Pluripotent stem cell-derived CD4-positive t cells, method for production thereof, and use thereof

Abstract

The present invention provides: a method for producing CD4-positive T cells, the method including a step for knocking out at least one gene selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3 and GIMAP5 and / or overexpressing RUNX1 gene in human artificial pluripotent stem cells (iPSCs); CD4-positive T cells produced by the method; and a pharmaceutical composition, such as an immunotherapeutic agent, which contains the cells.

Description

Pluripotent stem cell-derived CD4-positive T cells, their production method and use

[0001] The present invention relates to a method for producing CD4-positive T cells, particularly CD4 single positive (SP) T cells, via pluripotent stem cells, CD4-positive T cells obtainable by the method, and uses of the T cells, particularly their use as a cellular immunotherapeutic agent.

[0002] While adoptive immune cell therapy using T cells has been shown to be effective, it is generally performed by autologous transplantation, which has led to issues such as a limited number of cells that can be produced, time-consuming production, and T cell exhaustion. It has been reported that when antigen-specific T cells are induced, cloned, and then reprogrammed to produce induced pluripotent stem cells (iPSCs), the genomic structure of the rearranged T cell receptor (TCR) gene is inherited (Patent Documents 1 and 2). Previously, methods have been reported in which iPSCs produced from antigen-specific T cells are induced to differentiate into T cells exhibiting the same antigen specificity as the cells from which they were derived, thereby producing a large number of rejuvenated antigen-specific T cells (Patent Documents 1 and 2). It has also been reported that a large number of rejuvenated antigen-specific T cells can be produced from iPSCs produced from peripheral blood-derived T cells that lack antigen specificity. However, although these methods can consistently induce CD8-positive (CD8+) T cells, even when iPSCs (CD4T-iPSCs) are established from peripheral blood CD4-positive (CD4+) T cell clones and then induced to differentiate, they are unable to stably induce CD4+ T cells, and in reality, only by artificially introducing the CD4 gene into iPSCs can they be induced (Patent Document 3).CD8-positive cytotoxic T cells (CTLs) directly attack cancer, but CD4-positive helper T (Th) cells, particularly Th1 cells, which activate CTLs and suppress the proliferation and accumulation of regulatory T cells, one of the mechanisms of cancer immune escape, in an interferon (IFN)-γ-dependent manner, are extremely important in T cell immunotherapy.

[0003] Meanwhile, the present inventors established iPSCs (ATL-iPSCs) from adult T-cell leukemia (ATL) cells and attempted to induce differentiation of T cells from the ATL-iPSCs again, and succeeded for the first time in inducing CD4+CD25+FOXP3+ regulatory T cells (Patent Document 4). However, because these T cells are CD4+CD25+FOXP3+ and regulatory T cells, their applications are limited.

[0004] International Publication No. 2011 / 096482 Patent No. 6164746 International Publication No. 2018 / 168829 JP 2021-69322 A

[0005] The present invention aims to provide a method for efficiently producing non-regulatory CD4+ T cells via pluripotent stem cells, and to provide a novel and effective T cell immunotherapy using the CD4+ T cells.

[0006] The present inventors compared the gene expression of ATL-iPSC-derived CD4+ T cells described in Patent Document 3 with that of CD4T-iPSC-derived CD8+ T cells using single-cell RNA sequencing analysis. They found differences in the expression of six genes in CD4T-iPSC-derived CD8+ T cells compared to ATL-iPSC-derived CD4+ T cells. The present inventors then successfully induced differentiation into CD4+ T cells by knocking out one of these genes, RUNX3, in T-iPSCs. Furthermore, they confirmed that forced expression of RUNX3 in ATL-iPSCs led to differentiation into CD8+ T cells, demonstrating that the expression level of RUNX3 determines the induction of differentiation into CD4 / CD8 cells. They also confirmed that CD4+ T cells induced from T-iPSCs were not regulatory T cells. CD4+ T cells induced from CD8T-iPSCs were CD8-negative but maintained MHC class I-restricted antigen specificity, produced perforin and granzyme B, and exhibited antigen-specific cytotoxic activity. Meanwhile, these CD4+ T cells abundantly secreted various cytokines, including Th1 cytokines such as IFNγ and IL-2, and also exhibited a Th1 cell phenotype in expression analyses of transcription factors and cell surface molecules. When co-cultured with cancer antigen-specific CTLs or CAR-T cells, CD4+ T cells enhanced their cancer antigen-specific cytotoxic activity (tumor growth inhibitory effect) both in vitro and in vivo. Based on these findings, the present inventors conducted further research and completed the present invention.

[0007] That is, the present invention relates to the following [1] to

[18] . [1] A method for producing iPSC-derived CD4+ T cells, comprising the step of knocking out one or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 in human iPSCs and / or overexpressing the RUNX1 gene. [2] The method described in [1], wherein the iPSCs are derived from antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells, or fibroblasts. [3] The method described in [1] or [2], wherein the gene to be knocked out includes RUNX3. [4] The method described in any one of [1] to [3], wherein the CD4+ T cells are CD4 single-positive. [5] The method described in any one of [2] to [4], wherein the antigen-specific T cells are specific for an antigen peptide presented on cancer cells or virus-infected cells, or the peripheral blood-derived T cells or peripheral blood mononuclear cells are derived from a cancer patient or a virus-infected individual. [6] The method according to any one of [2] to [5], wherein the antigen-specific T cells or peripheral blood-derived T cells are CD8+ T cells. [7] The method according to any one of [2] to [5], wherein the antigen-specific T cells or peripheral blood-derived T cells are CD4+ T cells. [8] Human CD4+ T cells obtained by the method according to any one of [1] to [7]. [9] Human iPSC-derived T cells having the following properties: (a) being CD4 single-positive; (b) having a TCR specific to an antigen peptide presented on cancer cells or virus-infected cells; (c) exhibiting one or more of the following Th1 cell phenotypes; (c1) secreting IFNγ and IL-2 at high levels; (c2) expressing one or more genes selected from the group consisting of TBX21, STAT1, and STAT4 at high levels; (c3) expressing CD107a and CD366; (d) having cytotoxic activity specific to the antigen peptide of (b) alone; and (e) enhancing the proliferation and / or cytotoxic activity of cancer antigen- or virus antigen-specific CTL or CAR-T cells.

[10] The human iPSC-derived T cells according to [9], in which one or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3 and GIMAP5 have been knocked out, and / or the RUNX1 gene is overexpressed.

[11] A pharmaceutical composition comprising the human iPSC-derived CD4+ T cells of any one of [8] to

[10] .

[12] The pharmaceutical composition of

[11] , which is an immunotherapeutic agent.

[13] The pharmaceutical composition of

[12] , which is for treating cancer or a viral infection.

