CN116744947A

CN116744947A - Method for producing regenerated T cells through iPS cells

Info

Publication number: CN116744947A
Application number: CN202180088550.1A
Authority: CN
Inventors: 金子新; 五之坪良辅; 大泽光次郎; 等泰道; 山下和男; N·C·P·萨克斯
Original assignee: Hongtai Biotechnology Co ltd; Thyas Co ltd
Current assignee: Hongtai Biotechnology Co ltd; Thyas Co ltd
Priority date: 2021-01-04
Filing date: 2021-12-29
Publication date: 2023-09-12

Abstract

Disclosed is a method for producing regenerative T cells from iPS cells into which TCRs isolated from tumor tissue infiltrating CD 106-positive T cells have been introduced. [ solution ] A method for producing regenerative T cells by iPS cells, the method comprising: 1: a step of preparing cdnas encoding T cell receptor alpha and beta chains, respectively, in each individual cell from a population of T cells which are T cells obtained from a subject, are reactive to a tumor-associated antigen and are CD 106-positive; 2: initializing peripheral blood mononuclear cells or T cells of the subject from which the B cells and T cells have been removed into iPS cells, and selecting iPS cell clones having high differentiation efficiency into T cells from the iPS cells thus obtained; 3: a step of introducing the cDNA into the iPS cell clone, a hematopoietic stem cell differentiated from the iPS cell clone, an immature T cell differentiated from the hematopoietic stem cell, or a mature T cell differentiated from the immature T cell; and 4: a step of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell obtained in step (3) and into which the cDNA has been introduced into the mature T cell and proliferating the mature T cell.

Description

Method for producing regenerated T cells through iPS cells

Technical Field

The present invention relates to a method for producing tumor antigen-specific regenerative T cells by introducing T cell receptors obtained from a CD 106-positive T cell population into iPS cells derived from non-T non-B cells, monocytes or T cells, regenerative T cells produced by the method, cancer prevention or treatment agents comprising the regenerative T cells as an active ingredient, pharmaceutical compositions comprising the regenerative T cells, and methods for preventing or treating cancer using the pharmaceutical compositions.

Background

T cells play a central role in immune responses to foreign pathogens such as bacteria and viruses, and abnormal cells such as cancer cells. Thus, a decrease in T cell function is considered to be responsible for pathogen infection and carcinogenesis. T cell replacement therapy or regenerative therapy would be an extremely effective means of improving patient conditions and treating diseases in patients suffering from diseases caused by reduced T cell function.

In studies using humans and mice, it is known that in the case of T cell replacement therapy for infectious diseases or cancers, a high therapeutic effect can be achieved by using T cells that specifically recognize antigens possessed by foreign pathogens such as bacteria and viruses or abnormal cells such as cancer cells. On the other hand, in T cell replacement therapy, it is difficult to secure a sufficient amount of T cells and T cell failure such as a decrease in the proliferation ability of T cells and a decrease in immune response to antigens such as target cells has become an obstacle. Furthermore, how to obtain tumor-specific T cells is a big topic.

In order to overcome the above-described obstacle in T cell replacement therapy, a T cell replacement therapy has been proposed in which artificial pluripotent stem cells (iPS cells) are established from antigen-specific T cells, proliferated, and then regenerative T cells differentiated into T cells are used. Since a T Cell Receptor (TCR) for recognizing a target antigen is formed by gene reconstruction of a genome, the TCR gene of a T cell is preserved in iPS cells in which the T cell has been initialized before initialization. Therefore, by using T cells having a specificity for the target antigen as a raw material for producing iPS cells, regenerative T cells having the same antigen specificity as the original T cells can be produced (patent document 1 and non-patent document 1).

Stringent antigen specificity is essential for safe and effective T cell replacement therapies. However, it has been reported that, among regenerative T cells obtained from T cells by iPS cells as described above, the occurrence frequency of T cells having the same gene reconstruction pattern as that of the original T cells is not necessarily high (patent document 2). In addition, it has been reported that cd8αβ regenerative T cells obtained from human T cells by iPS cells lost antigen specificity due to additional reconstitution of TCR α chain genes at the CD8/CD4 double-positive stage (non-patent document 2).

Prior art literature

Patent literature

Patent document 1: WO2011/096482 text

Patent document 2: WO2013/176197 text

Non-patent literature

Non-patent documents 1.Nishimura T,et al.Generation of rejuvenated antigen to specific T cells by reprogramming to pluripotency and differentiation. Cell Stem cell.2013;12:114-126.

Non-patent document 2.Minagawa A,et al.Enhancing T cell receptor stability in rejuvenated iPSC-derived T cells improves their use in cancer immunotherapeutic.cell Stem cell.2018;23:850-858.

Disclosure of Invention

Problems to be solved by the invention

In cancer treatment using regenerative T cells (iPS-T cells) produced by iPS cells, the use of iPS-T cells that specifically recognize a target antigen present in tumor cells or tumor tissues is important for ensuring the safety and effectiveness of iPS-T cell replacement therapy in cancer treatment. Furthermore, when the onset or recurrence of cancer is detected, it is important to rapidly initiate iPS-T cell replacement therapy to improve the therapeutic outcome.

The object of the present invention is to provide a method for rapidly producing iPS-T cells into which TCRs isolated from tumor tissue infiltrating CD106 positive T cells have been introduced, and iPS-T cells produced according to the method. Furthermore, another object of the present invention is to provide iPS-T cells produced according to the method, a pharmaceutical composition comprising the iPS-T cells, and a method of preventing or treating cancer using the pharmaceutical composition.

In recent studies, it was found that even for one epitope, a plurality of T cell clones (5 to 10 clones) recognize antigens by using different TCRs, respectively. Currently, it is extremely difficult to maintain all antigen-specific T cell clones during the process of obtaining iPS-T cells from cells obtained from a patient. Furthermore, the likelihood of a clone differentiated into iPS-T cells as a T cell clone having the optimal TCR of killing target is affected by individual differences in the subject from which the cells for producing iPS-T cells were taken or by the lot from which iPS-T cells were produced. Furthermore, in order to isolate T cells that specifically recognize a tumor, it is necessary to administer a tumor antigen to a patient or to co-culture T cells and tumor antigen in vitro, but it is extremely difficult to employ such a method if the tumor antigen is unknown.

Currently, it takes a long time to produce iPS-T cells using iPS cell clones obtained in cells obtained from a patient. In order to rapidly provide iPS-T cell replacement therapy to patients in need thereof, shortening the production cycle is a major problem.

Means for solving the problems

The present inventors have found that a CD106 positive T cell population isolated from tumor tissue is a T cell population recognizing a tumor-associated antigen, can have specific aggressiveness to cancer cells, can obtain various TCR pools having antigen specificity from these T cell populations recognizing a tumor-associated antigen, can produce iPS-T cells by introducing TCRs into iPS cells established from non-T non-B cells or monocytes or T cells, can homogenize the quality of regenerated T cells and shorten the production cycle by screening iPS cell clones having good differentiation efficiency into T cells in advance, and can rapidly produce the iPS-T cells by adjusting the timing of obtaining the TCR pools and establishing iPS cells, and the TCR pools and the non-T non-B cells, monocytes or T cells can be derived from the same individual or from different individuals, thus completing the present invention.

Namely, the object of the present invention is achieved by the following invention.

