T cells specific to epstein-barr virus (EBV)

Abstract

The present disclosure relates to T cells specific to Epstein-Barr virus (EBV), particularly rejuvenated T cells (rejT) that allow allogeneic administration.

Description

T cells specific to Epstein-Barr virus (EBV)

[0001] This disclosure relates to T cells specific to Epstein-Barr virus (EBV).

[0002] Epstein-Barr virus (EBV)-associated lymphoma and chronic active EBV disease are diseases with a poor prognosis associated with EBV infection, exhibiting a latent state in which the nuclear antigen (EBNA) and latent membrane proteins (LMP1 / LMP2) are expressed.

[0003] It has been shown that iPSC technology can reactivate exhausted antigen-specific CTLs, resulting in T cells (rejT) that proliferate more vigorously than the original CTLs, exhibit a younger memory T cell phenotype, and possess antigen specificity. Similarly, virus-specific rejT can be obtained from virus-specific CTLs, exhibiting a potent antitumor effect (Nishimura T. et al., Cell Stem Cell, 2013;12(1):114-126, Ando M. et al., Stem Cell Reports, 2015;5(4):598-608, Ando M. et al., Haematology, 2020;105(3):796-807). WO2024190905A discloses a treatment method utilizing human papillomavirus (HPV)-specific rejT.

[0004] This disclosure provides T cells specific to Epstein-Barr virus (EBV) antigens (e.g., EBNA, latent membrane protein, etc.).

[0005] The present disclosure provides, for example, the following inventions: (1) A composition comprising a population of cells specific to the antigen of Epstein-Barr virus (EBV) (particularly CD4 / CD8DN T cells, CD4 / CD8 single-positive T cells, particularly CD8+ T cells, preferably cytotoxic T cells or CD4+ T cells, preferably Th1 cells). (2) The composition according to (1), wherein the T cells have reduced or absent HLA class I expression, and when co-cultured with allogeneic T cells, the CD107a expression level is lower compared to cells that do not have reduced or absent HLA class I expression. (3) The composition according to (1) or (2), wherein 5% or more of the T cells are stem cell memory T cells. (4) The composition according to any one of (1) to (3), wherein at least one HLA allele is exogenously introduced. (5) The composition according to any one of (1) to (4) above, wherein the T cells (especially CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) are T cells (i.e., rejT) (especially CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) obtained by re-inducing differentiation of the above-mentioned specific CD8+ T cells (first cells) obtained from a human patient having EBV infection after reprogramming. (6) The composition according to any one of (1) to (5) above, wherein the T cells (particularly CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) have lower expression of marker genes selected from the group consisting of PD-1, TIGIT, and LAG-3, and higher expression of factors selected from the group consisting of TIM-3, interferon-γ, and PRF1, compared with the first cells. (7) The composition according to any one of (1) to (6) above, which exhibits higher cytotoxicity to the antigen-expressing cells compared with the first cells.(8) The cell population includes T cells specific to the Epstein-Barr virus (EBV) antigen (particularly CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells), wherein the T cells have reduced or absent HLA class I expression, and when co-cultured with allogeneic T cells, the CD107a expression level is lower compared to cells that do not have reduced or absent HLA class I expression, and at least one HLA allele has been exogenously introduced, and 5% or more of the T cells are stem cell memory T cells, and the T cells (particularly CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) are T cells obtained by re-induction of differentiation after reprogramming of the above-mentioned specific CD8+ T cells (first cells) obtained from human patients with EBV infection (i.e., rejT) (particularly CD4 / CD8DN The composition according to any one of (1) to (7) above, wherein the T cells (especially CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) have lower expression of marker genes selected from the group consisting of PD-1, TIGIT, and LAG-3, and higher expression of factors selected from the group consisting of TIM-3, interferon-γ, and PRF1, compared to the first cells, and exhibit higher cytotoxicity against the antigen-expressing cells compared to the first cells. (9) The composition according to any one of (1) to (8) above, comprising an exogenously introduced HLA allele selected from the group consisting of HLA-A2402, HLA-A0201, HLA-A1101, and HLA-3303; HLA-A0101, HLA-A0201, HLA-A0301, and HLA-A1101; HLA-A3002, HLA-A2301, HLA-A0201, and HLA-A6801; HLA-A0201, HLA-A6802, and HLA-A2402; and HLA-A0101, HLA-A1101, HLA-A2601, and HLA-A2402.(10) The composition according to any one of (1) to (9) above, wherein the antigen is selected from the group consisting of LMP1 and LMP2. (11) The composition according to any one of (1) to (10) above, wherein T cells (particularly CD4 / CD8DN T cells, CD8+ T cells, preferably cytotoxic T cells, or CD4+ T cells, preferably Th1 cells) express the TCRα chain according to (12) and the TCRβ chain according to (13). (12) The amino acid sequence according to SEQ ID NO: 1 or 6, or having 1 to 3 amino acid mutations with respect to said amino acid sequence, which binds to LMP2, and the nucleic acid encoding said TCRα chain. (13) The amino acid sequence according to SEQ ID NO: 11, or having 1 to 3 amino acid mutations with respect to said amino acid sequence, which binds to LMP2, and the nucleic acid encoding said TCRβ chain. (14) T cells expressing the TCRα chain described in (12) and the TCRβ chain described in (13). (15) T cells described in (14) that are cytotoxic T cells. (16) A composition according to any one of (1) to (10) and (14) above for use in treating EBV infection in a patient having EBV infection.

[0006] (21) A method for producing a composition, wherein the composition comprises a cell population containing antigen-specific T cells, wherein 5% or more of the T cells are stem cell memory T cells, and the method comprises: inducing induced pluripotent stem cells from T cells obtained from a subject; inducing T cells from the obtained induced pluripotent stem cells (preferably clones thereof); confirming that 5% or more of the T cells are stem cell memory T cells; and recovering a cell population containing T cells, wherein 5% or more of the T cells in the cell population are stem cell memory T cells. (22) The method according to (21) above, wherein the induction of T cells is stopped when 5% or more of the T cells become stem cell memory T cells. (23) The method according to (21) or (22) above, wherein the composition is any of the compositions. (24) The method according to (21) or (22) above, wherein the composition is the composition described in (3) above. (25) The method according to (21) or (22) above, wherein the composition is the composition described in (8) above.

[0007] (31) A composition comprising a cell population including antigen-specific T cells, wherein 5% or more of the T cells are stem cell memory T cells. (32) The composition according to (32), wherein the T cells are cells derived from iPS cells derived from T cells. (33) The composition according to (31) or (32), wherein the T cells include CD27-positive, CD28-positive, and CCR7-positive T cells. (34) The composition according to any one of (31) to (33), wherein the T cells have lost HLA class I, and preferably have HLA-A and HLA-E reintroduced.

[0008] This section shows the expression of various HLAs in iPSCs in which the expression of HLA class I was suppressed by genome editing, and HLA-A2402 and HLA-E0103 were exogenously introduced and expressed. This section also shows the expression of various HLAs in T cells (rejT) induced from the iPSCs. This section shows the expression of perforin and granzyme B in parental clones (LMP2-CTL), unedited rejT (WT-LMP2-rejT), and edited rejT (HLA-edited-LMP2-rejT) established from a donor. This section also shows the expression of various markers in parental clones (clones), unedited rejT (WT-rejT), and edited rejT (HLA-edited-rejT) established from a donor. Finally, it shows the ratio of CD62L-positive and CD45RA-positive cells in parental clones (LMP2-CTL) and edited rejT (HLA-edited-LMP2-rejT) established from a donor. The study demonstrates cytotoxicity of recipient T cells against the parental clone (LMP2-CTL) and edited rejT cells (HLA-edited-LMP2-rejT). It also demonstrates cytotoxicity of NK cells against various cell types. The study shows cytotoxicity of extranodal NK / T-cell lymphoma and nasal type (ENKL) cells by parental clones (LMP2-CTL) and edited rejT cells (HLA-edited-LMP2-rejT) established from donors. The study demonstrates antitumor activity against ENKL cell lines in an in vivo model by parental clones (LMP2-CTL), unedited rejT cells (WT-rejT), and edited rejT cells (HLA-edited-rejT) established from donors. Figures 9 and 10 show the tumor volume at day 28 after T cell administration in an in vivo model of the ENKL cell line, using parental clones (LMP2-CTL), unedited rejT (WT-rejT), and edited rejT (HLA-edited rejT) established from donors. The survival curves from the experiments are shown. The figure shows the amount of HLA-edited-LMP2-rejT cells in the peripheral blood of individuals 35 days after administration, expressed by TCR expression levels. The positive control is the TCR expression level of cultured HLA-edited-LMP2-rejT. The results of single-cell analysis in the parental clones (LMP2-CTL) and edited rejT (HLA-edited-LMP2-rejT) from recipients are shown.This document shows the expression levels of interferon-γ and tumor necrosis factor α from HLA-edited LMP2-rej T cells. It also shows the results of a UMAP comparison of RUNX3, RUNX1, ZBTB7B, SOCS1, SOCS3, and GIMAP5 expression between CD4T-iPSC-derived CD8+ T cells and ATL-iPSC-derived CD4+ T cells. The document shows the flow cytometry analysis results of RUNX3 KO CD4T-iPSC-derived CD4+ T cells using anti-CD45RA and FoxP3 antibodies. It demonstrates the effect of RUNX3 knockout on CD4+ T cell differentiation. It shows the antigen-specific cytotoxic activity of RUNX3 KO LMP2-iPSC-T cells. It shows that RUNX3 KO LMP2-iPSC-T cells secrete IFNγ in response to antigen stimulation. This shows that RUNX3 KO LMP2-iPSC-T cells secrete perforin and granzyme B in response to antigen stimulation. This shows that RUNX3 KO LMP2-iPSC-T cells secrete IFNγ and TNF-α, as well as IL-2, which is secreted by Th1 cells, and also secrete IL-4 and IL-10. This shows the expression of TBX21, STAT1, and STAT4, which are characteristic of Th1 cells, in RUNX3 KO LMP2-iPSC-T cells. This shows the expression of CD107a and CD366, which are characteristic of Th1 cells, in RUNX3 KO LMP2-iPSC-T cells. This shows the enhancement of cytotoxicity of cytotoxic T cells by RUNX3 KO HPV16E6-iPSC-T cells. 1 x 10 CD8-positive GD2-specific CAR-T cells (GD2-CART) and RUNX3 KO LMP2-iPSC-T cells (CD4rejT). ６ Compared to conditions where each culture was incubated for 72 hours, the co-culture conditions (1:1 = 0.5 × 10) ６ : 0.5 × 10 ６ This study shows a significant increase in cell number. It demonstrates that RUNX3 KO LMP2-iPSC-T cells (CD4rejT) enhance the cytotoxicity of GD2-specific CAR-T cells (GD2-CART). It also shows the antitumor effect of combining GD2-CART and CD4rejT in a GD2-positive EBV-associated lymphoma cell-carrying model.