[14] The pharmaceutical composition of

[13] , which is combined with cancer antigen- or viral antigen-specific CTLs or CAR-T cells.

[15] The pharmaceutical composition of

[13] , which is combined with antigen-presenting cells and / or antigen peptides presented on cancer cells or virally infected cells.

[16] A method for treating cancer or a viral infection in a subject, comprising administering to the subject an effective amount of the human iPSC-derived CD4+ T cells of any one of [8] to

[10] .

[17] The human iPSC-derived CD4+ T cells of any one of [8] to

[10] for use in treating cancer or a viral infection.

[18] Use of the human iPSC-derived CD4+ T cells of any one of [8] to

[10] for the manufacture of a therapeutic agent for cancer or a viral infection.

[0008] According to the present invention, it is possible to stably produce CD4+ T cells, particularly CD4SP T cells, from peripheral blood T cells via iPSCs without introducing a CD4 gene. This allows efficient production of CD4+ T cells that can be used, for example, in cancer immunotherapy. Furthermore, the CD4+ T cells obtained according to the present invention possess both the Th1 cell phenotype and antigen-specific cytotoxic activity, and are capable of killing target cells (e.g., cancer cells, virus-infected cells) by themselves and activating target cell-specific CTLs. Therefore, the therapeutic effect can be further enhanced by using them in combination with target cell-specific CTLs or CAR-T cells.

[0009] 1 shows a comparison of the expression of RUNX3, RUNX1, ZBTB7B, SOCS1, SOCS3, and GIMAP5 between CD4T-iPSC-derived CD8+ T cells (HD-CD8rejT) and ATL-iPSC-derived CD4+ T cells (ATL-CD4rejT). This figure shows the time-course changes in the CD4 / CD8 ratio as determined by flow cytometry during induction of differentiation from RUNX3 KO CD4T-iPSC-derived hematopoietic progenitor cells. This figure shows the time-course changes in the CD4 / CD8 ratio as determined by flow cytometry during induction of differentiation from RUNX3 WT CD4T-iPSC-derived hematopoietic progenitor cells. This figure shows the time-course changes in the CD4 / CD8 ratio as determined by flow cytometry during induction of differentiation from RUNX3 overexpression ATL-iPSC-derived hematopoietic progenitor cells. This figure shows the results of flow cytometry analysis of CD4+ T cells derived from RUNX3 KO CD4T-iPSCs using anti-CD4, CD45RA, and Foxp3 antibodies. CD8SP T cells (left) induced to differentiate from LMP2-specific CTL-derived iPSCs, and CD4SP T cells (right) induced to differentiate from the iPSCs by knocking out RUNX3, both maintain class I-restricted LMP2 antigen specificity. This figure shows that RUNX3 KO LMP2-iPSC-Ts possess antigen-specific cytotoxic activity, although it is inferior to that of LMP2-iPSC-Ts. The line plots in each graph show, from top to bottom, LM2-iPSC-Ts (CD8+), RUNX3 KO LMP2-iPSC-Ts, and primary ATL cells (control). The effector cell:target cell (E:T) ratios are 20:1 and 10:1, respectively. This figure shows that RUNX3 KO LMP2-iPSC-Ts abundantly secrete IFNγ in response to antigenic stimulation. This figure shows that RUNX3 KO LMP2-iPSC-Ts abundantly produce perforin and granzyme B in response to antigenic stimulation. This figure shows that RUNX3 KO LMP2-iPSC-Ts highly secrete various cytokines in response to antigenic stimulation. **: p<0.01, ****: p<0.0001. This figure shows that RUNX3 KO LMP2-iPSC-Ts highly express TBX21, STAT1, and STAT4.1 shows that RUNX3 KO LMP2-iPSC-T are positive for CD107a and CD366. 1 shows that the cytotoxic activity of HPV16E6-iPSC-T is enhanced when CD8+ T cells induced to differentiate from HPV16E6-specific CTL-derived iPSCs are co-cultured with CD4+ T cells induced to differentiate from the iPSCs by knocking out RUNX3. Series 1: HPV16E6-iPSC-T + RUNX3 KO HPV16E6-iPSC-T (vs. antigen-positive cells), Series 2: HPV16E6-iPSC-T (vs. antigen-positive cells), Series 3: HPV16E6-iPSC-T + RUNX3 KO HPV16E6-iPSC-T (vs. antigen-negative cells), Series 4: HPV16E6-iPSC-T (vs. antigen-negative cells). The E:T ratios are 20:1 and 10:1 from left to right. (From top to bottom, series 1, 2, 3, and 4 are shown.) This figure shows that co-culture of GD2-specific CAR-T cells with RUNX3 KO T-iPSC-derived CD4+ T cells (A) promoted cell proliferation.

[0033] Figure 1 shows that co-culturing GD2-specific CAR-T cells with RUNX3 KO T-iPSC-derived CD4+ T cells enhances cytotoxic activity against GD2-positive EBV-associated lymphoma cells. Line plots, from top to bottom, show GD2-CARTs+CD4rej Ts, GD2-CARTs, and primary ATL cells (control).

[0034] Figure 1 shows that co-culturing GD2-specific CAR-T cells (CART) with RUNX3 KO T-iPSC-derived CD4+ T cells (CD4rej T) and administering the resulting mixture to mice transplanted with GD2-positive EBV-associated lymphoma cells enhances tumor growth suppression compared to administration of CAR-T cells alone.

[0010] The present invention provides a method for producing non-regulatory CD4+ T cells via pluripotent stem cells (hereinafter also referred to as "method for producing CD4+ T cells of the present invention" or "production method of the present invention"). One aspect of the method for producing CD4+ T cells of the present invention comprises the step of knocking out one or more genes selected from RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 and / or overexpressing the RUNX1 gene in iPSCs induced from human-derived somatic cells. In another aspect, the production method of the present invention comprises the step of inducing iPSCs from human-derived somatic cells (step (1)) prior to the step (step (2)). Steps (1) and (2) are described below.

[0011] (1-1) Isolation of Human-Derived Somatic Cells Human iPSCs used in the production method of the present invention can be derived from any human somatic cells. Examples of human somatic cells include tissue stem cells (somatic stem cells) such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and dental pulp stem cells; tissue progenitor cells; and differentiated cells such as lymphocytes (e.g., peripheral blood mononuclear cells, peripheral blood-derived T cells, tumor-infiltrating lymphocytes, and umbilical cord blood-derived mononuclear cells), fibroblasts (skin cells, etc.), epithelial cells, endothelial cells, muscle cells, hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes. Preferred examples include T cells (e.g., antigen-specific T cells, peripheral blood-derived T cells), peripheral blood mononuclear cells, and fibroblasts. The following provides a more detailed explanation using antigen-specific T cells or peripheral blood-derived T cells as examples; however, those skilled in the art can appropriately collect other human somatic cells using standard methods.