[1]

A method of producing regenerative T cells by iPS cells, comprising:

(1) A step of preparing cdnas encoding T cell receptor alpha and beta chains, respectively, in each individual cell from a T cell population that is a T cell obtained from a subject and is reactive to a tumor-associated antigen and positive for CD 106;

(2) Initializing peripheral blood mononuclear cells or T cells from which B cells and T cells have been removed as peripheral blood mononuclear cells of a subject into iPS cells, and screening iPS cell clones having high differentiation efficiency into T cells from the iPS cells thus obtained;

(3) A step of introducing the cDNA into the iPS cell clone, a hematopoietic stem cell differentiated from the iPS cell clone, an immature T cell differentiated from the hematopoietic stem cell, or a mature T cell differentiated from the immature T cell; and

(4) A step of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell obtained in step (3) and into which the cDNA has been introduced into a mature T cell and proliferating the mature T cell.

[2]

The method according to [1], wherein the step (2) is performed before the step (1) or in parallel with the step (1).

[3]

The method of [1] or [2], wherein the subjects in steps (1) and (2) are the same individual.

[4]

The method of [1] or [2], wherein the subjects in steps (1) and (2) are different individuals, and the subject in step (2) is a subject for cancer prevention or treatment.

[5]

The method of [1] or [2], wherein the subjects in steps (1) and (2) are different individuals, and the subject in step (1) is a subject for cancer prevention or treatment.

[6]

The method of [1] or [2], wherein the subjects in steps (1) and (2) are the same individual or different individuals, and subjects different from the subjects in steps (1) and (2) are subjects for cancer prevention or treatment.

[7]

The method according to [1] or [2], further comprising the steps of: selecting T cells which do not exhibit an alloreaction (alloreaction) to cells from a subject as a subject for cancer prevention or treatment from among T cells into which the cDNA obtained in step (4) is introduced.

[8]

The method according to [1] or [2], further comprising the steps of: screening the cDNA prepared in the step (1) for cDNA encoding a T cell receptor that does not induce an allogeneic response to cells from a subject being a subject for cancer prevention or treatment.

[9]

The method according to [7] or [8], wherein the cells from a subject as a subject for cancer prevention or treatment are peripheral blood mononuclear cells.

[10]

The method according to any one of [1] to [9], wherein the cDNA is introduced into the iPS cell clone, the hematopoietic stem cell clone differentiated from the iPS cell clone, the immature T cells differentiated from the hematopoietic stem cells, or the mature T cells differentiated from the immature T cells using a viral vector, a non-viral vector, or a genome editing technique in step (3).

[11]

The method of [10], wherein the genome editing technique is CRISPR/Cas9 or TALEN.

[12]

The method of [10], wherein the non-viral vector is a transposon vector.

[13]

The method of [12], wherein the transposon vector is a piggyBac (registered trademark) vector.

[14]

The method according to any one of [1] to [13], wherein in step (4), the iPS cell clone, the hematopoietic stem cell, or the immature T cell into which the cDNA is introduced is differentiated into a mature T cell in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of RetroNectin (registered trademark) and an anti-CD 3 antibody, or in the presence of an anti-CD 3 antibody and an anti-CD 28 antibody, and the mature T cell is proliferated.

[15]

The method of [14], wherein the feeder cells are autologous or allogeneic peripheral blood mononuclear cells.

[16]

The method of any one of [1] to [15], wherein the subject in step (1) or (2) is a patient suffering from hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, skin cancer such as malignant melanoma, bladder cancer, head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, large intestine cancer, brain tumor, sarcoma, or B-cell non-hodgkin's lymphoma.

[17]

The method of any one of [1] to [15], wherein the subject in step (1) or (2) is a highly immunogenic cancer patient.

[18]

The method of [17], wherein the highly immunogenic cancer is hepatocellular carcinoma, hepatoblastoma, colorectal cancer, lung cancer, or malignant melanoma.

[19]

The method of any one of [1] to [18], wherein the T cell population in step (1) is double positive for CD3/CD 106.

[20]

The method of any one of [1] to [19], wherein the hematopoietic stem cells are double positive for CD34/CD 43.

[21]

The method of any one of [1] to [20], wherein the immature T cells are CD8 a chain/β chain double positive.

[22]

The method according to any one of [1] to [21], wherein the iPS cell clone screened in step (2) that has high differentiation efficiency into T cells, or the hematopoietic stem cell differentiated from the iPS cell clone in step (3), the immature T cell differentiated from the hematopoietic stem cell, or the mature T cell differentiated from the immature T cell is stored to construct a master cell bank.

[23]

The method of [22], wherein the preservation is cryopreservation.

[24]

The method of [22], wherein the steps (3) and (4) are performed on the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell stored in the master cell bank.

[25]

The master cell bank of [22], comprising the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell.

[26]

A regenerative T cell produced by the method according to any one of [1] to [24 ].

[27]

A pharmaceutical composition comprising the regenerative T cell according to [26 ].

[28]

A method of preventing or treating cancer using the pharmaceutical composition according to [27 ].

Effects of the invention

In the method for producing regenerative T cells (iPS-T cells) by iPS cells of the present invention, peripheral blood mononuclear cells (non-T non-B cells or mononuclear cells) from which T cells and B cells have been removed or T cells are initialized, and iPS cells are obtained, and then iPS cell clones having excellent differentiation efficiency into T cells are selected in advance. Thus, a sufficient amount of iPS-T cells necessary for treatment can be ensured according to the timing required for treatment. In addition, in the method of the present invention, since TCR is introduced into iPS cell clone induced from non-T non-B cells or monocytes or T cells, hematopoietic stem cells differentiated from the iPS cell clone, immature T cells differentiated from the hematopoietic stem cells, or mature T cells differentiated from the immature T cells, in the case where these cells are expanded and cultured after differentiation into iPS-T cells, the reconstitution of TCR genes is more difficult to occur, and the antigen specificity of the introduced TCR is maintained. In addition, iPS-T cells with little failure due to expansion culture can be produced. Further, in the production of iPS-T cells, since iPS cell clones having good differentiation efficiency into T cells are selected and used, the influence of variations in yield and degree of differentiation between production batches of iPS-T cells and individual differences of subjects from which cells are taken on the quality and the like of the obtained iPS-T cells can be minimized. In addition, T cells having TCRs that recognize antigens and effectively kill targets can be efficiently produced.

According to the method of the present invention, since the cDNA encoding TCR is prepared from each individual cell in a T cell population having genetic diversity, cDNA populations having various antigen specificities can be prepared. The CD106 positive T cell population isolated from tumor tissue is a T cell population that recognizes tumor antigens, so isolating a tumor-specific TCR does not require prior immunization with tumor antigens or co-culture of T cells with tumor antigens in vitro. Therefore, the method of the invention can shorten the production period of the iPS-T cells, and can also produce the iPS-T cells with tumor specificity under the condition of unknown tumor antigen in cancer patients. Furthermore, since the preparation and preservation of iPS cell clones derived from non-T non-B cells or monocytes or T cells are performed before or in parallel with the preparation of cDNA encoding an antigen-specific TCR, iPS-T cells can be produced in a short time as compared with a method in which they are sequentially performed. Therefore, in the treatment of cancer using iPS-T cell replacement therapy, iPS-T cells specific to the expressed antigen can be prepared promptly for the case where the antigen of the tumor in the subject cancer patient is changed, or the expression resistance to the treatment or the cancer recurs.