[0009] In this specification, the subject may be a mammal, preferably a human. The subject may be a subject suffering from a tumor or cancer.

[0010] Epstein-Barr virus (EBV) is a type of virus belonging to the Gamma-herpesvirus subfamily of the Herpesviridae family, and is mainly transmitted through saliva. EBV is formally known as human herpesvirus 4 (HHV-4). EBV is the most common viral infection in humans, infecting approximately 90% of adults. The target cells are primarily B cells. Initial EBV infections are often asymptomatic or mild, but infections occurring after puberty can cause infectious mononucleosis, characterized by symptoms such as fever, sore throat, and swollen lymph nodes. After acute infection, EBV enters a latent state, remaining dormant within B cells. EBV can reactivate when immunity is weakened, leading to re-replication and infection of other cells. EBV can cause EBV-associated diseases. EBV uses latent membrane proteins (LMP1 and LMP2) to evade the immune system and avoid immune surveillance. To date, there is no treatment specifically for EBV.

[0011] In this specification, “treatment” includes prophylactic treatment and therapeutic treatment. Prophylactic treatment may be given to uninfected persons. Therapeutic treatment may be given to infected persons. Therapeutic treatment may be given to the infected virus, and prophylactic treatment may be given to prevent future infection, or to delay the onset of symptoms or signs of EBV infection by infection, or to reduce the symptoms of an EBV infection that has developed, delay the worsening of symptoms, or stop the worsening of symptoms, or to prevent severe illness. Therapeutic treatment may be given to patients with symptoms or asymptomatic carriers. Therapeutic treatment may be given to EBV-infected persons who do not have EBV-related disease, as well as to EBV-infected persons who have EBV-related disease.

[0012] In this specification, "EBV-related disease" refers to a disease caused by EBV infection. Examples of EBV-related diseases include cancer. Examples of EBV-related cancers include Burkitt lymphoma, nasopharyngeal cancer, Hodgkin lymphoma, T / NK cell lymphoma (especially extranodal NK / T cell lymphoma, nasal type), gastric cancer, leiomyosarcoma, and EBV-related lymphoproliferative disorders. EBV-related diseases may be characterized by being EBV-positive.

[0013] In this specification, "T-iPS cells" refers to iPS cells obtained by reprogramming T cells. In iPS cells obtained from T cells, the TCR allele on the genome undergoes recombination and has a different sequence from the germline, so they are sometimes referred to as T-iPS cells to distinguish them from ordinary iPS cells. More specifically, iPS cells obtained by reprogramming antigen-specific T cells have a gene encoding an antigen-specific T cell receptor, and this antigen specificity may be the same as the antigen specificity before reprogramming. By utilizing this, iPS cells having a gene encoding an antigen-specific T cell receptor can be induced from antigen-specific T cells. Furthermore, if iPS cells having a gene encoding an antigen-specific T cell receptor are further differentiated into T cells, T cells that maintain their original antigen specificity can be obtained from the resulting T cells. In this way, rejuvenated T cells (rejT) can be obtained from exhausted T cells.

[0014] In this specification, "CD4+ T cells" refer to CD4-positive T cells, and "CD8+ cells" refer to CD8-positive T cells. CD4 and CD8 double-positive T cells are referred to as CD4+ / CD8+ double-positive T cells. CD4+ / CD8+ T cells refer to either CD4+ T cells or CD8+ T cells. CD4 / CD8 double-negative (DN) T cells refer to T cells that do not express either CD4 or CD8. CD4 / CD8DN T cells can be used in CAR-T therapy by expressing a chimeric antigen receptor.

[0015] The T cell receptor (hereinafter referred to as TCR) is responsible for the antigen recognition function of T cells and is composed of proteins such as α, β, γ, and δ chains. Of these, a heterodimer of the TCRα chain protein (TCRα) and the TCRβ chain protein (TCRβ), or a heterodimer of the γ and δ chains, together with auxiliary molecules such as the CD3 complex (containing γ, δ, ε, and ζ), CD4, or CD8, forms the TCR complex.

[0016] TCRα and TCRβ have a variable region (V region + J region) and a constant region (C region). The V region within the variable region contains a complementation-determining region (CDR). Therefore, TCRs can be characterized by the amino acid sequence of the V region (including the CDR region) in TCRα and TCRβ, or by the V region and the J region. The V region includes CDR1-3, with CDR1 and CDR2 playing an important role in HLA restriction, and CDR3 playing a crucial role in antigen binding.

[0017] The T cells described herein can be produced by two main methods. One method involves creating iPS cells from patient T cells to obtain T cells, and the other involves obtaining T cells from hematopoietic stem cells or other blood cell lineage cells. In the latter method, it is necessary to introduce EBV-specific TCR or nucleic acid encoding it into cells to express EBV-specific TCR in the cells.

[0018] When administered to individuals of the same species but different lineage, the cells may contain mechanisms to evade the immune system. These mechanisms for evading the immune system are described below.

[0019] A method for obtaining T cells by creating iPS cells from patient T cells is described. The T cells of this disclosure (CD4 / CD8DN T cells, CD8+ T cells, or CD4+ T cells) can be obtained from healthy individuals or EBV-infected patients. CD8+ T cells obtained from healthy individuals or EBV-infected patients can be single-cell clones and obtained as clones that specifically respond to EBV antigens (e.g., EBNA, LMP1, or LMP2) (e.g., produce interferon-γ). In some embodiments, the CD8+ T cells of this disclosure include cytotoxic T cells (CTLs). In some embodiments, the CD4+ T cells of this disclosure include Th1 cells, Th2 cells, or Th17 cells, preferably Th1 cells. CD4+ T cells are preferably produced from CD4+ T cells via T-iPS cells, and CD8+ T cells or cytotoxic T cells are preferably produced from CD8+ T cells or cytotoxic T cells via T-iPS cells. CD4 / CD8DN T cells can be produced from CD4+ T cells or CD8+ T cells via T-iPS cells.

[0020] This disclosure describes how CD8+ T cells can be reprogrammed to be specific to EBV antigens (e.g., EBNA, LMP1, or LMP2). Reprogramming can be carried out by various methods, but is not limited to the above, including, for example, by introducing (e.g., transiently expressing) a combination of Yamanaka factor (e.g., Oct4, Sox2, Klf4, c-myc), other reprogramming factors (NANOG, LIN28), and SV40 large T antigen (SV40LT) into the CD8+ T cells. For example, (i) a combination of Yamanaka factor, NANOG, and LIN28, or (ii) a combination of Yamanaka factor and SV40LT can be preferably used as reprogramming factors.

[0021] When performing immunotherapy, it is preferable that the human T cells differentiated from iPS cells (rejT) have the same or substantially the same antigen specificity as the human T cells induced into iPS cells. Such human T cells can be obtained by the method described in US2013 / 0078226A.

[0022] T cells can be isolated from human tissue, for example, by known methods. Examples of human tissues include tissues containing T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesional tissue. Of these, peripheral blood is preferred from the viewpoint of being less invasive to humans and easy to prepare. Tumor-infiltrating lymphocytes (TILs) can be isolated from tumor tissue or peripheral blood. Known methods for isolating human T cells include, for example, magnetic selection using magnetic beads for cell separation, flow cytometry using antibodies against cell surface markers such as CD4 or CD8 and a cell sorter, and activated T cell induction methods using anti-CD3 antibodies and anti-CD28 antibodies. In addition, desired T cells can be isolated using cytokine secretion, expression of functional molecules, or signaling molecules such as PD-1 as indicators. Furthermore, cytotoxic T cells (CTLs) can be isolated using the secretion or production of granzymes or perforins as indicators. Furthermore, when isolating T cells with antigen specificity from human tissue, T cells with the desired antigen specificity can be purified from human tissue using polymerized MHC (major histocompatibility complex) conjugated with the desired antigen (e.g., "MHC tetramer," "Pro5® MHC Class I pentamer"). CD4+ T cells and CD8+ T cells can be obtained from healthy individuals (many healthy individuals have experience with EBV infection) or patients using conventional methods by those skilled in the art.

[0023] In the present invention, the genes (reprogramming factors) introduced to convert T cells into iPS cells are preferably combinations of at least four genes from among (a) Oct3 / 4 gene, (b) c-Myc gene, (c) Sox2 gene, (d) Klf4 gene, (e) NANOG gene, (f) LIN28 gene, and (g) SV40 large T (SV40LT). In particular, the combination of (i) Yamanaka factor, NANOG, and LIN28, or (ii) Yamanaka factor and SV40LT, can preferably be used as reprogramming factors for T cells.