[0012] The human-derived antigen-specific T cells or peripheral blood-derived T cells used may be those of a healthy individual or those of a patient with a viral infection, which is rich in T cells. Tissues, peripheral blood, etc., from a patient with a viral infection or a healthy individual who has previously been infected with a virus are preferred. The isolation of antigen-specific T cells or peripheral blood-derived T cells from a patient infected with a virus will be described below as an example. T cells from a patient infected with a virus can be isolated, for example, from the tissue of an ATL patient by known techniques. Examples of tissues from ATL patients include tissues containing T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissue. Among these, peripheral blood is preferred from the viewpoints of low invasiveness to humans and ease of preparation.

[0013] In a preferred embodiment, human peripheral blood-derived T cells can be isolated from the peripheral blood of the patient to be treated (e.g., a cancer patient or a virally infected person). When peripheral blood from a person other than the patient to be treated is used, peripheral blood from an individual whose HLA antigen matches the HLA-restricted HLA of the patient to be treated can be used. However, CD4+ T cells can also be produced in other cases.

[0014] There are no particular limitations on the antigen-specific T cells, as long as they are T cells in which a T cell receptor (TCR) has been rearranged so as to specifically bind to a particular antigen, but when the obtained CD4+ T cells are intended to be used as an immunotherapeutic agent, the antigen is preferably a disease-specific antigen peptide, for example, a cancer antigen peptide or a viral antigen peptide, etc. Antigen-specific T cells can be obtained, for example, by isolating lymphocytes that have infiltrated into a lesion (e.g., tumor, virus-infected tissue) of a patient, or by sensitizing T cells isolated from peripheral blood to an antigen or by introducing an antigen-specific TCR gene or a chimeric antigen receptor (CAR) gene to impart the desired antigen specificity.

[0015] Known techniques for isolating antigen-specific T cells or peripheral blood-derived T cells include, for example, magnetic selection using magnetic beads for cell separation, and flow cytometry using an antibody against a cell surface antigen, such as an anti-CD4 antibody or an anti-CD8 antibody, and a cell sorter.

[0016] The antigen-specific T cells or peripheral blood-derived T cells may be CD8+ T cells or CD4+ T cells. Regardless of which is used as the starting material, CD4+ T cells, particularly CD4SP cells, can be obtained by the production method of the present invention. However, when CD8+ T cells are used as the starting material, the obtained CD4+ T cells maintain class I-restricted antigen specificity despite not expressing the CD8 molecule, which is a coreceptor. On the other hand, when CD4+ T cells are used as the starting material, the obtained CD4+ T cells maintain class II-restricted antigen specificity.

[0017] (1-2) Establishment of iPSCs from Human Somatic Cells The isolated human somatic cells are reprogrammed to iPSCs. Here, "iPSCs" are also called induced pluripotent stem cells or induced pluripotent stem cells, and can be induced by introducing a cellular reprogramming factor into somatic cells such as antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells, or fibroblasts.The "cellular reprogramming factor" is not particularly limited as long as it is a factor that can confer pluripotency to somatic cells, such as antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells, or fibroblasts, either alone or in cooperation with other pluripotency factors, when introduced into the somatic cells. However, it 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, from the viewpoint of enabling efficient establishment of iPSCs with fewer factors among these proteins, it is more preferable to introduce the four factors Oct3 / 4, Sox2, Klf4, and c-Myc into the antigen-specific T cells or peripheral blood-derived T cells. When the CD4+ T cells obtained by the present invention are used as a pharmaceutical agent such as a human immunotherapeutic agent, it is also preferable to introduce the three factors excluding c-Myc or to use L-Myc instead of c-Myc in order to reduce the risk of tumorigenesis. Other preferred combinations of reprogramming factors are described, for example, in WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101 407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655 , WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WODescribed in 2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9, etc.

[0018] In the present invention, the method for introducing the cellular reprogramming factor into human somatic cells such as the antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells, or fibroblasts is not particularly limited, and can be appropriately selected from known methods and introduced in the form of a protein or nucleic acid. For example, when introducing the cellular reprogramming factor into human somatic cells such as the antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells, or fibroblasts in the form of a nucleic acid, the nucleic acid (e.g., cDNA, RNA) can be inserted into an appropriate expression vector (e.g., a viral vector, a non-viral vector) containing a promoter that functions in the somatic cells, and the expression vector can be introduced into the cells by viral infection, lipofection, liposome method, electroporation, calcium phosphate coprecipitation, DEAE-dextran method, microinjection, or the like.

[0019] Examples of such expression vectors include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as animal cell expression plasmids. However, it is preferable to use Sendai virus to introduce the nucleic acid encoding the cellular reprogramming factor into human somatic cells, as it is less likely to cause insertional mutagenesis, has high gene transfer efficiency, and allows for a large number of copies of the introduced gene. Examples of promoters used in such expression vectors include the SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, and HSV-TK promoter. 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). In addition to the promoter, the expression vector may also contain an enhancer, a poly(A) addition signal, a selectable marker gene (e.g., a neomycin resistance gene), an SV40 origin of replication, and the like.

[0020] Furthermore, when introducing each of these reprogramming factors, it is preferable to express SV40 large T antigen in the target cells together with each reprogramming factor, in order to increase the proliferation and survival rate required for reprogramming of the cells. Therefore, an optimal embodiment for introducing cellular reprogramming factors into the antigen-specific T cells or peripheral blood-derived T cells is to infect the antigen-specific T cells or peripheral blood-derived T cells with a Sendai virus vector into which nucleic acids encoding the four factors Oct3 / 4, Sox2, Klf4, and c-Myc have been inserted, and a Sendai virus vector into which nucleic acid encoding SV40 large T antigen has been inserted. However, the antigen-specific T cells or peripheral blood-derived T cells may also be established using a Sendai virus vector containing additional factors, or may be established without using a Sendai virus vector into which nucleic acid encoding SV40 large T antigen has been inserted. As a vector other than Sendai virus, the use of an episomal vector such as oriP / EBNA-1 is preferred. Alternatively, the reprogramming factors may be introduced in the form of mRNA, in which case they can be introduced using well-known and conventional techniques (nucleic acid modification, introduction by lipid nanoparticles) for mRNA vaccines and the like.

[0021] The medium for culturing the antigen-specific T cells or peripheral blood-derived T cells may be, for example, a known medium suitable for culturing T cells (more specifically, Roswell Park Memorial Institute (RPMI) 1640 medium, AIM V medium containing other cytokines and human serum). ＴＭ The medium may contain amino acids (e.g., L-glutamine) and antibiotics (e.g., streptomycin and penicillin) necessary for the culture.