According to the method of the present invention, the subject from which the T cells for producing the cDNA encoding TCR are taken may be the same individual as the subject of the cancer patient as the subject of the iPS-T cell replacement therapy or a different individual. Thus, TCR pools corresponding to multiple antigens can be prepared and screened for TCR with optimal antigen specificity for individual cancer patients.

Brief description of the drawings

FIG. 1 shows a schematic representation of the procedure for producing regenerative T cells from iPS cells in which non-T non-B cells or monocytes in peripheral blood have been initialized.

Fig. 2 shows details of the procedure for producing regenerative T cells from iPS cells that have been initialized to non-T non-B cells or monocytes of peripheral blood. In fig. 2, the upper graph shows a step of isolating TCR genes from CD106 positive T cells and a step of verifying antigen-specific reactivity, and the lower graph shows a step of producing iPS cells into which TCR genes are introduced and a step of producing regenerative T cells from the iPS cells.

FIG. 3 shows a procedure for introducing a TCR gene using piggyBac (registered trademark) transposon vector.

Fig. 4 shows the phenotype of iPS cell-induced regenerative T cells (tumor-associated antigen-specific CD 8-positive cytotoxic T cells).

FIG. 5 shows the results of analysis of telomeres as markers of cell aging in regenerative T cells.

FIG. 6 shows the results of analysis of the expression of PD-1 and TIGHT molecules as markers of cell failure in regenerative T cells.

FIG. 7 shows a comparison of the amounts of IL-2 and IFN-gamma produced in peripheral blood mononuclear cells (CD 8 alpha chain/beta chain biscationic cells) and in regenerated T cells of healthy subjects by stimulation with PMA and ionomycin.

FIG. 8 shows the expression of various markers in tumor infiltrating T cells. In each combination (panel), the cell staining described in the combination expressed positive. The cells are clustered according to expression pattern, and the encircled portions correspond to tumor-toxic T cells.

FIG. 9 shows IFN-gamma production upon autologous tumor stimulation of tumor infiltrating T cells in cell subsets classified according to marker expression. In only the CD106 positive cell subsets, autologous tumor stimulation produced IFN- γ, whereas in the marker positive cell subsets other than CD106, although autologous tumor stimulation produced IFN- γ, IFN- γ was also produced in the marker negative cell subsets, thus demonstrating inferior specificity to CD106.

FIG. 10 shows the phenotype of mature T cells from iPS cells into which the GPC3 antigen specific TCR gene has been introduced. The CD19 gene is a gene in tandem with the TCR gene in the same piggyBac (registered trademark) transposon vector, and serves as a marker for gene insertion and gene expression in the host chromosome.

FIG. 11 illustrates a method of selecting iPS cell clones having high differentiation efficiency into T cells in the step of producing regenerative T cells from iPS cells in which peripheral blood T cells have been initialized.

Fig. 12 illustrates a method of producing regenerative T cells by genome editing iPS cell clones selected as cells having high differentiation efficiency into T cells in the step of producing regenerative T cells from iPS cells in which peripheral blood T cells have been initialized.

FIG. 13 illustrates a method of producing regenerative T cells that recognize cancer antigens from host T cells.

Fig. 14 shows a schematic representation of the procedure for producing regenerative T cells from the same iPS cells that have been initialized with peripheral blood T cells.

Embodiments of the invention

[ T cells ]

In the present invention, a "T cell" obtained from a subject is a cell that expresses an antigen receptor called a T Cell Receptor (TCR) on the cell surface. TCRs include heterodimers consisting of alpha and beta chains and heterodimers consisting of gamma and delta chains. T cells with TCRs consisting of alpha and beta chains are referred to as alpha beta T cells, and T cells with TCRs consisting of gamma and delta chains are referred to as gamma delta T cells. In one embodiment of the invention, the T cells of the invention are preferably CD3/αβ type T cells, but may also be CD3/γδ type T cells. The TCR of the T cell of the invention is gene-transferred.

In the present invention, the term "T cell population" refers to a population of T cells in which the gene sequences of T Cell Receptors (TCRs) recognizing antigens are widely varied as a whole. Thus, T cells taken from a living body are a population of T cells specific for a variety of antigens. T cells present in vivo have TCR gene sequences different from single T cells and can elicit immune responses to any antigen, since random recombination of TCR genes occurs when T precursor cells develop and differentiate in the thymus. In the present invention, CD106 is a highly specific marker for identifying T cells that are aggressive to cancer cells, and individual CD106 positive T cells have TCRs that are tumor antigen specific. Thus, by having CD106 positive T cells as a population, it can be appreciated that the T cell population is immune to a variety of tumor antigens. Thus, a population of CD106 positive T cells taken from a living body is a population of T cells having genetic diversity.

In the present invention, the "tumor-associated antigen" is an antigen expressed in a tumor-specific or non-specific manner, and is an antigen derived from a protein overexpressed in tumor cells and mutants thereof, an antigen derived from a tumor virus, a differentiation antigen of a certain type, a novel tumor-associated antigen (neoantigen) due to gene mutation and splicing abnormality, or the like. T cells that respond to "tumor-associated antigens" are recognized as CD106 positive T cells. In the present specification, a tumor-associated antigen is sometimes referred to as a tumor antigen. In the case of a protein antigen, the protein antigen may be a peptide (peptide fragment) obtained by fragmenting the protein antigen. Antigens expressed in a tumor-specific or non-specific manner can be listed: WT1, GPC3, XAGE1, MUC5AC, MUC6, EGFRvIII, HER-2/neu, MAGE A3, MAGE A1, telomerase, PRAME, SSX2/4, PSCA, CTLA-4, gp100, GD2, GD3, fucose GM1, GM3, sLe (a), glycolipid F77, mesothelin, PD-L1, trp2, CD19, CD20, CD22, ROR1, CD33, c-Met, p53 mutant, NY-ESO-1, PSMA, ETV6-AML, CEA, PSA, AFP, hTERT, epCAM, ALK, androgen receptor, ephA2, CYP1B1, OY-TES-1, MAD-CT-2, melanA/MART1, survivin, ras mutant, erG, r-bcl, XBP1, etc., but is not limited thereto. Viral antigens may be listed: inactivated viruses such as HBV and HPV, and proteins derived from various viruses, for example, but not limited to, EBV LMP1, EBV LMP2, EBNA (EBV nuclear antigen), HPV E1, HPV E2, HPV E6, HPV E7, HBV HBs, HTLV-1Tax, and HBZ (HTLV-1 bZIP factor), etc.

In the present invention, "reactivity to a tumor-associated antigen" means that a T cell has a reaction in which T cells selectively bind/conjugate to an epitope peptide derived from a tumor-associated antigen presented by a major histocompatibility antigen (major histocompatibility complex: MHC) class I or class II on an antigen presenting cell by TCR, and means that T cells do not bind/conjugate to an epitope peptide other than the above epitope peptide. Examples of T cell responses that occur through binding/conjugation of TCR to epitope peptides derived from tumor-associated antigens presented by class I or class II MHC include cytotoxicity, production of IFN- γ and granzyme, expression of T cell activation markers, and activation of transcription factors such as NF-AT.