[0024] In the present invention, there are no particular limitations on the method for introducing the gene group into T cells, and known methods can be appropriately selected and used. For example, when introducing the gene group into T cells in the form of nucleic acids encoding the gene group, the nucleic acids encoding the gene group (e.g., cDNA, RNA) can be inserted into a suitable expression vector containing a promoter that functions in T cells, and the expression vector can be introduced into cells by infection, lipofection, liposome method, electroporation, calcium phosphate coprecipitation, DEAE dextran method, microinjection method, or electroporation method.

[0025] Among such expression vectors, using a stealth RNA expression vector containing the aforementioned gene group is more preferable in terms of reducing the risk of cancer and improving introduction efficiency. A stealth RNA expression vector is a vector designed to avoid entering chromosomes and to allow for sustained and stable gene expression in the cytoplasm rather than the nucleus. It can introduce large genes of 13,000 base pairs or more, and can introduce 10 genes simultaneously, does not damage cells, can be removed when the introduced gene is no longer needed, and possesses stealth properties that prevent cells from recognizing the vector as a foreign substance. Examples of such stealth RNA expression vectors include a complex consisting of a negative single-stranded RNA (A) containing the RNA sequences (1) to (8) below, a single-stranded RNA-binding protein (B), and RNA-dependent RNA polymerase, which does not activate the innate immune structure. (1) RNA sequences for the gene group; (2) RNA sequences derived from human mRNA constituting a non-coding region; (3) transcription initiation signal sequences recognized by RNA-dependent RNA polymerase; (4) transcription termination signal sequences recognized by RNA-dependent RNA polymerase; (5) RNA sequences containing replication origins recognized by RNA-dependent RNA polymerase; (6) RNA sequences encoding RNA-dependent RNA polymerase; (7) RNA sequences encoding proteins that regulate the activity of RNA-dependent RNA polymerase; (8) RNA sequences encoding single-stranded RNA-binding proteins.

[0026] Furthermore, when establishing T-iPS cells, it is preferable that the T cells be stimulated and activated with anti-CD3 antibody and anti-CD28 antibody in the presence of interleukin-2 (IL-2) or interleukin-7 (IL-7) and interleukin-15 (IL-15) before the introduction of the gene group. Alternatively, they may be stimulated and activated with at least one substance selected from the group consisting of phytohemagglutinin (PHA), interleukin-2 (IL-2), alloantigen-expressing cells, anti-CD3 antibody, and anti-CD28 antibody, and CD3 and CD28 agonists. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody to the culture medium and culturing the T cells for a certain period of time. The anti-CD3 antibody and anti-CD28 antibody may also be bound to magnetic beads or the like. Moreover, instead of adding these antibodies to the culture medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish on which the anti-CD3 antibody and anti-CD28 antibody are bound to 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 culture medium along with the feeder cells.

[0027] To provide such stimulation to the T cells, there are no particular restrictions on the concentration of PHA added to the culture medium, but it is preferably 1 to 100 μg / mL. Similarly, there are no particular restrictions on the concentration of IL-2 added to the culture medium, but it is preferably 1 to 200 ng / mL. Furthermore, there are no particular restrictions on the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the culture medium, but it is preferably 1 to 10 times the amount of T cells cultured. In addition, to provide such stimulation to the T cells, there are no particular restrictions on the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of the culture dish, but the concentration during coating is preferably 0.1 to 100 μg / mL for anti-CD3 antibody, preferably 1 to 100 μg / mL, and 0.1 to 10 μg / mL for anti-CD28 antibody.

[0028] Furthermore, the culture period for performing such stimulation is not particularly limited as long as it is a sufficient period to provide such stimulation to the T cells and is long enough to proliferate the T cells to the number of cells necessary for the introduction of the reprogramming factor, but it is usually 2 to 7 days, but from the viewpoint of gene transfer efficiency, it is preferably 3 to 5 days. It is preferable to infect the T cells by mixing them with the vector in a 15 mL tube, or to culture them on a culture dish coated with retronectin from the viewpoint of increasing gene transfer efficiency.

[0029] For culturing the T cells and adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, a known medium suitable for culturing T cells can be used, for example, Roswell Park Memorial Laboratory (RPMI) 1640 medium, AIM V™ medium, or NS-A2 containing other cytokines and human serum. In addition to PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody, the medium may also contain amino acids necessary for culture (e.g., L-glutamine) and antibiotics (e.g., streptomycin, penicillin). It is also preferable to add IL-7 and IL-15 to the medium instead of IL-2. There are no particular restrictions on the concentration of IL-7 and IL-15 added, but it is preferable that each is 1 to 100 ng / mL.

[0030] Furthermore, there are no particular restrictions on the conditions when introducing the reprogramming factor into the T cells or the conditions thereafter, but it is preferable to culture the T cells that have been introduced with the reprogramming factor under feeder-free conditions. Examples include wells coated with iMatrix-511 solution, which is a laminin 511E8 fragment, or with vitronectin. Culturing under feeder cell conditions is also possible, and examples of feeder cells include mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation or antibiotic treatment.

[0031] Furthermore, in the process of inducing T cells into T-iPS cells, it is preferable to add iPS cell culture medium from the following day. Thereafter, it is preferable to replace half of the culture medium every other day, gradually replacing the T cell culture medium with iPS culture medium.

[0032] Furthermore, it is preferable to gradually replace the culture medium from a known medium suitable for culturing T cells to a medium suitable for culturing iPS cells in accordance with the transition from T cells to iPS cells. As such a medium suitable for culturing iPS cells, any known medium can be appropriately selected and used. For example, StemFit AK03N is preferred when iMatrix coated, or Essential 8 Medium when coated with vitronectin. On feeder cells such as MEF cells, Dulbecco's modified Eagle medium / F12 medium (human iPS cell medium) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, and b-FGF is preferred.

[0033] The selection of T-iPS cells obtained in this manner can be carried out by appropriately selecting known methods. Such known methods include, for example, selecting ES cell / iPS cell-like colonies by observing their morphology under a microscope. On the other hand, in the case of T-iPS cells established from single-cell CTL clones, since their properties are often similar, there is also a method of subculturing all established colonies without selecting each T-iPS cell colony.

[0034] Confirmation that the cells selected in this manner are T-iPS cells can be performed, for example, by detecting the expression of undifferentiated cell-specific markers (ALP, SSEA-4, Tra-1-60, and Tra-1-81, etc.) in the selected cells using immunohistochemistry or RT-PCR, or by transplanting the selected cells into mice and observing teratoma formation. Furthermore, confirmation that the cells selected in this manner are derived from the aforementioned T cells can be performed by detecting the state of TCR gene rearrangement using RT-PCR.

[0035] When selecting and collecting these cells, it is preferable to observe the growth state of the colonies and then collect them. Generally, it is 10 to 40 days, preferably 14 to 28 days, after introducing the gene group containing the initialization factor into the T cells. As the culture environment, unless otherwise specified above, it is preferably under the conditions of 5% CO2, 35 to 38 °C, more preferably 37 °C.

[0036] Next, EBV antigen-specific T cells are induced to differentiate from the established T-iPS cells. When preparing CD4 single-positive T cells, it is preferable to knock out RUNX3 in the T-iPS cells.

[0037] As this redifferentiation induction method, a method of differentiating T-iPS cells into CD4 / CD8+ single-positive T cells is preferable, and a method of differentiating T-iPS cells into CD4 / CD8 double-negative T cells and then differentiating the CD4 / CD8 double-negative T cells into CD4 / CD8+ single-positive T cells is more preferable.

[0038] Furthermore, as described in US2013 / 0078226A, T-iPS cells are differentiated into CD4 / CD8 double-negative cells, a substance that stimulates the T cell receptor is added to stimulate the CD4 / CD8 double-negative cells, and then the CD4 / CD8 double-negative cells stimulated by the T cell receptor are preferably obtained by differentiating them into CD8 single-positive T cells in the presence of cytokines of IL-7 and IL-15, although not particularly essential. Alternatively, the CD4 / CD8 double-negative cells stimulated by the cell receptor are preferably differentiated into CD4+ T cells in the absence or under the expression suppression condition of RUNX3.

[0039] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, it is preferable to culture the T-iPS cells on feeder cells (preferably mouse stromal cells) in a medium containing cytokines, serum (e.g., fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine, α-monothioglycerol, ascorbic acid, etc.

[0040] As the stromal cells to be used, it is preferable to use OP9 cells or 10T1 / 2 cells (C3H10T1 / 2 cells) that have been treated with radiation or the like. The cytokine added to the medium is preferably at least one cytokine selected from the group consisting of VEGF, SCF, TPO, and FLT3L, and more preferably VEGF, SCF, and TPO, or VEGF, SCF, and FLT3L.

[0041] Further, examples of the medium include X-VIVO medium, Iscove's modified Dulbecco's medium (IMDM medium), α-MEM, and DMEM. From the viewpoint of facilitating the formation of a T-iPS sac (a bag-like structure containing hematopoietic progenitor cells), the IMDM medium is preferable. The culture period of these T-iPS cells is preferably 8 to 14 days, more preferably 10 to 14 days, after starting the culture of T-iPS cells. The culture environment is not particularly limited, but is preferably under the conditions of 5% CO2, 35 to 38°C, more preferably 37°C. Further, it is more preferable to culture for about one week under low oxygen concentration conditions (oxygen concentration: for example, 5 to 20%).