[0022] Furthermore, there are no particular limitations on the conditions for or after introducing the cell reprogramming factor into the antigen-specific T cells or peripheral blood-derived T cells, but the antigen-specific T cells or peripheral blood-derived T cells into which the factor has been introduced are preferably cultured under feeder-free conditions. Examples include wells coated with iMatrix-511 solution, which is a laminin 511E8 fragment, or vitronectin or Matrigel. Cultures can also be established under feeder cell conditions. Examples of feeder cells include mouse embryonic fibroblasts (MEFs), STO cells, and SNL cells whose cell division has been arrested by irradiation or antibiotic treatment.

[0023] Furthermore, in the process of inducing T-iPSCs from the antigen-specific T cells or peripheral blood-derived T cells, it is preferable to add an iPSC medium from the next day. After that, it is preferable to gradually replace the CTL medium with the iPS medium by replacing half of the medium every other day.

[0024] Furthermore, it is preferable to gradually replace a known medium suitable for T cell culture with a medium suitable for iPSC culture as the antigen-specific T cells or peripheral blood-derived T cells transition to iPSCs. The medium suitable for iPSC culture can be appropriately selected from known media, and examples of suitable media include StemFit AK03N for iMatrix-coated cells or Essential 8 Medium for vitronectin-coated cells, mTeSR for Matrigel, and Dulbecco's modified Eagle's medium / F12 medium (human iPSC medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like for feeder cells such as MEF cells.

[0025] Selection of T-iPSCs induced from the antigen-specific T cells or peripheral blood-derived T cells in this manner can be performed by appropriately selecting a known method. Examples of such known methods include a method in which iPSC-like colonies are selected by observing their morphology under a microscope. Alternatively, all established T-iPSC colonies may be passaged as they are, without selecting each colony.

[0026] The fact that the selected cells are T-iPSCs can be confirmed, for example, by detecting the expression of undifferentiated cell-specific markers (SSEA-4, Tra-1-60, Tra-1-81, etc.) in the selected cells by q-PCR or by ALP staining, or by transplanting the selected cells into mice and observing the formation of teratomas.

[0027] The timing for 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 introduction of the cell reprogramming factor into the antigen-specific T cells or peripheral blood-derived T cells. ２ The conditions are 35 to 38° C., and more preferably 37° C. Efficient growth of colonies can be observed even in a low-oxygen culture environment (oxygen concentration: for example, 5%).

[0028] (2) Manipulation of Gene Expression from Established T-iPSCs In this step, target genes (RUNX3, ZBTB7B, SOCS1, SOCS3, and / or GIMAP5) are knocked out and / or RUNX1 is overexpressed from established T-iPSCs. These target genes have been suggested to be involved in CD4 / CD8 lineage selection. In the Examples described below, the present inventors have found for the first time that ATL-iPSC-derived CD4+ T cells have reduced expression of these genes (RUNX3, ZBTB7B, SOCS1, SOCS3, and / or GIMAP5) and increased expression of RUNX1, compared to CD8+ T cells obtained by inducing differentiation from conventional T-iPSCs.

[0029] The target gene to be knocked out is not particularly limited as long as it is one or more of the above five genes, but preferably includes RUNX3. Knocking out these genes or overexpressing RUNX1 can be achieved by gene transfer techniques using viral vectors of known target genes, genome editing techniques, etc., with genome editing techniques being preferred. To avoid the risk of insertional mutagenesis, overexpression of the RUNX1 gene can be achieved, for example, by knocking in the RUNX1 gene under the control of a constitutively expressible promoter or a T cell-specific promoter at the RUNX1 locus endogenous to iPSCs, or by introducing the RUNX1 gene using a viral vector such as Sendai virus, lentivirus, or retrovirus, or another episomal vector. Preferably, the RUNX1 gene is knocked in.

[0030] Genome editing technologies include ZFN, TALEN, and CRISPR-Cas9, with CRISPR-Cas9 being more preferred. CRISPR-Cas9 is composed of a guide RNA (gRNA) that specifically recognizes and binds to the target DNA sequence and the nuclease Cas9. Cas9, guided by the gRNA, cleaves the double strand of DNA, and knocks out the target gene when the cleaved double-stranded DNA is repaired. gRNA can design appropriate candidate sequences based on the sequence information of each target gene (provided by various public databases such as NCBI, and this information is easily accessible to those skilled in the art). Various commercially available tools (e.g., Chop Chop Harvard, CRISPR Design, etc.) can also be used as gRNA design tools. Knock-in of the RUNX1 gene can be performed by introducing donor DNA containing the RUNX1 gene under the control of the constitutively expressible promoter or the T cell-specific promoter into iPSCs together with gRNA.

[0031] Genome-edited T-iPSCs are cultured as single cells. Genomic DNA is extracted from the resulting single colonies and subjected to genotyping PCR. The PCR products are purified and Sanger sequenced to confirm that the genomic DNA from each single-cell colony has acquired a frameshift mutation, resulting in an amino acid sequence completely different from the wild-type after the insertion / deletion, and that a stop codon has also been acquired along the way.

[0032] In cells in which the target gene has been knocked out or knocked in, the state of TCR gene rearrangement can be detected by genomic PCR, thereby confirming that the cells from which the T-iPSCs were derived are derived from antigen-specific T cells.

[0033] The genetically modified iPSCs thus obtained can be induced to differentiate into T cells by methods known per se, such as those described in Patent Documents 1 to 4. For example, when inducing T cells from iPSCs, the cells may be activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in the presence of interleukin-2 (IL-2), interleukin-7 (IL-7), or interleukin-15 (IL-15), or by stimulation with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 antibody, anti-CD28 antibody, CD3 agonist, and CD28 agonist. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody, and / or anti-CD28 antibody to a medium and culturing the cells undergoing differentiation induction for a certain period of time. The anti-CD3 antibody and / or anti-CD28 antibody may be bound to magnetic beads or the like, and instead of adding these antibodies to the medium, the cells may be stimulated by culturing them for a certain period of time on a culture dish to which the anti-CD3 antibody and / or anti-CD28 antibody is bound on the surface. More specifically, the method described in Reference Example 1(3) below is a preferred example, but is not limited to this.

[0034] The period of the differentiation-inducing culture of iPSCs into T cells is not particularly limited as long as a sufficient CD4+ fraction is obtained, but is, for example, 3 weeks or more, preferably 5 weeks or more. The upper limit of the culture period is not particularly limited, but may be, for example, 15 weeks or less, preferably 12 weeks or less. The increase in the proportion of the CD4+ T cell population, preferably the CD4SP T cell population, can be confirmed, for example, by collecting a portion of the cells during the differentiation-inducing culture and performing flow cytometry using an anti-CD4 antibody, preferably an anti-CD8 antibody.