In the present invention, the T cells are preferably αβ T cells. Because of low invasiveness, the preferred T cell sampling source is peripheral blood, but is not limited thereto. Other preferred sources of collection include cancer tissue or tumor tissue, lymph nodes or other tissues or organs, or all sources of collection in the body such as blood, cord blood, lymph fluid, interstitial fluid (interstitial fluid, intercellular fluid and interstitial fluid), body cavity fluid (ascites fluid, pleural fluid, pericardial fluid, cerebrospinal fluid, synovial fluid and aqueous humor). In one embodiment of the invention, the preferred T cells are T cells derived from tumor tissue. T cells derived from tumor tissue are typically tumor infiltrating T cells.

In the case where the subject is a cancer patient, the cancer of the cancer patient is selected from the group consisting of hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, skin cancer such as breast cancer, ovarian cancer, malignant melanoma, bladder cancer, head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, brain tumor, sarcoma, and B-cell non-hodgkin lymphoma. The cancer is preferably hepatocellular carcinoma or hepatoblastoma.

[ iPS cell cloning ]

In the present invention, iPS cells are preferably prepared by initializing non-T non-B cells, monocytes or T cells, but are not limited to non-T non-B cells, monocytes or T cells. "non-T non-B cells" refers to monocytes that are classified as neither T cells nor B cells. In the present invention, non-T non-B cells or monocytes may be prepared by taking peripheral blood monocytes from a subject and then removing T cells and B cells contained in the monocytes. Peripheral blood mononuclear cells can be isolated from human whole blood by a mononuclear cell isolation solution. As the monocyte isolation solution, lymphoprep (registered trademark) can be exemplified. For removing B cells and T cells from monocytes, antibodies to the surface antigens CD19, CD20, CD22 or B cell receptor possessed by B cells and the surface antigens CD3, CD4 or CD8 possessed by T cells may be used, and for example, magnetic beads such as flow cytometry or MACS (registered trademark) beads may be used. T cells can be isolated from human whole blood by a monocyte isolation solution. As the monocyte isolation solution, lymphoprep (registered trademark) can be exemplified. The purification of T cells can be performed using the surface antigens CD3, CD4 or CD8 or T cell receptors possessed by T cells. For example, magnetic beads such as flow cytometry or MACS (registered trademark) beads may be used.

Methods for producing iPS cells are known in the art. In the present invention, iPS cells may be preferably induced by introducing a cell initializing factor into non-T non-B cells, monocytes or T cells. Examples of cell initializing factors include genes or gene products such as Oct3/4, sox2, sox1, sox3, sox15, sox17, klf4, klf2, c-Myc, N-Myc, L-Myc, nanog, lin, fbx, ERas, ECAT15-2, tcl1, β -catenin, lin28b, sall1, sall4, esrrb, nr5a2, tbx3, and Glis 1. These cell initializing factors may be used alone or in combination. Of these cell initializing factors, oct3/4, sox2, klf4, and c-Myc (so-called factor 4 in mountain) are preferably introduced into non-T non-B cells, monocytes, or T cells from the viewpoint of efficient establishment of iPS cells.

The method for introducing the cell-initiating factor into the non-T non-B cells, monocytes or T cells is not particularly limited, and methods known in the art may be employed. For example, in the case where a gene encoding the cell-initializing factor is introduced into the non-T non-B cell or the monocyte, the gene encoding the cell-initializing factor (e.g., cDNA) may be inserted into an expression vector comprising a promoter functioning in the non-T non-B cell, the monocyte or the T cell, and the expression vector may be introduced into the non-T non-B cell, the monocyte or the T cell by infection, a lipofection method, a liposome method, a calcium phosphate coprecipitation method, a DEAE dextran method, a microinjection method or an electroporation method. When the cell-initiating factor is in the form of a protein and the protein is introduced into the non-T non-B cell, monocyte or T cell, a method using a protein-introducing agent, a method using a protein-introducing domain fusion protein, an electroporation method, and a microinjection method can be exemplified. In the case where the cell initializing factor is in the form of messenger RNA (mRNA) and the mRNA is introduced into the non-T non-B cells or monocytes, a method using an mRNA introducing reagent and a method of adding to a medium may be exemplified.

Examples of the expression vector used for introducing a gene by infection include a viral vector such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, sendai virus, and animal cell expression plasmid, however, it is preferable to introduce a gene encoding the cell-initiating factor into the non-T non-B cell, monocyte, or T cell using sendai virus in view of difficulty in occurrence of insertion mutagenesis, high efficiency of gene introduction, and large replication number of the introduced gene.

Examples of the promoter used for introducing the gene encoding the cell-initiating factor into an expression vector other than T-cell, monocyte or T-cell include SR. Alpha. Promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, ubiquitin promoter and the like. Depending on the presence or absence of a drug such as tetracycline, these promoters may be capable of controlling the expression of genes inserted downstream of the promoter. These expression vectors may include, in addition to promoters, enhancers, poly A addition signals, selectable marker genes (e.g., neomycin resistance genes), SV40 origins of replication, and the like.

The medium for culturing iPS cells obtained by initializing non-T non-B cells, monocytes or T cells is not particularly limited, and may be prepared by taking a medium for culturing animal cells as a basal medium and adding cytokines thereto to maintain the undifferentiated potential of iPS cells. Examples of the basal Medium include Iscove 'sModified Dulbecco's Medium, IMDM, medium 199, eagle minimum essential Medium (Eagle's Minimum Essential Medium, EMEM), alpha MEM, dulbecco's modified Eagle's Medium, DMEM, ham's F, RPMI 1640, fischer, neurobasal Medium (Life Technologies), stemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co.Inc.), and mixed media thereof. Serum or serum-free medium may be added. As the cytokine, bFGF is preferably mentioned, and its concentration in the medium is, for example, 1 to 100. Mu.g/mL (preferably 50. Mu.g/mL).

In one embodiment of the present invention, the method for culturing iPS cells may be an adherent culture or a suspension culture, preferably an adherent culture. Examples of the method for isolating iPS cells include a physical isolation method using a cell scraper or the like, and a separation method using a dissociation solution having protease activity, a dissociation solution having collagenase activity, or a dissociation solution having protease activity and collagenase activity (for example, accutase (registered trademark) and Accumax (registered trademark) or the like).

In one embodiment of the invention, when iPS cells reach a cell density of 1×10 ³ Up to 1X 10 ⁴ Cells/cm ² 、1×10 ⁴ Up to 1X 10 ⁵ Cells/cm ² Or 1X 10 ⁵ Up to 1X 10 ⁶ Cells/cm ² When subculturing is preferably performed in another culture vessel. The number of passages may be any number as long as a desired amount of iPS cells can be obtained, and is preferably 1 to 5 times or 5 to 10 times.

The iPS cells produced consisted of a large number of iPS cell clones. As described below, in order to screen iPS cell clones having high differentiation efficiency into T cells, iPS cells are preferably cloned by colony picking method. The colony picking method is not particularly limited, and a method using a pipette under a microscope, a limiting dilution analysis method, a method using a fully automatic colony selector, and the like can be used. The obtained iPS cell clones may be expanded and cultured to construct a master cell bank. The "master cell pool" consisting of the iPS cell clones is a single pool of each individual iPS cell clone that is dispensed and accumulated in separate cell storage containers. In the master cell bank, iPS cell clones are preferably cryopreserved. Methods of cryopreservation of cells are well known to those skilled in the art. For example, cultured iPS cell clones may be collected and washed with a buffer or a medium, and after counting the number of cells, enriched by centrifugation or the like, suspended in a freezing medium (e.g., a medium containing 10% DMSO), and then cryopreserved. The master cell bank of the present invention may be stored in any device or reservoir capable of cryopreservation. In the case of cryopreservation, the preservation temperature is not particularly limited as long as it is suitable for preservation of cells. Examples thereof include-20 ℃, -80 ℃ and-120 ℃ to-196 ℃, preferably-150 ℃ or less.