[0042] To differentiate T-iPS cells into CD4 / CD8 double-negative cells, it is preferable to further culture the cells contained in the T-iPS sac on feeder cells (preferably stromal cells, more preferably human stromal cells) in a culture medium containing cytokines and serum (e.g., FBS), but use well with cytokine coating under feeder-free conditions. The cells present inside the T-iPS sac can be separated, for example, by passing them through a sterile sieve-like device (e.g., a cell strainer). From the viewpoint of inducing differentiation into T cells via notch signaling, it is preferable to use OP9-DL1 cells, OP9-DL4 cells, 10T1 / 2 / DL4 cells, or 10T1 / 2 / DL1 cells that have been treated with radiation or the like as stromal cells for this culture. Examples of cytokines to be added to the culture medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and IL-15. Examples of culture media include α-MEM medium, DMEM medium, and IMDM medium, but α-MEM medium is preferred. In addition to IL-7 and FLT3L, the culture medium may also contain amino acids necessary for cultivation (e.g., L-glutamine) and antibiotics (e.g., streptomycin, penicillin).

[0043] The culture period for the cells contained in this T-iPS sac is preferably the period until T cell receptors (TCRs) are expressed on the cell surface of the CD4 / CD8 double-negative cells obtained by differentiation in this manner, and is preferably 14 to 28 days from the start of culturing the cells contained in the T-iPS sac. There are no particular restrictions on the culture environment, but it is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0044] Furthermore, whether or not T cell receptors (TCRs) are expressed on the cell surface of CD4 / CD8 double-negative cells can be evaluated by flow cytometry using anti-TCRαβ antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.

[0045] In a method for producing antigen-specific human CD4 / CD8 single-positive cells, by introducing stimulation to the TCR signaling pathway in CD4 / CD8 double-negative cells derived from T-iPS cells, for example, by stimulating CD4 / CD8 double-negative cells derived from T-iPS cells via the TCR complex expressed on their cell surface, further rearrangement of the TCRA gene can be suppressed. Consequently, the frequency of T cells having the same TCR gene rearrangement pattern as the original human T cells can be made extremely high in the redifferentiated CD4 / CD8 single-positive cells. TCR rearrangement is carried out by RAG, and similarly, knockout of RAG1 and / or RAG2 can also make the frequency of T cells having the same TCR gene rearrangement pattern as the original human T cells extremely high.

[0046] A preferred method for stimulating the T cell receptor of CD4 / CD8 double-negative cells derived from T-iPS cells is to contact the CD4 / CD8 double-negative cells derived from T-iPS cells with at least one substance selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, an antigen peptide to which human T cells that are the origin of T-iPS cells specifically bind, cells expressing a complex with HLA that is restrictive to the T cell receptor, and an MHC multimer to which the antigen peptide is bound. From the viewpoint of providing physiological stimulation, a method of contacting the cells with cells expressing a specific peptide / HLA complex is more preferred. Furthermore, from the viewpoint of emphasizing the uniformity of stimulation, a method of contacting the cells with antibodies or reagents is more preferred.

[0047] The contact method can be, for example, by adding PHA or the like to the culture medium and culturing the T cells for a certain period of time. Furthermore, the anti-CD3 antibody and anti-CD28 antibody may be conjugated with magnetic beads or the like. Alternatively, instead of adding these antibodies to the culture medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish to which the anti-CD3 antibody and anti-CD28 antibody are conjugated. Furthermore, the antigen peptide may also be added to the culture medium along with feeder cells to provide stimulation.

[0048] To stimulate the TCR of CD4 / CD8 double-negative cells, the concentration of PHA added to the culture medium is preferably 1 to 100 μg / ml. Furthermore, the concentrations of anti-CD3 antibody and anti-CD28 antibody added to the culture medium are preferably 1 to 10 times the amount of T cells cultured. Additionally, to stimulate the TCR of CD4 / CD8 double-negative cells, the concentrations of anti-CD3 antibody and anti-CD28 antibody bound to the surface of the culture dish during coating are preferably 0.1 to 100 μg / ml for anti-CD3 antibody and 0.1 to 10 μg / ml for anti-CD28 antibody.

[0049] The culture period for the cells contained in this T-iPS sac is preferably the period necessary for T cell receptors (TCRs) to be expressed on the cell surface of the CD4 / CD8 double-negative cells obtained by differentiation in this manner, and is preferably 7 to 29 days from the start of culturing the cells contained in the T-iPS sac. The culture environment is preferably 5% CO2, 35 to 38°C, more preferably 37°C.

[0050] In the present invention, in order to differentiate CD4 / CD8 double-negative cells stimulated with T cell receptors into CD4 / CD8 single-positive cells, it is preferable to culture the CD4 / CD8 double-negative cells in a culture medium containing cytokines and serum (e.g., human serum). The cytokines added to the culture medium can be any cytokines that can differentiate CD4 / CD8 double-negative cells into CD4 / CD8 single-positive cells, such as IL-7 and IL-15. Among these, it is preferable to add a combination of IL-7 and IL-15 in order to select the CD8 lineage during differentiation into CD8 single-positive cells and to facilitate the generation of memory-type CD8+ T cells. The concentration of IL-7 and IL-15 added is preferably 1 to 20 ng / ml. Examples of culture media include RPMI-1640 medium, X-VIVO medium, DMEM medium, and α-MEM medium, but RPMI-1640 medium or X-VIVO medium is preferred. Furthermore, the culture medium may also contain amino acids necessary for culture (e.g., L-glutamine), antibiotics (e.g., streptomycin, penicillin), and cytokines other than IL-7 and IL-15.

[0051] In such cultures, CD4 / CD8 double-negative cells may be co-cultured with feeder cells. Peripheral blood mononuclear cells (PBMCs) are preferred as the feeder cells. Preferably, these PBMCs are allogeneic (allogeneic) to the CD4 / CD8 double-negative cells. Furthermore, from the viewpoint of continuously stimulating the TCR and suppressing further TCR rearrangement, it is even more preferable to use peripheral blood mononuclear cells that present antigen peptides to which human T cells, the origin of the CD4 / CD8 double-negative cells, specifically bind.

[0052] The culture period for differentiating these CD4 / CD8 double-negative cells into CD4+ / CD8+ T cells is preferably 2 to 4 weeks. The culture environment is preferably 5% CO2, 35 to 38°C, and more preferably 37°C.

[0053] Confirmation that these differentiated CD4+ / CD8+ T cells are derived from T-iPS cells, and that these T-iPS cells originated from T cells, can be performed, for example, by detecting the state of TCR gene rearrangement using genomic PCR.

[0054] Furthermore, the CD4 / CD8DN T cells and CD4+ / CD8+ T cells obtained in this manner can be isolated by appropriately selecting known methods. Such known methods include, for example, flow cytometry using an antibody against the cell surface marker of CD4 / CD8 and a cell sorter. For example, in the case of CD4 / CD8DN T cells and CD4+ / CD8+ T cells, purification methods can be employed using an affinity column immobilized with the antigen recognized by the T cells from which the CD4+ / CD8+ cells originated, or using an MHC multimer (e.g., MHC tetramer) to which the antigen is conjugated.

[0055] Furthermore, the CD4 / CD8DN T cells and CD4+ / CD8+ T cells obtained by this disclosure, for example, do not express PD-1 but express CCR7, and express CCR7 together with CD27 and CD28, which are representative of the central memory T cell phenotype, and also have longer telomeres compared to the original T cells, and possess high self-renewal capacity. Therefore, according to the present invention, it is possible to produce CD4 / CD8DN T cells and CD4+ / CD8+ T cells that have the same TCR gene rearrangement pattern as the original T cells, but do not express PD-1 and express CD27, CD28 and CCR7. Human T cells differ from the obtained T cells in that they express PD-1 and have a low proportion of immature memory phenotypes. The CD8 single-positive cells obtained by this disclosure may also express TIM-3.

[0056] To maintain the CD4+ / CD8+ T cells obtained in this manner, the cells may be stimulated every one to two weeks. Such stimulation may include contact with at least one substance selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, IL-2, IL-7, IL-15, antigens recognized by the CD4+ / CD8+ T cells, MHC multimers conjugated with such antigens, feeder cells allo-positive to the CD8 single-positive cells, and feeder cells auto-positive to the CD4+ / CD8+ T cells.

[0057] The present disclosure provides a T cell receptor (TCR) having a specific amino acid sequence, cytotoxic T cells (CTLs) expressing the TCR, and compositions and pharmaceutical compositions containing the CTLs.

[0058] The T cell receptor (TCR) comprises a V region, a J region, and a C region. According to this disclosure, a TCRα chain having the amino acid sequence described in SEQ ID NO: 4 is provided. Furthermore, according to this disclosure, a TCRα chain having the amino acid sequence described in SEQ ID NO: 9 is provided. Furthermore, according to this disclosure, a TCRβ chain having the amino acid sequence described in SEQ ID NO: 14 is provided. According to this disclosure, a TCRα chain having the amino acid sequence described in SEQ ID NO: 4 or 9 can associate with a TCRβ chain having the amino acid sequence described in SEQ ID NO: 14 and bind to LMP2. Thus, the TCRα and β chains of this disclosure include LMP2 binding means as identified by the above SEQ ID NOs, thereby binding to LMP2.

[0059] The TCRα chain of this disclosure may have the amino acid sequence described in SEQ ID NO: 3 or 8. The TCRβ chain of this disclosure may have the amino acid sequence described in SEQ ID NO: 13.

[0060] The TCRα chain of this disclosure may have the amino acid sequence described in SEQ ID NO: 1 or 6. The TCRβ chain of this disclosure may have the amino acid sequence described in SEQ ID NO: 11.

[0061] The TCRα chain of this disclosure may have mutations (i.e., substitutions, insertions, or deletions) of 1 to 3 amino acids, 1 amino acid, 2 amino acids, or 3 amino acids from the amino acid sequence described in SEQ ID NO: 1 or 6, while maintaining antigen specificity to LMP2. The mutations may be located in a portion other than CDR3. In some embodiments, the mutations may be located within CDR3. In some embodiments, the mutations within CDR3 may be 1-amino acid mutations (i.e., substitutions, insertions, or deletions).