[0035] The CD4+ T 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 CD4 and a cell sorter. Furthermore, whether the obtained CD4+ T cells are regulatory can be confirmed by analyzing the expression of a surface antigen marker characteristic of regulatory T cells, such as Foxp3, by flow cytometry using an antibody against the marker.

[0036] Furthermore, the present inventors confirmed that when RUNX3-overexpressing ATL-iPSCs were induced to differentiate by forced expression of RUNX3, they failed to differentiate into CD4+ T cells, demonstrating that the expression level of RUNX3 determines the induction of differentiation into CD4 / CD8 cells. Similarly, differential expression analysis of ATL-iPSC-CD4+ T cells and CD4T-iPSC-CD8+ T cells identified ZBTB7B, SOCS1, SOCS3, and GIMAP5 as having decreased expression in the former, and RUNX1 as having increased expression in the former, and by controlling their expression, the induction of differentiation into CD4 / CD8 cells can be controlled.

[0037] The T-iPSC-derived CD4+ T cells obtained by the production method of the present invention may be any type as long as they are induced by any of the methods described above and are CD4 positive, particularly CD4 single positive (SP), but preferably have the following properties (a) to (e): (a) are CD4 single positive; (b) have a TCR specific to an antigen peptide presented on cancer cells or virus-infected cells; (c) exhibit one or more of the following Th1 cell phenotypes; (c1) secrete high levels of IFNγ and IL-2; (c2) highly express one or more genes selected from the group consisting of TBX21, STAT1, and STAT4; (c3) express CD107a and CD366; (d) have cytotoxic activity specific to the antigen peptide of (b) alone; and (e) enhance the proliferation and / or cytotoxic activity of cancer antigen- or virus antigen-specific CTL or CAR-T cells.

[0038] When the original antigen-specific T cells were CD8+ T cells, the rearranged TCR was inherited even during reprogramming, and thus CD4+ T cells obtained via iPSCs are class I-restricted. Because CD4SP T cells do not express CD8 molecules, one would expect reduced TCR binding to antigen peptide-class I molecule complexes and reduced activation capacity due to the lack of CD8 stimulation. However, surprisingly, the resulting CD4SP T cells abundantly produce cytotoxic cytokines such as IFNγ, TNFα, perforin, and granzyme B, and retain their original antigen-specific cytotoxic activity.

[0039] Furthermore, these CD4SP T cells secrete abundant cytokines and function as helper T cells, activating antigen-presenting cells and activating CD8+ T cells (CTLs), thereby further enhancing the cytotoxic activity of CTLs. These CD4SP T cells abundantly secrete Th1 cytokines such as IFNγ and IL-2, and exhibit a Th1 cell phenotype based on the expression profiles of transcription factors and surface antigens. Therefore, they are expected to function as Th1 cells, activating target cells such as macrophages, CTLs, and NK cells, thereby providing infection protection and anti-tumor immunity. One of the reasons why cancer immunotherapy using peripheral blood CTLs is not as effective as expected is that CD8+ CTLs have low IL-2 production, which may lead to their own death before killing cancer cells. However, the CD4SP cells obtained by the present invention have a high IL-2 secretion ability, which is expected to promote the survival and proliferation of CTLs and further enhance their cytotoxic activity. Furthermore, Th1 cell therapy is believed to be capable of suppressing the increase and accumulation of regulatory T cells, which are induced in tumors and regional lymph nodes as cancer grows, in an IFNγ-dependent manner, thereby suppressing cancer immune escape.

[0040] In a preferred embodiment, the CD4SP cells obtained by the present invention secrete high levels of cytokines such as IL-4 and IL-10 in addition to Th1 cytokines.

[0041] iPSC-derived CD4SP T cells having the above characteristics are novel T cells that have not been previously known. Accordingly, the present invention also provides human iPSC-derived T cells (hereinafter also referred to as "CD4+ T cells of the present invention") having the following properties: (a) being CD4 single-positive; (b) having a TCR specific for an antigenic peptide presented on cancer cells or virus-infected cells; (c) exhibiting one or more of the following Th1 cell phenotypes: (c1) secreting IFNγ and IL-2 at high levels; (c2) expressing one or more genes selected from the group consisting of TBX21, STAT1, and STAT4 at high levels; (c3) expressing CD107a and CD366; (d) independently possessing cytotoxic activity specific to the antigenic peptide of (b); and (e) enhancing the proliferation and / or cytotoxic activity of cancer antigen- or virus antigen-specific CTL or CAR-T cells.

[0042] The CD4+ T cells of the present invention may be obtained by any method as long as they have the above-mentioned properties, but preferably, in the T cells, expression of one or more genes selected from RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 is suppressed and / or expression of the RUNX1 gene is enhanced. Preferably, in the CD4+ T cells of the present invention, expression of at least the RUNX3 gene is suppressed. There are no particular limitations on the form of suppression of expression of these genes, but preferably, the genes are knocked out or overexpressed (preferably knocked in) by genetic manipulation.

[0043] It was confirmed that the CD4+ T cells obtained by the production method of the present invention are not regulatory T cells. It was also confirmed that the CD4+ T cells exhibit a Th1 cell phenotype. Therefore, the CD4+ T cells of the present invention, including the CD4+ T cells obtained by the production method of the present invention, can be used, for example, in various cancer immunotherapies and immunotherapies for infectious diseases, preferably viral infections. Therefore, the present invention provides a pharmaceutical composition, preferably an immunotherapeutic agent, comprising the CD4+ T cells obtained by the production method of the present invention or the CD4+ T cells of the present invention.

[0044] The pharmaceutical composition of the present invention can be a drug for preventing or treating cancer or infectious diseases (e.g., viral infections). Cancers to which the pharmaceutical composition is applied are not particularly limited, and examples thereof include acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia), and the like. , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; cancer of the peritoneum, omentum and mesentery; pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, etc. Furthermore, the infectious diseases to which the pharmaceutical composition is applied are not particularly limited, and examples thereof include viral infections (e.g., RNA viruses, DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C viruses, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV)), parasitic infections (e.g., Plasmodia species, Protozoan and metazoan pathogens such as Leishmania species, Schistosoma species, and Trypanosoma species), bacterial infections (e.g., Mycobacteria, especially M. tuberculosis, Salmonella, Streptococci, E. coli, and Staphylococci), fungal infections (e.g., Candida species, Aspergillus species, and Pneumocystis carinii), and the like.