"differentiation efficiency" refers to the ratio of the presence of hematopoietic stem cells, immature T cells, and mature T cells in all living cells, respectively, in each differentiation stage from iPS cells to hematopoietic stem cells, from hematopoietic stem cells to immature T cells, and from immature T cells to mature T cells. Differentiated cells at each stage of differentiation can be identified by FACS analysis of surface markers. The efficiency of differentiation into hematopoietic stem cells is expressed as the ratio of CD34/CD43 biscationic cells in all living cells, the efficiency of differentiation into immature T cells is expressed as the ratio of CD4/CD8 biscationic cells or CD5 positive cells in all living cells, and furthermore, the efficiency of differentiation into mature T cells is expressed as the ratio of cells positive for all of CD8 a chain, CD8 β chain, TCR a chain and TCR β chain in all living cells.

"high differentiation efficiency" or "good differentiation efficiency/excellent" means that the ratio of CD34/CD43 double positive cells as hematopoietic stem cells is 5 to 15% or more in the process of differentiating from iPS cells into hematopoietic stem cells; in the differentiation from hematopoietic stem cells into immature T cells, the ratio of CD4/CD8 double positive cells or CD5 positive cells as immature T cells is 10% or more or 50% or more, respectively; the ratio of cells positive for all of CD8 a chain, CD8 β chain, TCR a chain and TCR β chain in differentiating from immature T cells into mature T cells is 50% or more.

The regenerative T cells of the present invention are preferably manufactured by first clonally differentiating the iPS cells into hematopoietic stem cells, then differentiating the hematopoietic stem cells into immature T cells, and finally differentiating the immature T cells into CD8 single positive T cells, i.e., mature T cells.

In the present invention, the "hematopoietic stem cells" are cells which can differentiate into hematopoietic cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes and megakaryocytes. Hematopoietic stem cells and Hematopoietic Progenitor Cells (HPCs) are not distinguished from each other and refer to the same cell unless otherwise indicated. Hematopoietic stem/progenitor cells are recognized by, for example, the double positivity of the surface antigens CD34 and CD 43.

In the present invention, "immature T cells" refer to T cells at various stages from the stage of T cells in which neither TCR alpha chain nor beta chain is expressed to the stage of CD8 single positive cells which express TCR alpha and beta chains, which have undergone CD4/CD8 double positive cells. The immature T cells are preferably double positive for CD8 alpha/beta chains.

In the present invention, "mature T cells" refer to T cells that express TCR α and β chains and have progressed through CD4/CD8 double positive cells to CD8 single positive cells. Mature T cells are preferably CD8 alpha/beta double positive.

[ culture of hematopoietic Stem cells and immature T cells ]

Hematopoietic stem cells are preferably produced by culturing iPS cells in a medium supplemented with vitamin C. The term "vitamin C" as used herein refers to L-ascorbic acid and its derivatives. "L-ascorbic acid derivative" refers to a substance that is converted to vitamin C by an in vivo enzymatic reaction. L-ascorbic acid derivatives include, for example, vitamin C phosphate, ascorbyl glucoside, ethyl ascorbic acid, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbyl-2-phosphate-6-palmitate. Preferred L-ascorbic acid derivatives are vitamin C phosphates, and examples thereof include sodium phosphate-L-ascorbate and magnesium phosphate-L-ascorbate phosphate. For example, the medium contains vitamin C at a concentration of 5 to 500. Mu.g/mL.

The medium for producing hematopoietic stem cells is not particularly limited, and can be prepared by using a medium for culturing animal cells as a basal medium, and adding vitamin C and the like thereto. As the basal medium, for example, iscove's Modified Du Medium (IMDM) medium, medium 199, eagle Minimum Essential Medium (EMEM), alpha MEM medium, dulbecco's Modified Eagle Medium (DMEM), ham's F12 medium, RPMI 1640 medium, fischer medium, neurobasal medium (Life Technologies), stemPro34 (Life Technologies) and a mixed medium thereof can be cited. The medium may contain serum or be serum-free. If necessary, the basal medium may contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol, lipids, amino acids, L-glutamine, nonessential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like.

In the medium for producing hematopoietic stem cells, cytokines selected from BMP4 (bone morphogenic protein 4), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), TPO (thrombopoietin) and FLT3L (FLT 3 ligand) may be further added. The concentration is, for example, 1 to 100ng/mL for BMP4, 1 to 100ng/mL for VEGF, 1 to 100ng/mL for bFGF, 10 to 100ng/mL for SCF, 1 to 100ng/mL for TPO, and 1 to 100ng/mL for FLT 3L.

Tgfβ inhibitors may be added to the culture medium of hematopoietic stem cells. "TGF-beta inhibitor" is a low molecular weight inhibitor that interferes with the signaling of the TGF-beta family, and examples include SB431542 and SB202190 (R.K. Lindemann et al, mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908 and SD208 (Scios), and LY2109761, LY364947 and LY580276 (Lilly Research Laboratories). The addition concentration in the medium is preferably 0.5 to 100. Mu.M.

The iPS cells can be co-cultured with feeder cells such as C3H10T1/2 (Takayama N.et al, J Exp Med.2817-2830, 2010) or heterologous stromal cells (Niwa A et al, J Ce11 physiol.2009nov;221 (2): 367-77).

The method of culturing iPS cells in hematopoietic stem cell production may be either adherent culture or suspension culture. However, suspension culture is preferred. For example, iPS cells may be used for suspension culture after releasing colonies cultured to 80% confluence in a culture dish used and separating them into individual cells. Examples of the method for isolating iPS cells include a physical isolation method using a cell scraper or the like, and a separation method using a dissociation solution having protease activity and collagenase activity (for example, accutase (registered trademark) and Accumax (registered trademark) or the like) or using a dissociation solution having collagenase activity.

Suspension culture is a method in which cells are cultured in a state of not adhering to a culture vessel. Suspension culture is not particularly limited, and may be performed by using a culture vessel which has not been subjected to artificial treatment (e.g., coating treatment with an extracellular matrix or the like) for improving adhesion to cells, or by using a culture vessel which has been subjected to artificial treatment to inhibit adhesion (e.g., coating treatment with polyhydroxyethyl methacrylate (poly-HEMA) or nonionic surfactant polyol (Pluronic F-127 or the like)). During suspension culture, embryoid Bodies (EBs) are preferably formed and cultured. When embryoid bodies are cultured in suspension to obtain hematopoietic stem cells, they are preferably isolated as single cells and then subjected to adherent culture.

Hematopoietic stem cells may also be prepared from a vesicle-like structure (also referred to as iPS-sac) obtained by culturing iPS cells. Here, the "vesicle-like structure" is a three-dimensional sac-like (having an internal space) structure derived from iPS cells, is formed of an endothelial cell population or the like, and internally contains hematopoietic stem cells.