[0062] The TCRβ chain of this disclosure may have mutations (i.e., substitutions, insertions, or deletions) of 1 to 3 amino acids, 1 amino acid, 2 amino acids, or 3 amino acids from the amino acid sequence described in SEQ ID NO: 11, while maintaining antigen specificity to LMP2. The mutations may be located in a portion other than CDR3. In some embodiments, the mutations may be located within CDR3. In some embodiments, the mutations within CDR3 may be 1-amino acid mutations (i.e., substitutions, insertions, or deletions).

[0063] The present disclosure provides T cells expressing a TCR having the above-described specific amino acid sequence. These T cells are preferably CD4 / CD8DN T cells or CD4 / CD8 monopositive T cells, and may, for example, be CD8 monopositive cells or CD4 monopositive cells, and more preferably cytotoxic T cells (CTLs) or Th1 cells.

[0064] TCRs expressed on CD8+ T cells are considered to be HLA class I-restricted, while TCRs expressed on CD4+ T cells are considered to be HLA class II-restricted.

[0065] Differentiating T-iPS cells obtained from the target into CD4+ T cells is useful. While CD8-positive cytotoxic T cells (CTLs) directly attack cancer, 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 immune evasion in cancer) in an interferon (IFN)γ-dependent manner, are also useful in T cell immunotherapy and CAR-T cell therapy. The resulting CD4+ T cells may be Th1 cells. The resulting CD4+ T cells exhibit one or more of the following Th1 cell phenotypes: (1) They secrete IFNγ and IL-2, preferably in high secretion; (2) They express one or more genes selected from the group consisting of TBX21, STAT1, and STAT4, preferably in high expression; (3) They express CD107a and CD366.

[0066] To effectively differentiate T-iPS cells into CD4+ T cells, the differentiation induction process from T-iPS cells to CD4+ T cells can be carried out in the absence of expression of one or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5, preferably in the absence of RUNX3 gene expression. The CD4+ T cells obtained in this way may express IL-2. Furthermore, the CD4+ T cells obtained in this way may secrete IFNγ upon antigen stimulation. In addition, the obtained CD4+ T cells may express one or more genes selected from the group consisting of TBX21, STAT1, and STAT4. The absence of gene expression can be achieved, for example, by gene knockout. Gene knockout can be achieved by methods well known to those skilled in the art. The CD4+ T cells obtained in this way may include Th1 cells. The obtained Th1 cells may be isolated or purified from the CD4+ T cells. CD4+ T cells may have an HLA class I-restricted TCR and can be generated from CD8+ T cells via T-iPS cells.

[0067] Such CD4+ T cells (especially Th1 cells) and CTLs can be obtained from T-iPS cells, or from hematopoietic lineage cells such as hematopoietic stem cells through differentiation induction. To obtain T cells expressing the above TCR, the gene encoding the TCR can be introduced into the cells at either the hematopoietic stem cell stage or a subsequent differentiation stage. In one embodiment, the endogenous TCR of the cells may be disrupted, and cells having a TCR with the above amino acid sequence can be provided by disrupting the gene encoding the endogenous TCR.

[0068] The mechanisms of immune evasion may be as follows: Cells may have HLA class I and / or HLA class II disrupted, or their expression levels reduced or deleted, to prevent removal by the host. Disruption of HLA class I and / or HLA class II can be carried out using known techniques. Furthermore, the reduction or deletion of HLA class I and / or HLA class II expression levels can be achieved by disruption or reduction of β2-microglobulin (B2M) and / or disruption or reduction of CIITA, respectively.

[0069] The cells may preferably express one of the HLAs. The HLA may be HLA-A, for example, HLA-A01, HLA-A02, HLA-A03, HLA-A11, HLA-A-23, HLA-A24, HLA-A26, HLA-A30, HLA-A33, or HLA-A68. For example, HLA-A24 binds to KIR3DL1 on NK cells and is useful in evading attack on the cells of this disclosure by NK cells. HLA may also include exogenously introduced HLA alleles selected from the group consisting of HLA-A2402, HLA-A0201, HLA-A1101, and HLA-3303; HLA-A0101, HLA-A0201, HLA-A0301, and HLA-A1101; HLA-A3002, HLA-A2301, HLA-A0201, and HLA-A6801; HLA-A0201, HLA-A6802, and HLA-A2402; and HLA-A0101, HLA-A1101, HLA-A2601, and HLA-A2402. This HLA allele may be the same as the HLA allele of rejT. This allows them to evade innate immunity.

[0070] Cells may also possess means of evading innate immunity to prevent removal by the host. These means of evading innate immunity may include, for example, the expression of one or more selected from the group consisting of HLA-E, a fusion protein of HLA-E and β2-microglobulin (B2M), HLA-G, a fusion protein of HLA-G and B2M, NKG2D ligand, and CD47. In particular, in cells with disrupted or reduced B2M expression, the fusion proteins of HLA-E and β2-microglobulin (B2M) and HLA-G and B2M may be effective in evading innate immunity. For example, HLA-E binds to NKG2A and is useful in evading attack on the cells of this disclosure by NK cells.

[0071] The immunological techniques described above may help enhance the effectiveness of cells when they are administered not only to the self but also to allogeneic organisms.

[0072] To confirm whether the obtained iPS cell-derived T cells or T cells possess EBV antigen-specific cytotoxic activity, the antigen-bound MHC pentamer, MHC tetramer, etc., is used to confirm that they retain the same antigen specificity as the original peripheral blood-derived CTLs.

[0073] Furthermore, EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) can also be produced by genetic recombination using the gene encoding the TCR of the present invention. That is, for example, the gene encoding the TCR of the present invention can be introduced into host T cells, and EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) can be selected. To introduce the gene encoding the TCR into host T cells, it is preferable to incorporate the gene into various viral vectors. When producing CAR-T cells, instead of the TCR described above, a gene encoding a chimeric antigen receptor (CAR) that binds to the EBV antigen can be introduced into the T cells.

[0074] The selection of EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells) may be based on the confirmation of EBV antigen-specific cytotoxic activity, or on the detection of cytokine production capacity such as IFN-γ.

[0075] The resulting EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells) or T-iPS cells are useful as pharmaceutical compositions or cell-mediated drugs.

[0076] The resulting EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells) or T-iPS cells possess excellent EBV-specific cytotoxic activity (and in the case of CD4+ T cells, it may promote cytotoxicity), making them particularly useful as therapeutic agents for EBV infection and EBV-induced cancers.

[0077] Among the EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) of the present invention, EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) that are rich in tissue-resident memory T cells are particularly excellent EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells), and are therefore particularly useful as therapeutic agents for EBV infection and EBV-induced cancer. EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) that are rich in tissue-resident memory T cells are also provided.

[0078] The antigen-specific T cells of this disclosure (in particular EBV antigen-specific T cells) (e.g., CD4 / CD8DN T cells, CD4+ T cells, in particular Th1 cells, or CD8+ cells, in particular cytotoxic T cells) include stem cell memory T cells. Stem cell memory T cells (SCMs) can be determined as CD62L-positive and CD45RA-positive cells. In some embodiments, SCM may constitute 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, or 25% or more of antigen-specific T cells (especially EBV antigen-specific T cells) (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells). In some embodiments, SCMs may constitute 1-50%, 5-40%, or 10-30% of antigen-specific T cells (especially EBV antigen-specific T cells) (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells). SCMs are the most undifferentiated group of memory T cells that survive in the body for a long period after being activated in response to an antigen. The higher the amount of SCMs, the stronger the antitumor effect of antigen-specific T cells (especially EBV antigen-specific T cells) (e.g., CD4 / CD8DN T cells, CD4+ T cells, especially Th1 cells, or CD8+ cells, especially cytotoxic T cells) can be expected to be.

[0079] Accordingly, the present disclosure may provide a composition comprising a cell population including antigen-specific T cells, wherein 5% or more of the T cells are stem cell memory T cells. Here, the stem cell memory T cells may preferably have the same antigen specificity, and more preferably may be cells (e.g., clones) derived from iPS cells derived from T cells. In this embodiment, the antigen may be, for example, a cancer antigen or a pathogen antigen, but preferably an EBV antigen. In this embodiment, the T cells may also be, for example, CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells. The T cells may also be preferably PD-1 negative and CCR7 positive, more preferably CD27 positive, CD28 positive, and CCR7 positive. In one preferred embodiment, the T cells have lost HLA class I (e.g., by disruption of the β2 microglobulin gene), and more preferably HLA-A and HLA-E have been reintroduced. The composition of this disclosure can be obtained by harvesting a cell population in which 5% or more of the T cells are stem cell memory T cells, and preferably by confirming that 5% or more of the T cells are stem cell memory T cells.

[0080] The present disclosure provides a method for producing the following composition: a composition comprising a cell population containing antigen-specific T cells, wherein 5% or more of the T cells are stem cell memory T cells. The method of the present disclosure may include inducing induced pluripotent stem cells from T cells obtained from a subject, inducing T cells from the obtained induced pluripotent stem cells (preferably clones thereof), and recovering a cell population containing T cells. The induction of T cells can be stopped when 5% or more of the T cells have become stem cell memory T cells. The method of the present disclosure may further include confirming that 5% or more of the T cells are stem cell memory T cells.

[0081] In one embodiment, EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) are observed in the peripheral blood of subjects 35 days after administration. In another embodiment, the TCR of EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) is observed in greater numbers in the peripheral blood of subjects 35 days after administration compared to the TCR of cultured EBV antigen-specific CD4 / CD8 single-positive T cells.