[0045] The pharmaceutical composition of the present invention can be used in combination with cancer antigen- or viral antigen-specific CTLs or CAR-T cells. The CD4+ T cells of the present invention themselves exhibit cytotoxic activity specific to the antigen recognized by the TCR of the original T cell, and can proliferate and activate other CTLs or CAR-T cells, thereby enhancing their cytotoxic activity. Therefore, the combined use of both can achieve a higher therapeutic effect than the administration of CTLs or CAR-T cells alone.

[0046] The pharmaceutical composition of the present invention can be used in combination with antigen-presenting cells (e.g., dendritic cells) and / or antigen peptides presented on cancer cells or virus-infected cells. The CD4+ T cells of the present invention can activate antigen-presenting cells to activate CTLs specific to the antigen peptide, and therefore, by using both in combination, cancer antigen- or virus antigen-specific CTLs can be efficiently induced in a subject.

[0047] The pharmaceutical composition of the present invention can be prepared by formulating the CD4+ T cells obtained by the production method of the present invention or the CD4+ T cells of the present invention using a known pharmaceutical method, and can be administered primarily parenterally, for example, as an injection (intravenous injection, drip infusion injection, etc.), capsule, liquid, film-coated agent, etc.

[0048] In preparing these formulations, the composition may be appropriately combined with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline for stably maintaining cells, phosphate buffered saline (PBS), culture medium (RPMI, AIM-V, X-VIVO10, etc.), vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer (e.g., human serum albumin), vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, solubilizing agent, or other additives. The composition may also be used in combination with known pharmaceutical compositions or immunostimulants used in the treatment or prevention of the aforementioned diseases.

[0049] The CD4+ T cells of the pharmaceutical composition of the present invention may be cultured in an appropriate medium before administration to a subject. Stimulatory molecules may also be added to the medium to maintain and / or amplify T cell activation and / or proliferation. Furthermore, serum or plasma may be added to the medium. The amount of these substances added to the medium is not particularly limited, but examples include 0% to 20% by volume. The amount of serum or plasma used can be varied depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources. While autologous sources are preferred from the viewpoint of reducing immune rejection, non-autologous sources are also useful in terms of rapid preparation and ensuring sufficient cell numbers.

[0050] The pharmaceutical composition of the present invention is preferably administered parenterally to a subject. Examples of parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, body weight, age, symptoms, and type of composition of the subject. Generally, the number of cells is 1×10 per administration for a subject weighing 60 kg. ６ ~1 x 10 １０ Preferably, 1×10 ７ ~1 x 10 ９ 5×10 ７ ~5 x 10 ８ The dose may be administered once or multiple times.

[0051] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0052] Reference Example 1 Production of CD4-positive T cells (regulatory T cells) from HTLV-1-infected CD4-positive T cells (1) Isolation of HTLV-1-infected CD4-positive T cells Peripheral blood mononuclear cells were separated from the peripheral blood of an ATL patient, and then CD4-positive T cells were isolated by positive selection using CD4 beads. The phenotype of the obtained T cells was analyzed by flow cytometry using various cell surface markers and confirmed to be strongly positive for CD3CD4CD25.

[0053] (2) Establishment of T-iPSCs The HTLV-1-infected CD4+ T cells obtained in (1) were transfected with a Sendai virus (SeV) vector (SeVp [KOSM302L]) containing nuclear reprogramming factors (Oct4, Sox2, Klf4, c-Myc) and a SeV vector containing a nucleic acid encoding the SV40 large T antigen. The transfected T cells were transferred to a 6-well plate coated with iMatrix and incubated in a T cell medium (medium composition: RPMI, 10% human AB serum) supplemented with IL-2 for 24 hours at 20°C. ２ The cells were cultured in an incubator. The day after gene transfer, an equal volume of iPS medium (StemFitAK03N) was added, and then half the volume was replaced with StemFitAK03N every other day. T-iPSC colonies could be observed after 21 days, after which colonies were picked.

[0054] (3) Induction of Differentiation from CD4 T-iPSCs to CD4+ Cells. The T-iPSC colonies obtained in (2) were finely disrupted and cultured on 10T1 / 2 feeder cells in a medium containing VEGF, FBS, insulin, transferrin, L-glutamine, α-monothioglycerol, ascorbic acid, etc., for the first week in hypoxic culture and for the second week in an incubator at 20% oxygen concentration. The cells were then cultured on 10T1 / 2 feeder cells expressing Notch Ligand with cytokines (IL-7, FLT3L, SCF) for an additional 4 weeks, with replacement of the feeder cells with new ones once a week. After 4 weeks, all floating cells were collected and T cell receptor (TCR) stimulated. TCR stimulation was performed using X-ray irradiated allogeneic peripheral blood mononuclear cells and PHA or anti-CD3 / CD28 antibodies approximately every 2 weeks.

[0055] The phenotype of T cells obtained by differentiation induction from CD4 T-iPSCs was analyzed by intracellular staining and flow cytometry. As a result, CD4+ T cells induced from HTLV-1-infected CD4 T-iPSCs were FOXP3-positive and CD25-positive, and exhibited a regulatory T cell-like phenotype.

[0056] Example 1: Gene expression of ATL-iPSC-derived CD4+ T cells obtained in the above Reference Example was compared with that of CD4T-iPSC-derived CD8+ T cells established from CD4T cells of a healthy donor using single-cell RNA sequencing analysis. For single-cell RNA sequencing analysis, emulsions were prepared and RNA was extracted from ATL-iPSC-derived CD4+ T cells and CD4T-iPSC-derived CD8+ T cells using Chromium Next Gem Single Cell 3' Reagent Kits v3.1 (Dual Index) and a 10x Chromium Controller System according to the 10x Genomics protocol, and barcodes were assigned. Library preparation was then performed, and samples were evaluated on a Bioanalyzer 2100 before being externally sequenced using HiseqX (Illumina, San Diego, CA). FastQ files were aligned to GRCh38 using Cell Ranger (v7.0.0) (10x Genomics). RNA data normalization and downstream analysis were performed using the Seurat R package (version 4.3.0), which allows for integrated processing of multimodal single-cell datasets.

[0057] Figure 1 shows the results of comparing the expression of RUNX3, RUNX1, ZBTB7B, SOCS1, SOCS3, and GIMAP5 in CD4T-iPSC-derived CD8+ T cells and ATL-iPSC-derived CD4+ T cells using UMAP. In ATL-iPSC-derived CD4+ T cells, RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 were expressed at low levels, while RUNX1 was expressed at high levels. In contrast, in CD4T-iPSC-derived CD8+ T cells, these five genes were expressed at high levels, while RUNX1 was expressed at low levels.

