TRAJ10*01 SEQ ID NO:139 <223> TRAV1-2*01 TRAJ16*01 SEQ ID NO:140 <223> TRAV8-1*01 TRAJ6*01 SEQ ID NO:141 <223> TRAV12-1*01 TRAJ6*01 SEQ ID NO:142 <223> TRAV12-1*01 TRAJ9*01 SEQ ID NO:143 <223> TRAV6*01 TRAJ13*01 SEQ ID NO:144 <223> TRAV12-1*01 TRAJ27*01 SEQ ID NO:145 <223> TRBV7-9*01 TRBD1*01 TRBJ2-5*01 SEQ ID NO:146 <223> Reproductive system TRBD1*01 TRBJ2-7*01 SEQ ID NO:147 <223> TRBV29-1*02 TRBD1*01 TRBJ2-2*01 SEQ ID NO:148 <223> Reproductive system TRBD2*01 / *02 TRBJ2-7*01 / *02 SEQ ID NO:149 <223> TRBV29-1*01 TRBD1*01 TRBJ2-2*01

Claims

1. A method for preventing a change in antigen specificity of a T cell receptor (TCR) in a differentiation process of an iPS cell obtained from a T cell having antigen specificity into a T cell having the same antigen specificity as the T cell, in a process for producing a pharmaceutical composition, comprising: The manufacturing process includes: Obtaining CD4 / CD8 double negative T cells from iPS cells obtained from T cells having antigen specificity; Inducing CD4 / CD8 double positive T cells from the obtained CD4 / CD8 double negative T cells; Obtaining CD4 or CD8 single positive T cells from the obtained CD4 / CD8 double positive T cells; Obtaining a pharmaceutical composition comprising the obtained CD4 or CD8 single positive T cells and a pharmacologically acceptable carrier or vehicle; Including, The method comprises: inhibiting further assembly of TCR genes in at least CD4 / CD8 double negative T cells; wherein inhibiting further assembly of the TCR gene is carried out by stimulating the T cell receptor of a CD4 / CD8 double negative T cell.

2. The method described in claim 1, wherein stimulating the T cell receptor is carried out by providing a TCR signal or a TCR signal-like signal to the CD4 / CD8 double negative T cell.

3. A method for producing a pharmaceutical composition comprising CD8 single positive T cells having the same antigen specificity as a T cell having an antigen specificity from an iPS cell obtained from the T cell, the method comprising: (a) obtaining CD4 / CD8 double negative T cells from iPS cells obtained from T cells having antigen specificity; (b) inhibiting further assembly of TCR genes in at least CD4 / CD8 double negative T cells; and (c) inducing CD4 / CD8 double positive T cells from CD4 / CD8 double negative T cells; (d) inducing CD8 single positive T cells from the obtained CD4 / CD8 double positive T cells; and (e) obtaining a pharmaceutical composition comprising the induced CD8 single positive T cells; wherein inhibiting further assembly of the TCR gene is carried out by stimulating the T cell receptor of a CD4 / CD8 double negative T cell.

4. (b) is carried out in (c); The method according to claim 3.

5. The method according to claim 3 or 4, wherein suppressing further assembly of the TCR gene is carried out by providing a TCR signal or a TCR signal-like signal to the CD4 / CD8 double negative T cell.

6. A method for producing a pharmaceutical composition comprising CD4 single positive T cells having the same antigen specificity as a T cell having an antigen specificity from an iPS cell obtained from the T cell, the method comprising: (a) obtaining CD4 / CD8 double negative T cells from iPS cells obtained from T cells having antigen specificity; (b) inhibiting further assembly of TCR genes in at least CD4 / CD8 double negative T cells; and (c) inducing CD4 / CD8 double positive T cells from CD4 / CD8 double negative T cells; (d) inducing CD4 single positive T cells from the obtained T cells; and (e) obtaining a pharmaceutical composition comprising the induced CD8 single positive T cells; wherein inhibiting further assembly of the TCR gene is carried out by stimulating the T cell receptor of a CD4 / CD8 double negative T cell.

7. (b) is carried out in (c); The method according to claim 6.

8. The method according to claim 6 or 7, wherein suppressing further assembly of the TCR gene is carried out by providing a TCR signal or a TCR signal-like signal to the CD4 / CD8 double negative T cell.

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