[0082] In one embodiment, EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) cause regression of EBV-related tumors 28 days after administration to the subject.

[0083] The abundance of tissue-resident memory T cells can be confirmed by flow cytometry using labeled antibodies that recognize CD3, CD8, CD62L, CCR7, CD103, and CD69. Here, "abundant" means that CD3+, CD8+ T cells are CD62L-, CCR7-, CD103+, or CD69+, and more preferably CD3+, CD8+ T cells are CD62L-, CCR7-, CD103+, and CD69+. The proportion of tissue-resident memory T cells is preferably at least 30%, more preferably 50%, even more preferably 60%, and even more preferably 70%.

[0084] Another aspect of the present invention is a composition for use in treating EBV infection and / or EBV-induced cancer, comprising EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) or T-iPS cells having the T cell receptor or a functional fragment thereof.

[0085] EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) can target EBV antigens expressed in target cells after EBV infection and destroy EBV-infected cells within the target body. A reduction or loss of infected cells has medical and technical significance.

[0086] Another aspect of the present invention is the use of EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) or T-iPS cells having the T cell receptor or a functional fragment thereof, for the manufacture of pharmaceuticals for use in treating EBV infection and / or EBV-induced cancer.

[0087] Another aspect of the present invention is EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) or T-iPS cells having the T cell receptor or a functional fragment thereof, for treating EBV infection and / or EBV-induced cancer. Furthermore, another aspect of the present invention is a method for treating EBV infection and / or EBV-induced cancer in a subject requiring such treatment, characterized by administering EBV antigen-specific T cells (e.g., CD4 / CD8DN T cells, CD4+ T cells, particularly Th1 cells, or CD8+ cells, particularly cytotoxic T cells) or T-iPS cells having the T cell receptor or a functional fragment thereof.

[0088] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the T cells of the present invention. Examples of such carriers include physiological saline and Ringer's solution. Furthermore, the pharmaceutical composition of the present invention may optionally contain known pharmaceutically acceptable additives such as preservatives and colorants.

[0089] The pharmaceutical composition of the present invention is preferably in the form of an injectable preparation, and more preferably in the form of an injectable preparation for T-cell infusion therapy. The pharmaceutical composition of the present invention is preferably administered to EVB-related cancer patients by T-cell infusion at intervals of several weeks.

[0090] Furthermore, the pharmaceutical composition of the present invention is also useful for treating EBV-infected cells and EBV-positive cells. In particular, it is useful for treating EBV-related diseases such as nasopharyngeal cancer, Hodgkin lymphoma, T / NK cell lymphoma (especially extranodal NK / T cell lymphoma, nasal type), leiomyosarcoma, and EBV-associated lymphoproliferative disorders, which are cancers caused by EBV infection. EBV-related diseases may be characterized by being EBV-positive. EBV positivity can be confirmed, for example, by in situ hybridization.

[0091] In this study, to avoid rejection by the patient's immune cells, CRISPR / Cas9 gene editing was used to generate HLA class I edited LMP2-specific rejT cells from a donor. First, donor iPSCs (referred to as T-iPS) were established from HLA-A2402-restricted LMP2-CTL clones. Next, HLA class I antigen expression was eliminated to prevent attack by recipient T cells, and at the same time, the HLA-A2402 and HLA-E genes were forced to be expressed to avoid attack by recipient NK cells. To avoid NK cell attack via the bw4 epitope / KIR3DL1, the HLA-A2402 gene was introduced following the endogenous B2M gene. Furthermore, to avoid NK cell attack via HLA-E / NKG2A binding, a trimer peptide (B2M-HLA-E) fusion gene was introduced to prevent the expression of endogenous B2M. These HLA-A2402 genes and trimer peptide (B2M-HLA-E) fusion genes were heterozygously knocked in (KI) (Cell Reports Medicine 2023).

[0092] HLA expression in the obtained cells was analyzed by flow cytometry. Figure 1 shows that in the edited iPSCs, HLA class I expression was lost, and exogenous HLA-A2402 and HLA-E0103 were expressed. T cells (rejT) were induced from the edited iPSCs. Figure 2 shows that in the rejT cells, HLA class I expression was also lost, and exogenous HLA-A2402 and HLA-E0103 were expressed.

[0093] The obtained rejT cells were further analyzed. Specifically, LMP2-rejT cells were differentiated from iPSCs with genome editing of HLA class I antigen and from WT iPSCs before genome editing. Intracellular staining for the cytotoxic proteins granzyme B and perforin was performed, including on the original LMP2-CTLs, and their expression was confirmed by flow cytometry. RejT cells derived from edited iPSCs showed higher perforin expression and higher granzyme B expression compared to untreated T cells (LMP2-CTL clones) obtained from donors (see Figure 3).

[0094] The expression of TIGIT, PD-1, LAG-3, and TIM-3 in the obtained rejT cells was confirmed by flow cytometry. As a result, the edited iPSC-derived rejT cells showed lower TIGIT expression, lower PD-1 expression, and lower LAG-3 expression compared to untreated T cells (LMP2-CTL clones) obtained from donors (see Figure 4). Furthermore, the edited iPSC-derived rejT cells showed higher TIM-3 expression compared to untreated T cells (LMP2-CTL clones) obtained from donors (see Figure 4).

[0095] CD3 + CD8 + Cells were gated, and CD62L and CD45RA expression was confirmed in the resulting fractions. Edited iPSC-derived rejT cells contained more CD62L and CD45RA-expressing cells compared to untreated T cells (LMP2-CTL clones) obtained from donors. This confirmed that edited iPSC-derived rejT cells are rich in stem cell memory T cells (SCMs) exhibiting a young memory phenotype (see Figure 5). This result also suggests that stem cell memory T cells were abundant at the CD4 / CD8DN T cell stage.

[0096] Alloreactivity was investigated. Specifically, HLA-edited iPSC mixed lymphocytes were mixed in a 1:1 ratio with CD3-positive T cells from a healthy HLA-A2402-positive donor (presumably the host patient) and co-cultured for 20 hours. Subsequently, flow cytometry was performed to compare CD107 expression in CD8 T cells and CD4 T cells, respectively. Cell Stimulation Cocktail was used for the positive control culture, and Protein Transport Inhibitor Cocktail was used for the negative control co-culture. In these HLA-edited iPSC mixed lymphocyte reaction assays, HLA-edited iPSC mixed lymphocytes suppressed alloreactivity when co-cultured with CD8+ T cells. When co-cultured, a comparison of HLA-edited rejT cells with unedited rejT cells showed that CD107a expression levels in CD8+ T cells from the same host were significantly lower in HLA-edited rejT cells (see Figure 6, p = 0.0421).

[0097] We prepared HLA-edited LMP2-rejT cells (dual KI) that overexpress HLA-A2402 and HLA-E, single KI-A24-rejT cells that overexpress only HLA-A2402, and single KI-E-rejT cells that overexpress only HLA-E. In addition to these rejT cells, we prepared WT LMP2-rejT cells and CD8-positive T cells from the NK cell donor themselves. We then compared these cells with positive controls. 51 After labeling with Cr and co-culturing with NK cells from HLA-A2402-positive healthy donors for 6 hours, 51 The cytotoxic activity of NK cells was measured by Cr release. Effector:Target ratios were 9:1, 3:1, 1:1, and 0.33:1 for all target cells. Dual knock-in (KI) rejT of HLA-A24 and HLA-E significantly suppressed NK cell cytotoxicity compared to single KI-A24-rejT (p < 0.0001) or single KI-E01-rejT (p < 0.0001) (cytotoxicity rate at effector / target (E:T) ratio of 9:1: KI-A24&E-rejT 19.2%, KI-A24-rejT 31.4%, KI-E-rejT 45.4%) (see Figure 7).

[0098] To analyze the antitumor effect of HLA-edited LMP2-rejT against ENKL, an in vitro cytotoxicity assay was performed. Specifically, the cytotoxic activities of HLA-edited-LMP2-rejT and LMP2-CTL against NK-YS and ENKL-J1, which are tumor cell lines of extranodal NK / T lymphoma, nasal type (ENKL), an EBV-related lymphoma, were 51 verified by a Cr release test. After labeling the ENKL cell lines with 51 Cr, they were co-cultured with HLA-edited-LMP2-rejT or LMP2-CTL for 6 hours, and the cytotoxic activity was measured. The effector:target ratios were 20:1, 10:1, 5:1 for HLA-edited-LMP2-rejT and 10:1, 5:1 for LMP2-CTL due to insufficient cell numbers caused by reduced proliferative capacity. HLA mismatched LCL was used as the target control. The cytotoxicity of HLA-edited LMP2-rejT was higher against ENKL cells (NK-YS and ENKL-J1) than the original LMP2-CTL (NK-YS: 81.7% vs 72.1%, ENKL-J1: 80.0% vs 41.5%, E:T ratio 5:1) (see Figure 8).

[0099] Next, the tumor regression effects of HLA-edited-LMP2-rejT, WT LMP2-rejT, and LMP2-CTL against the tumor cell line NK-YS of extranodal NK / T lymphoma, nasal type (ENKL), an EBV-related lymphoma, were verified in vivo. NK-YS labeled with luciferase was intraperitoneally administered 4 days before treatment (0.3×10 6 cells / mouse). On the day of starting treatment, it was divided into an untreated group and three treatment groups (LMP2-CTL administration group, WT LMP2-rejT administration group, HLA-edited-LMP2-rejT administration group) and treated. On days 0, 7, and 14, 8.0×10 6Cells were administered individually. Tumor growth was observed using the IVIS Imaging System on days 0, 7, 14, 21, and 28. Survival time was also observed. On day 35, peripheral blood was collected from mice administered with HLA-edited LMP2-rejT, RNA was extracted, and TCR was detected by qPCR. HLA-edited LMP2-rejT in culture was used as a positive control for TCR expression, and its TCR expression level was set to 1 for comparison. Nuclease-free water was used as the negative control.