[0058] Example 2: Because the genes identified by the differential expression analysis in Example 1 were suggested to be related to CD4 / CD8 lineage selection, the inventors hypothesized that knocking out or knocking in these genes in T-iPSCs might enable differentiation of T-iPSCs into CD4+ T cells. Therefore, using the genome editing method described above, one of the identified genes, RUNX3, was knocked out by nucleofection of gRNA using CRISPR / Cas9. After knockout, T-iPSCs were cultured for 7-10 days and then reseeded to form single cells. Colonies derived from single cells were picked, cultured, and genomic DNA was extracted. Genotyping PCR was performed using the extracted genomic DNA as a template, and the resulting PCR products were Sanger sequenced. The information obtained by Sanger sequencing confirmed that single-base deletion or two-base insertion, or two-base deletion or single-base insertion, resulted in amino acid mutations downstream of the insertion or deletion site. Furthermore, due to the occurrence of a stop codon during the process, the gene was shorter than the wild-type gene, and the amino acid mutations resulted in incomplete translation. The resulting RUNX3 knockout T-iPSCs were induced to differentiate into T cells using a method similar to that described in Reference Example 1 (3). The expression of CD4 and CD8 was monitored over time by flow cytometry after the initiation of differentiation induction. The results are shown in Figure 2. At 3 weeks after the initiation of differentiation induction, CD4- / CD8- and CD4+ / CD8- cell populations were present, with the CD4+ / CD8- cell population subsequently increasing. Flow cytometry at 10 weeks showed the state after CD4 selection using CD4 beads, and CD4+ / CD8- iPSC-derived CD4+ T cells were successfully obtained.

[0059] For comparison, Figure 3 shows the time course of the CD4 / CD8 ratio as determined by flow cytometry during differentiation induction of hematopoietic progenitor cells derived from wild-type CD4 T-iPSCs. After extraction of the hematopoietic progenitor cells, they were continuously cultured on feeder cells, and flow cytometry was performed weekly using a T cell surface antigen marker. Three weeks after the start of differentiation induction, the CD4- / CD8- cell population was predominant, and from week 8 onwards, a predominantly CD8+ cell population emerged.

[0060] Example 3 To demonstrate that the expression level of RUNX3 determines the induction of differentiation into CD4 / CD8, RUNX3 was overexpressed in ATL-iPSCs, which originally had low RUNX3 expression and which could be induced to differentiate into CD4+ T cells, and we investigated whether CD4 or CD8 cells were obtained by differentiation induction. ATL-iPSCs were induced to differentiate, and RUNX3 was overexpressed using a retroviral vector at the stage of hematopoietic progenitor cells. Differentiation was then induced in the same manner as before, and the CD4 / CD8 ratio was analyzed over time by flow cytometry.

[0061] The results are shown in Figure 4. Three weeks after the start of differentiation induction, the CD4- / CD8- cell population was predominant, but from week 9 onwards, a predominantly CD8+ cell population emerged. That is, without overexpression of RUNX3, ATL-iPSCs differentiated into CD4+ T cells, whereas overexpression of RUNX3 induced differentiation into CD8+ T cells. This demonstrates that high levels of RUNX3 expression induce differentiation into CD8+ T cells, whereas low levels of RUNX3 expression induce differentiation into CD4+ T cells.

[0062] Example 4 CD4+ T cells obtained by differentiation induction from RUNX3-knockout T-iPSCs were analyzed by flow cytometry for Foxp3, a characteristic of regulatory T cells, and CD45RA, a naive cell marker, using antibodies against each antigen. Figure 5 shows the results of flow cytometry analysis of CD4+ T cells derived from RUNX3 KO CD4T-iPSCs using anti-CD45RA and anti-Foxp3 antibodies. CD4+ T cells derived from RUNX3 KO CD4T-iPSCs were CD4+, CD45RA-, and Foxp3-. On the other hand, CD4+ T cells derived from ATL-iPSCs were observed as a CD4+, CD45RA-, and Foxp3+ fraction, as well as a CD4+, CD45RA+, and Foxp3low fraction. Specifically, RUNX3-knockout T-iPSC-derived CD4+ T cells did not exhibit the naive regulatory T cell (CD45RA+, Foxp3low) or activated regulatory T cell (CD45RA-, Foxp3high) fractions seen in ATL-iPSC-CD4+ T cells. These findings confirm that RUNX3-knockout T-iPSC-derived CD4+ T cells are not regulatory T cells.

[0063] Example 5: LMP2-specific CTLs were isolated from peripheral blood mononuclear cells derived from healthy individuals by standard methods. iPSCs were induced from the LMP2-specific CTLs by the same method as in Reference Example 1, and RUNX3 was knocked out by the same method as in Example 2. iPSCs derived from wild-type LMP2-specific CTLs and iPSCs derived from RUNX3-knocked-out LMP2-specific CTLs were each induced to differentiate into T cells by the same method as in Reference Example 1(3), and analyzed by flow cytometry using an anti-CD4 antibody or anti-CD8 antibody and an LMP2-class I tetramer.

[0064] The results are shown in Figure 6. When T cells are induced to differentiate from iPSCs derived from antigen-specific LMP2-CTLs, they usually differentiate into CD8 SP cells (Figure 6, left). However, knocking out RUNX3 led to differentiation into CD4 SP cells (Figure 6, right). The original LMP2-specific CTLs were class I-restricted CD8 SP CTLs, but knocking out RUNX3 allowed them to differentiate into CD4 SP cells while maintaining class I-restricted LMP2 antigen specificity.

[0065] Example 6 The antigen-specific cytotoxic activity of RUNX3 KO LMP2-iPSC-T cells prepared in Example 5 was evaluated by chromium assay using three types of LMP2-expressing cells (lymphoma-derived NK-YS, ENKL-J1, and EBV-infected T lymphocytes, CAEBV). LMP2-iPSC-T cells (CD8+) were used as a positive control, and primary ATL cells were used as a negative control. The results are shown in Figure 7. RUNX3 KO LMP2-iPSC-T cells possessed antigen-specific cytotoxic activity, although it was inferior to that of LMP2-iPSC-T cells.

[0066] Therefore, the IFNγ secretion ability of RUNX3 KO LMP2-iPSC-T cells was evaluated by ELISPOT assay. Furthermore, the production of the cytotoxic cytokines perforin and granzyme B was evaluated by flow cytometry. The results are shown in Figure 8. RUNX3 KO LMP2-iPSC-T cells secreted as much IFNγ as LMP2-iPSC-T cells in response to antigen stimulation (Figure 8A). Furthermore, they were shown to produce perforin and granzyme B in response to antigen stimulation (Figure 8B).