The temperature conditions in the culture process for producing hematopoietic stem cells from iPS cells are not particularly limited, for example, about 37 ℃ to about 42 ℃, and preferably about 37 ℃ to about 39 ℃. Further, the culture time may be appropriately determined by one skilled in the art while monitoring the number of hematopoietic stem cells or the like. The number of days of culture is not particularly limited as long as hematopoietic stem cells can be obtained, for example, at least 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more, and preferably 14 days. The long culture period does not cause problems in the production of hematopoietic stem cells. In addition, cultivation under low oxygen conditions is also possible, and in one embodiment of the present invention, the oxygen concentration under low oxygen conditions may be exemplified by 15%, 10%, 9%, 8%, 7%, 6%, 5% or less.

"CD4/CD8 double positive T cells" are cells in which the surface antigens CD4 and CD8 are both positive (CD8+CD4+) in T cells, and since T cells can be recognized by positive for the surface antigens CD3 and CD45, CD4/CD8 double positive T cells can be identified as cells in which CD4, CD8, CD3 and CD45 are positive. The CD4/CD8 double positive T cells can be differentiated into CD4 single positive cells or CD8 single positive cells by induction.

CD4/CD8 double positive T cells can be produced by a method comprising the step of culturing hematopoietic stem cells in a medium supplemented with a p38 inhibitor and/or SDF-1.

"p38 inhibitor" is defined as a substance that inhibits the function of the p38 protein (p 38MAP kinase). Examples of the p38 inhibitor include chemical inhibitors of p38, dominant negative mutants of p38, and nucleic acids encoding the same, but are not limited thereto.

Chemical inhibitors of p38 can be listed: SB203580 (4-fluorophenyl) -2- (4-methylsulfonylphenyl) -5- (4-pyridyl) -1H-imidazole) and its derivatives, SB202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole) and its derivatives, SB239063 (trans-4- [4- (4-fluorophenyl) -5- (2-methoxy-4-pyrimidinyl) -1H-imidazol-1-yl ] cyclohexanol) and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SClO-469, SCIO-323, VX-702 or FR167653, but are not limited thereto. These compounds are commercially available, for example, SB203580, SB202190, SC239063, SB220025 and PD169316 are available from Calbiochem, inc., and SClO-469 and SCIO-323 are available from SCIOs, inc., etc. The p38 inhibitor is added to the medium in a range of, for example, about 1. Mu.M to about 50. Mu.M.

Dominant negative mutants of p38 may be exemplified by p38T180A in which threonine at position 180 in the DNA binding domain of p38 is point mutated to alanine; or p38Y180F, wherein tyrosine 182 of p38 is point mutated to phenylalanine, etc. in human and mouse.

SDF-1 (stromal cell derived factor 1) refers not only to SDF-1α or its mature form, but also to SDF-1β, SDF-1 gamma, SDF-1 delta, SDF-1 epsilon orAn isoform or mature form thereof, or a mixture thereof in any ratio. Preferably, SDF-1 alpha is used. SDF-1 may also be referred to as CXCL-12 or PBSF.

SDF-1 may have one or more amino acids in its amino acid sequence substituted, deleted and/or added, and the sugar chain may be substituted, deleted and/or added as long as it has activity as a chemokine. Amino acid mutations are acceptable if at least four cysteine residues (Cys 30, cys32, cys55, and Cys71 in the case of human SDF-1 alpha) remain in SDF-1 and they have more than 90% identity to the amino acid sequence in its native form. SDF-1 can be mammalian, e.g., human, non-human, e.g., monkey, sheep, cow, horse, pig, dog, cat, rabbit, rat, or mouse, etc. For example, a protein registered in GenBank with a registration number NP-954637 may be used as human SDF-1α, and a protein registered in GenBank with a registration number NP-000600 may be used as SDF-1β.

As SDF-1, a commercially available product can be used. Products purified from natural products may be used, or products produced by peptide synthesis or genetic engineering techniques may be used. SDF-1 is added to the medium in a range of, for example, about 10ng/mL to about 100 ng/mL.

The medium used to produce CD4/CD8 double positive T cells is not particularly limited. Can be prepared by taking a medium for culturing animal cells as a basal medium and adding a p38 inhibitor and/or SDF-1 thereto and further preferably adding vitamin C. The type of vitamin C used to produce CD4/CD8 double positive T cells is, for example, as described above, and the concentration of vitamin C is, for example, 5 to 200. Mu.g/mL. As the basal medium, for example, iscove's Modified Du Medium (IMDM), medium 199, eagle Minimum Essential Medium (EMEM), alpha MEM medium, dulbecco's Modified Eagle Medium (DMEM), ham's F medium, RPMI 1640 medium, fischer's Neurobasal medium (Life Technologies) and mixed medium thereof can be cited. The medium may be supplemented with serum or serum-free. If necessary, the basal medium may contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, nonessential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like.

Cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT3L and IL-7 may be further added to the medium used to produce CD4/CD8 double positive T cells. The concentration is, for example, 10 to 100ng/mL for SCF, 10 to 200ng/mL for TPO, 1 to 100ng/mL for FLT3L, and 1 to 100ng/mL for IL-7.

Hematopoietic stem cells may be cultured by adherent culture or suspension culture, however, adherent culture is preferred. In the case of the adhesion culture, the culture vessel may be coated for use. Examples of coating agents include Matrigel (Niwa A et al PLos one.6 (7): e22261,2011), collagen, gelatin, laminin, heparan sulfate proteoglycan, fibronectin, fc-DLL4 or nestin, and combinations thereof.

The culture temperature conditions for culturing hematopoietic stem cells to produce CD4/CD8 double positive T cells are not particularly limited, but, for example, preferably about 37 ℃ to about 42 ℃, and more preferably about 37 ℃ to about 39 ℃. In addition, the culture time can be appropriately determined by one skilled in the art while monitoring the number of CD4/CD8 double positive T cells, etc. The number of days of culture is not particularly limited as long as the CD4/CD8 double positive T cells can be obtained, for example, at least 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 22 days or more, or 23 days or more, and preferably 23 days.

The resulting CD4/CD8 double positive T cells may be isolated and used, or as a cell population comprising other cell types. In the case of isolation, methods known to those skilled in the art can be used. Examples thereof include a method of labeling with an antibody against CD4, CD8, CD3 and/or CD45 and separating the labeled antibody by a flow cytometer, and a method of purifying the labeled antibody by an affinity column for immobilizing an antigen of interest.

"CD8 single positive T cells", i.e., mature T cells, are T cells in which the surface antigen CD8 is positive (CD 8 ⁺ CD4 ^- ) Also known as cytotoxic T cells. Since T cells can be recognized by positive for the surface antigens CD3 and CD45, CD8 single positive T cells can be identified as cells positive for CD8, CD3 and CD45 and negative for CD 4.

CD8 single positive T cells can be produced by a method comprising the step of culturing CD4/CD8 double positive T cells in a medium supplemented with adrenocortical hormone.

The adrenocortical hormone is preferably a glucocorticoid or a derivative thereof, and for example, cortisone acetate, cortisol, fludrocortisone acetate, prednisolone (prednisolone), triamcinolone, methylprednisolone (methylprednisolone), dexamethasone (dexamethasone), betamethasone (betamethasone), or beclomethasone dipropionate (beclomethasone dipropionate) may be mentioned. Preferably, the adrenocortical hormone is dexamethasone. Its concentration in the medium is, for example, 1 to 100nM.