[0100] As shown in Figure 9, the bioluminescence signal in mice treated with the original CTL was not significantly different from that of the untreated group (p = 0.3919), but the signal in mice treated with HLA-edited LMP2-rejT was significantly lower than that of the untreated group on day 28 (p = 0.0219). Comparing the tumor luminescence intensity of each group on day 28, the HLA-edited-LMP2-rejT group and the WT LMP2-rejT group had significantly lower tumor luminescence intensity signals compared to the original LMP2-CTL group (Figure 10). The HLA-edited-LMP2-rejT group and the WT LMP2-rejT group had significantly longer survival times compared to the untreated group and the LMP2-CTL group (Figure 11). On day 35, we confirmed that HLA-edited-LMP2-rejT remained in the peripheral blood of mice administered with HLA-edited-LMP2-rejT (Figure 12).

[0101] To compare gene expression between the original CTLs and EBV-rejT cells, single-cell RNA sequencing was performed. As shown in Figure 13, EBV-rejT cells showed higher expression of cytotoxicity-related genes (IFNG, PRF1) and lower expression of exhaustion-related genes (TIGIT, LAG-3) compared to the original CTLs.

[0102] A cytometric bead array was performed, and cytokines (IFN-γ, TNF-α) secreted by HLA-edited LMP2-rejT cells stimulated with LMP2 antigen peptide were quantitatively analyzed by flow cytometry. The positive control was the cytokine expression level from HLA-edited LMP2-rejT cells stimulated with PHA-L. As a result, HLA-edited LMP2-rejT cells showed high INFγ and TNFα expression (see Figure 14).

[0103] 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 isolated from the peripheral blood of ATL patients, and CD4-positive T cells were isolated by positive selection with 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 CD3, CD4, and CD25.

[0104] (2) Establishment of T-iPSCs HTLV-1 infected CD4-positive T cells obtained in (1) were infected with a Sendai virus (SeV) vector (SeVp [KOSM302L]) containing nuclear reprogramming factors (Oct4, Sox2, Klf4, c-Myc) and an SeV vector containing nucleic acid encoding the SV40 large T antigen, and then genetically modified. The genetically modified T cells were moved to iMatrix-coated 6-well plates and cultured in a CO2 incubator in T cell medium (medium composition: RPMI, 10% human AB serum) supplemented with IL-2. The day after genetic modification, an equal amount of iPS medium (StemFitAK03N) was added, and thereafter, half the amount was replaced with StemFitAK03N every other day. T-iPSC colonies were observed 21 days later, and then colony picking was performed.

[0105] (3) Differentiation induction from CD4 T-iPSCs to CD4-positive cells The T-iPSC colonies obtained in (2) were finely crushed 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 under hypoxic conditions, followed by the next week in an incubator with a 20% oxygen concentration. Subsequently, they were cultured for another 4 weeks on 10T1 / 2 feeder cells expressing Notch Ligand with cytokines (IL-7, FLT3L, SCF), transferring to new feeder cells once a week. After 4 weeks, all suspended cells were collected and stimulated with T cell receptors (TCRs). TCR stimulation was performed approximately every 2 weeks with X-ray irradiated allogeneic peripheral blood mononuclear cells and PHA, or with anti-CD3 / CD28 antibody.

[0106] The phenotype of T cells obtained by differentiation induction from CD4T-iPSCs was analyzed by intracellular staining and flow cytometry. The results showed that CD4-positive T cells induced from HTLV-1 infected CD4T-iPSCs were FOXP3-positive and CD25-positive, exhibiting a regulatory T cell-like phenotype.

[0107] Reference Example 2 The gene expression of ATL-iPSC-derived CD4+ T cells obtained in the above reference example was compared with the gene expression of CD4T-iPSC-derived CD8+ T cells established from healthy human CD4 T cells using single-cell RNA sequencing analysis. For single-cell RNA sequencing analysis, emulsion preparation and RNA extraction were performed using a 10x Chromium controller system, following the 10x Genomics protocol, using Chromium Next Gem Single Cell 3' Reagent Kits v 3.1 (Dual Index) from both ATL-iPSC-derived CD4+ T cells and CD4T-iPSC-derived CD8+ T cells, and then barcode assignment was performed. Subsequently, library preparation was performed, and after evaluation of the samples using a Bioanalyzer 2100, sequencing was outsourced to an external laboratory using HiseqX (Illumina, San Diego, California). The fastq files were aligned to GRCh38 using Cell Ranger (v7.0.0) (10x Genomics). RNA data normalization and downstream analysis were performed using the Seulat R package (version 4.3.0)19, which enables integrated processing of multimodal single-cell datasets.

[0108] Figure 15 shows the results of a UMAP comparison of the expression of RUNX3, RUNX1, ZBTB7B, SOCS1, SOCS3, and GIMAP5 in CD4T-iPSC-derived CD8+ T cells and ATL-iPSC-derived CD4+ T cells. In ATL-iPSC-derived CD4+ T cells, RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 were low-expressed, while RUNX1 was high-expressed. On the other hand, in CD4T-iPSC-derived CD8+ T cells, these five genes were high-expressed, while RUNX1 was low-expressed.

[0109] Reference Example 3: CD4+ T cells obtained by differentiation induction from RUNX3 knockout T-iPSCs were analyzed by flow cytometry using antibodies against FoxP3, which is characteristic of regulatory T cells, and CD45RA, a naive cell marker. Figure 16 shows the flow cytometry analysis results of CD4+ T cells derived from RUNX3 KO CD4T-iPSCs using anti-CD45RA and 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 showed fractions of CD4+, CD45RA-, and FoxP3+, and fractions of CD4+, CD45RA+, and FoxP3low. In other words, CD4+ T cells derived from T-iPSCs with RUNX3 knocked out did not show fractions of naive regulatory T cells (CD45RA+, Foxp3low) or activated regulatory T cells (CD45RA-, Foxp3high) as seen in ATL-iPSC-CD4+ T cells. From the above, it was confirmed that CD4+ T cells derived from T-iPSCs with RUNX3 knocked out are not regulatory T cells.

[0110] Reference Example 4: LMP2-specific CTLs were isolated from peripheral blood mononuclear cells derived from healthy individuals using a standard method. iPSCs were induced from LMP2-specific CTLs using the same method as in Reference Example 1, and RUNX3 was knocked out. iPSCs derived from wild-type LMP2-specific CTLs and iPSCs derived from LMP2-specific CTLs with RUNX3 knocked out were differentiated into T cells, and analyzed by flow cytometry using an anti-CD4 antibody or an anti-CD8 antibody and an LMP2-class I tetramer.

[0111] The results are shown in Figure 17. When T cells were differentiated from antigen-specific LMP2-CTL-derived iPSCs, they normally differentiated into CD8SP cells (Figure 17 left), but knocking out RUNX3 resulted in differentiation into CD4SP cells (Figure 17 right). The original LMP2-specific CTLs were class I-restricted CD8SP CTLs, but when RUNX3 was knocked out, they maintained class I-restricted LMP2 antigen specificity even while differentiating into CD4SP cells. One or more genes selected from the group consisting of RUNX3, ZBTB7B, SOCS1, SOCS3, and GIMAP5 were highly expressed in CD4+ T cells, similar to RUNX3, and a similar effect may be observed with the reduction of these genes.

[0112] Reference Example 5: The antigen-specific cytotoxic activity of RUNX3 KO LMP2-iPSC-T cells prepared in Reference Example 4 was evaluated by chromosome assay using three types of LMP2-expressing cells (NK-YS, ENKL-J1, and CAEBV from EBV-infected T lymphocytes derived from lymphoma). 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 18. RUNX3 KO LMP2-iPSC-T cells possessed antigen-specific cytotoxic activity, although it was inferior to that of LMP2-iPSC-T cells.

[0113] Therefore, the IFNγ secretion capacity of RUNX3 KO LMP2-iPSC-T cells was evaluated using the ELISPOT assay. Furthermore, the production of the cytotoxic cytokines perforin and granzyme B was evaluated by flow cytometry. The results are shown in Figure 19. RUNX3 KO LMP2-iPSC-T cells secreted IFNγ at a high level comparable to that of LMP2-iPSC-T cells in response to antigen stimulation (Figure 19A). They also demonstrated the production of perforin and granzyme B in response to antigen stimulation (Figure 19B).

[0114] Reference Example 6: The secretion capacity of various cytokines in RUNX3 KO LMP2-iPSC-T cells was investigated. The results are shown in Figure 20. In response to antigen stimulation, RUNX3 KO LMP2-iPSC-T cells highly secreted cytokines such as IFNγ and TNF-α, which are highly secreted by CTLs, as well as IL-2, which is secreted by Th1 cells, and also highly secreted IL-4 and IL-10.

[0115] Reference Example 7: To further evaluate whether RUNX3 KO LMP2-iPSC-T cells exhibit the phenotype of Th1 cells, the expression of transcription factors and cell surface molecules characteristic of Th1 cells was examined. The results are shown in Figures 21A and 21B. RUNX3 KO LMP2-iPSC-T cells highly expressed TBX21, STAT1, and STAT4 (Figure 21A), and were also positive for CD107a and CD366 (Figure 21B). From these results, it became clear that RUNX3 KO LMP2-iPSC-T cells exhibit the phenotype of Th1 cells.