[0067] Example 7: The ability of RUNX3 KO LMP2-iPSC-T cells to secrete various cytokines was investigated. The results are shown in Figure 9. In response to antigen stimulation, RUNX3 KO LMP2-iPSC-T cells secreted high levels of IL-2, secreted by Th1 cells, as well as IL-4 and IL-10, in addition to cytokines secreted at high levels by CTLs, such as IFNγ and TNF-α.

[0068] Example 8 To further evaluate whether RUNX3 KO LMP2-iPSC-T cells exhibit a Th1 cell phenotype, we examined the expression of transcription factors and cell surface molecules characteristic of Th1 cells. The results are shown in Figure 10. RUNX3 KO LMP2-iPSC-T cells highly expressed TBX21, STAT1, and STAT4 (Figure 10A), and were positive for CD107a and CD366 (Figure 10B). These results demonstrate that RUNX3 KO LMP2-iPSC-T cells exhibit a Th1 cell phenotype.

[0069] Example 9: HPV16E6-specific CTLs were isolated from peripheral blood mononuclear cells of healthy individuals by standard methods. iPSCs were induced from HPV16E6-specific CTLs by the same method as in Reference Example 1, and RUNX3 was knocked out as in Example 2. RUNX3 KO HPV16E6-iPSCs were induced to differentiate into T cells by the same method as in Reference Example 1(3). The cytotoxic activity of the resulting RUNX3 KO HPV16E6-iPSC-T cells in combination with HPV16E6-iPSC-T cells (CD8+) induced to differentiate from wild-type HPV16E6-iPSCs was evaluated by chromium assay as in Example 6. The results are shown in Figure 11. HPV16E6-iPSC-T cells exhibited specific cytotoxic activity against the HPV16E6-expressing cervical cancer cell line SiHa (series 2), and this activity was further enhanced by coculture with RUNX3 KO HPV16E6-iPSC-T cells (series 1).

[0070] Example 10: GD2-targeting chimeric antigen receptor T cells (GD2-CAR-T) were prepared from peripheral blood mononuclear cells derived from healthy individuals by a standard method (Kinoshita et al., Cancer Research Communications 2024). The cytotoxic activity of the RUNX3 KO LMP2-iPSC-T cells prepared in Example 5 in combination with the GD2-CAR-T cells obtained above was evaluated by chromium assay in the same manner as in Example 6. In addition, cell proliferation was compared between when each cell type was cultured alone and when the two were co-cultured.

[0071] The results are shown in Figure 12. GD2-specific CAR-T cells (GD2-CARTs) and RUNX3 KO LMP2-iPSC-T cells (CD4rejTs) were cultured at 1 x 10 ６ Compared with the condition where the cells were cultured separately for 72 hours, the co-culture condition (1:1 = 0.5 × 10 ６ : 0.5 x 10 ６) significantly increased the cell number (Figure 12A). Furthermore, when the cytotoxic activity against GD2-positive EBV-associated lymphoma cells was evaluated by chromium assay in GD2-CARTs cultured for 72 hours and in CD4rej Ts co-cultured with GD2-CARTs (1:1) for 72 hours, the cytotoxic activity was enhanced in the co-culture compared to CAR-T cells alone (Figure 12B).

[0072] Example 11 The combined effect of GD2-specific CAR-T cells and RUNX3 KO LMP2-iPSC-T cells was confirmed by animal experiments. 1 x 10 GD2-positive EBV-associated lymphoma cells were injected into NOG mice. ６ Each cell was transplanted intraperitoneally, and 5 days later, effector cells obtained by culturing GD2-CARTs alone for 72 hours or co-culturing GD2-CARTs + CD4rej Ts (1:1) for 72 hours were added to the mice at a concentration of 4 × 10 ６ The cells were administered by intraperitoneal injection, and tumor growth was observed over time. The results are shown in Figure 13. At least three weeks after effector cell administration, the tumor growth inhibitory effect was more pronounced in mice administered GD2-CARTs + CD4rejTs than in mice administered GD2-CARTs alone.

[0073] The iPSC-derived CD4+ T cells obtained by the present invention retain their own cytotoxic activity and function as Th1 cells, and are therefore highly useful as immunotherapeutic agents for the treatment of cancer and infectious diseases, either alone or in combination with CAT-T, TCR-T cells, or CTLs. This application is based on Japanese Patent Application No. 2023-089785 (filing date: May 31, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing CD4+ T cells derived from human induced pluripotent stem cells (iPSCs), comprising the steps of knocking out one or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3 and GIMAP5, and / or overexpressing the RUNX1 gene in iPSCs.

2. The method of claim 1, wherein the iPSCs are derived from antigen-specific T cells, peripheral blood-derived T cells, peripheral blood mononuclear cells or fibroblasts.

3. The method according to claim 1 or 2, wherein the gene to be knocked out comprises RUNX3.

4. The method according to any one of claims 1 to 3, wherein the CD4+ T cells are CD4 single positive.

5. The method according to any one of claims 2 to 4, wherein the antigen-specific T cells are specific to an antigen peptide presented on cancer cells or virus-infected cells, or the peripheral blood-derived T cells or peripheral blood mononuclear cells are derived from a cancer patient or a virus-infected individual.

6. The method according to any one of claims 2 to 5, wherein the antigen-specific T cells or peripheral blood-derived T cells are CD8+ T cells.

7. The method according to any one of claims 2 to 5, wherein the antigen-specific T cells or peripheral blood-derived T cells are CD4+ T cells.

8. Human CD4+ T cells obtained by the method according to any one of claims 1 to 7.

9. A human iPSC-derived T cell having the following properties: (a) being CD4 single positive; (b) having a TCR specific to an antigen peptide presented on cancer cells or virus-infected cells; (c) exhibiting one or more of the following Th1 cell phenotypes; (c1) secreting IFNγ and IL-2 at high levels; (c2) highly expressing one or more genes selected from the group consisting of TBX21, STAT1, and STAT4; (c3) expressing CD107a and CD366; (d) having cytotoxic activity specific to the antigen peptide of (b) alone; and (e) enhancing the proliferation and / or cytotoxic activity of cancer antigen- or virus antigen-specific CTL or CAR-T cells.

10. The human iPSC-derived T cells of claim 9, in which one or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3 and GIMAP5 are knocked out, and / or the RUNX1 gene is overexpressed.

11. A pharmaceutical composition comprising the human iPSC-derived CD4+ T cells according to any one of claims 8 to 10.

12. The pharmaceutical composition of claim 11, which is an immunotherapeutic agent.

13. The pharmaceutical composition according to claim 12, for use in treating cancer or viral infections.

14. The pharmaceutical composition according to claim 13, which is combined with cancer antigen- or viral antigen-specific CTL or CAR-T cells.

15. The pharmaceutical composition according to claim 13, which is combined with an antigen-presenting cell and / or an antigen peptide presented on a cancer cell or a virus-infected cell.

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