The medium for producing CD8 single positive T cells is not particularly limited, and may be prepared by taking a medium for culturing animal cells as a basal medium and adding adrenocortical hormone thereto. As the basal medium, for example, iscove's Modified Du Medium (IMDM), medium 199, eagle Minimum Essential Medium (EMEM), alpha MEM medium, dulbecco's Modified Eagle Medium (DMEM), ham's F medium, RPMI 1640 medium, fischer's Neurobasal medium (Life Technologies) and mixed medium thereof can be cited. The medium may be supplemented with serum or serum-free. If necessary, the basal medium may contain one or more substances selected from, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol, lipids, amino acids, L-glutamine, nonessential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like.

The medium used to produce CD8 single positive T cells preferably further contains anti-CD 3 antibodies, retinoids or cytokines. Examples of cytokines include IL-2, IL-7, IL-15, and IL-21.

The anti-CD 3 antibody is not particularly limited as long as it is an antibody specifically recognizing CD 3. For example, antibodies produced by OKT 3 clones can be cited. The concentration of anti-CD 3 antibody in the medium is, for example, 10 to 1000ng/mL.

The vitamin C species used to produce CD8 single positive T cells are, for example, those described above, and can be used under the same conditions as described above.

The concentration of cytokines in the medium used to produce CD8 single positive T cells is, for example, 10 to 1000U/mL for IL-2 and 1 to 100ng/mL for IL-7.

The temperature conditions during the culture of the CD4/CD8 double positive T cells for producing CD8 single positive T cells are not particularly limited, for example, preferably about 37 ℃ to about 42 ℃, and more preferably about 37 ℃ to about 38 ℃. In addition, the culture time can be appropriately determined by one skilled in the art while monitoring the number of CD8 single positive T cells, etc. The number of days of culture is not particularly limited as long as CD8 single positive T cells can be obtained, for example, at least 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more, and preferably 3 days.

[ preparation of cDNA encoding TCR ]

In the present invention, it is preferable to prepare cDNAs encoding the TCR alpha chain and the beta chain, respectively, from each single cell. T cells taken from the subject are genetically diverse T cell populations as a whole, but are identified as CD106 positive T cells without determining the antigen or peptide sequence recognized by the TCR possessed by each T cell, with different antigen specificities for each T cell. To screen TCRs that are optimal for various tumor-associated antigens, i.e., TCRs that are highly reactive to various tumor-associated antigens, cdnas are preferably prepared from individual cells.

The CD106 may also be used as a marker, and a T cell population responsive to a tumor-associated antigen may be isolated as a single cell from T cells obtained from a subject by a cell sorter or the like. In addition to CD106, cell surface CD137 may be utilized as a marker for isolated cells. Known methods for isolating human T cells can be exemplified by flow cytometry using antibodies against T cell surface markers such as CD106, CD3, and CD137, and cell sorters. Gene cloning can be performed from the obtained single T cells using a PCR method, and cDNA encoding the alpha chain and beta chain of TCR, respectively, can be amplified.

[ construction of vector ]

PCR fragments amplified using the cDNA of the isolated TCR as a template can be integrated into viral or non-viral vectors using, for example, a Gibson assembly system. Specifically, the isolated TCR α chain gene and TCR β chain gene are linked by a T2A sequence downstream of the ubiquitin promoter, and, downstream thereof, to a marker gene such as EGFR (EGFRt, truncated EGFR) from which the ligand binding site and intracellular domain for the IRES (internal ribosome entry site) sequence have been removed or CD19 from which the intracellular domain has been deleted, is linked, and the construct is integrated into a viral vector or a non-viral vector. Viral vectors and non-viral vectors may be used as vectors, however, non-viral vectors are preferred. As the non-viral vector, a transposon vector is preferable, and a piggyBac (registered trademark) transposon vector is more preferable. The transposon method is an inexpensive and safe next generation gene transfer method compared with the conventional viral vector method.

[ cell introduction of TCR cDNA ]

As a method of introducing a cDNA pair encoding a TCR α chain and a β chain, respectively, into an iPS cell clone obtained from a non-T non-B cell or a monocyte or a T cell which has a good differentiation state into a T cell, the hematopoietic stem cell differentiated from the iPS cell clone, the immature T cell differentiated from the hematopoietic stem cell, or the mature T cell differentiated from the immature T cell, a method using a viral vector or a non-viral vector, or a method using a genome editing technique instead of TCR may be employed. Examples of the viral vector include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus and sendai virus, and animal cell expression plasmids, but retrovirus or lentivirus are preferable. When retrovirus or lentivirus infection is performed, a rotavirus infection method or the like is preferably used. When non-viral vectors are used, transposon methods are preferred. To replace TCRs using genome editing techniques, CRISPR/Cas9 methods, CRISPR/MAD methods, and CRISPR/Cas3 methods may be used. Examples of the method for introducing genes of non-viral vectors or the method for introducing guide RNA and donor DNA for genome editing include a lipid transfection method, a liposome method, a calcium phosphate coprecipitation method, a DEAE dextran method, a microinjection method and an electroporation method. The PCR product can be introduced directly into cells without the use of a vector. Electroporation is preferably used to introduce transposon vectors or PCR products into cells. As the electroporation device, the gene transfer device ExPERT (registered trademark) system (MaxCyte corporation) is preferable. The method of introducing cDNA pairs encoding TCR alpha and beta chains, respectively, may use genome editing techniques such as CRISPR/Cas9 and TALEN. The method of introducing TCR genes by CRISPR/Cas9 is, for example, to introduce guide RNAs designed in the genes of endogenous TCR α and β chains (guide RNAs for sense and antisense strands), and target TCR α and β chain genes having homologous recombination portions of endogenous TCR α chains on the 5 'and 3' sides, into target cells together with vectors encoding Cas9, by a gene linked by P2A sequences (self-cleaving sites). In this case, endogenous promoters and enhancers can be used for expression of the target TCR α and β chain genes.

Examples of promoters used in the expression vector for introducing the cDNA pair into the cell include: EF1 alpha promoter, SR alpha promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter, ubiquitin promoter, etc. Depending on the presence or absence of a drug such as tetracycline, these promoters may be capable of controlling the expression of genes inserted downstream of the promoter. In addition to promoters, expression vectors may include enhancers, poly A addition signals, selectable marker genes (e.g., neomycin resistance genes), SV40 origins of replication, and the like.

By culturing the iPS cell clone into which the cDNA pair is introduced, the hematopoietic stem cell differentiated from the iPS cell clone, the immature T cell differentiated from the hematopoietic stem cell, or the mature T cell differentiated from the immature T cell under the culture conditions such as the above-described medium, medium composition, culture temperature, etc., it is possible to obtain a regenerative T cell expressing a TCR composed of the α chain and β chain encoded by the cDNA pair, respectively. In this culture, the proliferation of these cells is preferably performed in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of retroNectin (registered trademark) and anti-CD 3 antibodies, or in the presence of anti-CD 3 antibodies and anti-CD 28 antibodies. The feeder cells are preferably autologous or allogeneic peripheral blood mononuclear cells. "autologous" refers to the same subject from which the peripheral blood mononuclear cells and the iPS cell clones were derived, and "allogenic" refers to the different subjects from which the peripheral blood mononuclear cells and the iPS cell clones were derived.