[0116] Reference Example 8 HPV16E6-specific CTLs were isolated from peripheral blood mononuclear cells of healthy individuals using a standard method. iPSCs were induced from the HPV16E6-specific CTLs using the same method as in Reference Example 1, and RUNX3 was knocked out using the same method as in Reference Example 3. T cells were induced from the RUNX3-KO HPV16E6-iPSCs using the same method as in Reference Example 1(3). The cytotoxic activity of the obtained RUNX3-KO HPV16E6-iPSC-T cells in combination with HPV16E6-iPSC-T cells (CD8+) differentiated from wild-type HPV16E6-iPSCs was evaluated by a chromium assay, as in Reference Example 5. The results are shown in Figure 22. HPV16E6-iPSC-T cells showed cytotoxic activity specific to the cervical cancer cell line SiHa expressing HPV16E6 (Series 2), but this cytotoxic activity was further enhanced by co-culturing with RUNX3 KO HPV16E6-iPSC-T cells (Series 1).

[0117] Reference Example 9: GD2-targeted chimeric antigen receptor T cells (GD2-CAR-T) were generated from peripheral blood mononuclear cells derived from healthy individuals using a standard method (Kinoshita et al., Cancer Research Communications 2024). The cytotoxic activity of the RUNX3 KO LMP2-iPSC-T cells generated in Reference Example 4 and the CD8-positive GD2-CAR-T cells obtained above was evaluated by a chromium assay, similar to Reference Example 5. Furthermore, cell proliferation was compared when each cell type was cultured alone versus when both were co-cultured.

[0118] The results are shown in Figure 23. CD8-positive GD2-specific CAR-T cells (GD2-CART) and RUNX3 KO LMP2-iPSC-T cells (CD4rejT) were divided into 1 x 10⁻¹⁰ cells. 6 Compared to conditions where each sample was cultured for 72 hours, the co-culture conditions (1:1 = 0.5 x 10) 6 : 0.5 x 10 6 In this case, the number of cells increased significantly (Figure 23A). Furthermore, when the cytotoxic activity against GD2-positive EBV-associated lymphoma cells was evaluated by chromosome assay using GD2-CART cultured for 72 hours and CD4rejT and GD2-CART (1:1) co-cultured for 72 hours, the co-cultured group showed enhanced cytotoxic activity compared to CAR-T cells alone (Figure 23B).

[0119] Reference Example 10: The combined effect of GD2-specific CAR-T cells and RUNX3 KO LMP2-iPSC-T cells was confirmed in animal experiments. NOG mice were given 1 x 10⁶ GD2-positive EBV-associated lymphoma cells. 6 The cells were transplanted into the peritoneal cavity one by one, and five days later, the effector cells, which had been cultured alone with GD2-CART for 72 hours or co-cultured with GD2-CART + CD4rejT (1:1) for 72 hours, were divided into 4 x 10⁻¹⁴ cells. 6 The cells were administered by intraperitoneal injection, and tumor growth was observed over time. The results are shown in Figure 24. More than three weeks after administration of effector cells, the tumor growth inhibitory effect was more pronounced in mice treated with GD2-CART + CD4rejT compared to mice treated with GD2-CART alone.

[0120] Reference Example 11 The obtained rejT cells were αβT cells, and their TCRα chain had the amino acid sequence described in SEQ ID NO: 1 or 6, and their TCRβ chain had the amino acid sequence described in SEQ ID NO: 11. Analysis revealed that the two TCRα and β chains had the following structures.

[0121]

[0122]

[0123]

[0124] In conclusion, we successfully generated HLA class I edited LMP2-rejTs from healthy donors with minimal immunogenicity, thereby enhancing T cell function while avoiding alloimmune responses.

[0125] Sequence listing text Sequence ID 1: TCRα chain (No. 1) MMKCPQALLAIFWLLLSWVSSEDKVVQSPLSLVVHEGDTVTLNCSYEVTNFRSLLWYKQEKKAPTFLFMLTSSGIEKKSGRLSSILDKKELFSILNITATQTGDSAIYLCAVTPHFGNEKLTFGTGTRLTIIPNIQNPDP Sequence ID 2: L region of TCRα chain (No. 1) MMKCPQALLAIFWLLLSWVSS Sequence ID 3: V region of TCRα chain (No. 1) EDKVVQSPLSLVVHEGDTVTLNCSYEVTNFRSLLWYKQEKKAPTFLFMLTSSGIEKKSGRLSSILDKKELFSILNITATQTGDSAIYLCAVTPHFGNEKLTF Sequence ID 4: CDR3 region of TCRα chain (No. 1) CAVTPHFGNEKLTF Sequence ID 5: J region of TCRα chain (No. 1) GTGTRLTIIP Sequence ID 6: TCRα chain (No. 2) METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTAPGGTDKLIFGTGTRLQVFPNIQNPDP Sequence ID 7: L region of TCRα chain (No. 2) METLLKVLSGTLLWQLTWVRS Sequence ID 8: V region of TCRα chain (No. 2) QQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTAPGGTDKLIF SEQ ID NO: 9: CDR3 region of TCRα chain (No. 2) CGTAPGGTDKLIF SEQ ID NO: 10: J region of TCRα chain (No. 1) FGTGTRLQVFP SEQ ID NO: 11: TCRβ chain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPRDRGINEQFFGPGTRLTVLEDLKNVFPPEVAVFE SEQ ID NO: 12: L region of TCRβ chain MGTSLLCWMALCLLGADHASEQ ID NO: 13: V region of TCRβ chain DTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYL SEQ ID NO: 14: CDR3 region of TCRβ chain CASSPRDRGINEQFF SEQ ID NO: 15: J region of TCRβ chain GPGTGRLTVL

Claims

1. A composition comprising a cell population including T cells specific to the antigen of Epstein-Barr virus (EBV).

2. The composition according to claim 1, wherein the T cells have reduced or absent HLA class I expression, and when co-cultured with allogeneic T cells, the CD107a expression level is lower compared to cells that do not have reduced or absent HLA class I expression.

3. The composition according to claim 1 or 2, wherein 5% or more of the T cells are stem cell memory T cells.

4. The composition according to any one of claims 1 to 3, wherein at least one allele of HLA is introduced exogenously.

5. The composition according to any one of claims 1 to 4, wherein the T cells are T cells (rejT cells) obtained by re-inducing differentiation of CD4 / CD8 monopositive T cells (first cells) having the above specificity obtained from a human individual having EBV infection after reprogramming.

6. The composition according to any one of claims 1 to 5, wherein the T cells, compared to the first cells, have lower expression of marker genes selected from the group consisting of PD-1, TIGIT, and LAG-3, higher expression of factors selected from the group consisting of interferon-γ and PRF1, and a high stem cell memory fraction.

7. The composition according to any one of claims 1 to 6, which exhibits higher cytotoxicity to the antigen-expressing cells compared to the first cells.

8. A composition according to any one of claims 1 to 7, comprising a cell population including T cells specific to the antigen of Epstein-Barr virus (EBV), wherein the T cells have reduced or absent HLA class I expression, and when co-cultured with allogeneic T cells, their CD107a expression level is lower compared to cells that do not have reduced or absent HLA class I expression, and at least one HLA allele is exogenously introduced, the T cells are T cells (rejT) obtained by re-induction of the above-mentioned specificity CD4+ / CD8+ T cells (first cells) obtained from a human patient with EBV infection after reprogramming, the T cells have lower expression of marker genes selected from the group consisting of PD-1, TIGIT, and LAG-3, and higher expression of factors selected from the group consisting of interferon-γ and PRF1 compared to the first cells, 5% or more of the T cells are stem cell memory T cells, and exhibit higher cytotoxicity against the antigen-expressing cells compared to the first cells.

9. The composition according to any one of claims 1 to 8, comprising an exogenously introduced HLA allele selected from the group consisting of HLA-A2402, HLA-A0201, HLA-A1101, and HLA-3303; HLA-A0101, HLA-A0201, HLA-A0301, and HLA-A1101; HLA-A3002, HLA-A2301, HLA-A0201, and HLA-A6801; HLA-A0201, HLA-A6802, and HLA-A2402; and HLA-A0101, HLA-A1101, HLA-A2601, and HLA-A2402.

10. The composition according to any one of claims 1 to 9, wherein the antigen is selected from the group consisting of LMP1 and LMP2.

11. The composition according to any one of claims 1 to 10, expressing a TCRα chain having the amino acid sequence described in SEQ ID NO: 1 or 6, or having 1 to 3 amino acid mutations relative to said amino acid sequence and binding to LMP2, and a TCRβ chain having the amino acid sequence described in SEQ ID NO: 11, or having 1 to 3 amino acid mutations relative to said amino acid sequence and binding to LMP2.

12. A TCRα chain having the amino acid sequence described in Sequence ID No. 1 or 6, or having 1 to 3 amino acid mutations relative to said amino acid sequence, and binding to LMP2, and a nucleic acid encoding said TCRα chain.

13. A TCRβ chain having the amino acid sequence described in Sequence ID No. 11, or having 1 to 3 amino acid mutations relative to said amino acid sequence, which binds to LMP2, and a nucleic acid encoding said TCRβ chain.

14. T cells expressing the TCRα chain and the TCRβ chain according to claim 11.

15. The T cell according to claim 14, which is a cytotoxic T cell.

16. The composition according to any one of claims 1 to 12 for use in treating EBV infection in a patient having EBV infection.

17. A method for producing a composition, wherein the composition comprises a cell population containing antigen-specific T cells, wherein 5% or more of the T cells are stem cell memory T cells, and the method comprises: inducing induced pluripotent stem cells from T cells obtained from a subject; inducing T cells from the obtained induced pluripotent stem cells (preferably clones thereof); confirming that 5% or more of the T cells are stem cell memory T cells; and recovering a cell population containing T cells, wherein 5% or more of the T cells in the cell population are stem cell memory T cells.

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