The iPS cells, the hematopoietic stem cells, the immature T cells, or the mature T cells into which the cDNA pair is introduced may be cloned. For screening of clones, cloning by colony picking is preferred. The colony picking method is not particularly limited, and examples thereof include a method using a pipette under a microscope, a limiting dilution method, and a method using a fully automatic colony picker.

For example, whether the target TCR is expressed can be confirmed based on TCRV beta gene bank analysis using the TCRV beta assay kit (BECKMAN COULTER Co., catalog number IM-3497). Since marker genes such as EGFR (truncated EGFR) and CD19t (truncated CD 19) are integrated into a vector, expression of TCR alpha chain and beta chain can be presumed by analyzing expression of the marker genes.

The iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell may construct a master cell bank after expansion culture. The "master cell bank" as described herein is that the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell are dispensed into separate cell storage containers for each individual pool of individual clones and pooled. In general, it is preferable to proliferate the cells within a range where the cell characteristics are not changed, and to dispense them into a plurality of cell storage containers. The cells stored in the master cell bank are used as cells of a starting material for producing regenerative T cells by introducing TCR genes or CAR (chimeric antigen receptor ) genes, and for each production of the regenerative T cells, a cell storage container containing a desired number of the cells can be removed from the master cell bank. Thus, the regenerative T cells of the present invention can be repeatedly provided with the same quality.

In the master cell bank, it is preferable that the cells dispensed into a cell storage container are cryopreserved. Methods of cryopreservation of cells are well known to those skilled in the art. For example, individual clones of cells after expansion culture can be recovered, washed with a buffer solution or a medium, concentrated by centrifugation or the like after counting the number of cells, suspended in a freezing medium (for example, a medium containing 10% DMSO), and then cryopreserved. For example, it is possible to obtain a beverage by incorporating 1X 10 per bottle ⁶ Up to 1X 10 ⁷ Cell storage containers of individual cells accumulate 200 to 1000 flasks in a cell storage container store (storage) or the like to construct a master cell bank. The master cell bank of the present invention may be stored in any device or reservoir capable of cryopreservation. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is suitable for preservation of cells. For example, -20 ℃, -80 ℃ and-120 ℃ to-196 ℃, preferably-150 ℃ or less.

[ production of regenerated T cells from iPS cells ]

Each epitope is said to have 5 to 10 TCR pairs corresponding to the tumor-associated antigen, each TCR pair being predicted to have a different sensitivity to point mutant of the tumor-associated antigen. Thus, for example, even when a point mutation occurs in a tumor-associated antigen, there is a high possibility that a TCR capable of recognizing the epitope is present, and an antigen-specific regenerative T cell can be rapidly prepared for the point mutation mutant. In addition, even when tumor-associated antigens are completely altered due to mutation, even when resistance occurs in cancer treatment using regenerative T cell replacement therapy, the responsiveness of a patient to various tumor antigens can be analyzed, TCRs responsive to these tumor-associated antigens can be produced starting from already obtained iPS cells having a high ability to differentiate toward T cells or differentiated cells derived from iPS cells, and thus, regenerative T cells can be produced rapidly.

[ T cells as a TCR acquisition Source ]

T cells that are the source of the TCR pair collection corresponding to the tumor-associated antigen are preferably collected from tumor tissue. The harvested T cells can be expanded in vitro by co-culturing with tumor tissue to expand a tumor responsive cell population, followed by cell isolation using CD106 as a marker.

When the subject from which the T cells for obtaining the TCR pairs are taken and the subject of the cancer treatment subject as regenerative T cell replacement therapy are different individuals, it is not necessary to take antigen-specific T cells from the subject of the treatment subject. Therefore, since T cells for obtaining a TCR pair can be taken in advance at any period independent of the treatment period of regenerative T cell replacement therapy, regenerative T cell replacement therapy can be started quickly.

When the subject from which the T cells for obtaining the TCR pair are taken and the subject as a subject of the cancer treatment for regenerative T cell replacement therapy are different individuals, it is necessary to confirm that the obtained TCR pair does not exhibit an allogeneic response to the cells of the subject of the treatment. The method of confirmation is carried out by known methods. For example, it can be confirmed that the obtained T cells having the TCR pair do not respond to cells of the subject to be treated by mixed lymphocyte culture (MLR) or the like. Alternatively, it was confirmed that the obtained T cells having TCR pairs did not respond to an antigen in which one or more residues of the constituent amino acids of the antigen were replaced with other amino acids.

[ pharmaceutical composition ]

Pharmaceutical compositions containing regenerative T cells of the invention are useful in treating subjects with cancer. The pharmaceutical composition of the present invention can be produced by a method commonly used in the field of formulation technology (for example, a method described in Japanese pharmacopoeia, etc.). The pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of the additive include a cell culture medium, physiological saline, and a suitable buffer (e.g., phosphate buffer).

The pharmaceutical compositions of the invention may be prepared by suspending the regenerative T cells in physiological saline or a suitable buffer (e.g., phosphate bufferLiquid), and the like. In order to exhibit the desired therapeutic effect, the amount per dose preferably comprises, for example, 1X 10 ⁷ More than one cell. More preferably, the cell content is 1X 10 ⁸ More than one, and more preferably 1X 10 ⁹ More than one. The cell content can be appropriately adjusted in consideration of the sex, age, weight, affected area state, cell state, and the like of the administration subject. In order to protect cells, the pharmaceutical composition of the present invention may contain dimethyl sulfoxide (DMSO), serum albumin, and the like, in addition to regenerative T cells. In addition, it may contain antibiotics or the like for preventing bacterial contamination, vitamins, cytokines or the like for promoting activation and differentiation of cells. In addition, the pharmaceutical compositions of the present invention may contain other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preserving agents, preservatives, physiological saline, etc.).

The pharmaceutical composition containing the regenerative T cells of the present invention as an active ingredient can be stored frozen. In the case of cryopreservation, the storage temperature is not particularly limited as long as it is suitable for preservation of cells. Examples thereof include-20 ℃, -80 ℃ and-120 to-196 ℃, preferably-150 ℃ or less. In the case of cryopreservation, the cells are preferably stored in an appropriate container such as a freezing vial or a freezing bag. Methods for minimizing the risk of cell damage to regenerative T cells during freezing and thawing are well known to those skilled in the art.

In the cryopreservation of regenerative T cells of the present invention, the T cells are recovered from the medium, washed with a buffer or medium, enriched by centrifugation or the like after counting the number of cells, suspended in a freezing medium (e.g., a medium containing 10% DMSO), and then cryopreserved at a low temperature. The regenerative T cells may be stored as clones or as a mixture of clones. Pharmaceutical compositions containing regenerative T cells of the invention may, for example, contain 5X 10 per container (e.g., frozen vials, frozen bags, etc.), for example ⁴ From 9 to 10 ¹⁰ However, it may be changed according to the type of target cancer, the administration subject, the administration route, etc.

Examples of the route of administration of the pharmaceutical composition containing the regenerative T cells of the present invention include injection, intratumoral injection, arterial injection, portal intravenous injection, intraperitoneal administration, and the like. However, the administration route is not limited thereto, as long as regenerative T cells, which are the active ingredients in the pharmaceutical composition of the present invention, can be delivered to the affected part. For the administration schedule, both single administration and multiple administration are possible. As for the time of the multiple administrations, for example, a method of repeating the administration once every 2 to 4 weeks or a method of repeating the administration once every 6 months to 1 year may be employed. The sex, age, weight, condition, etc. of the subject patient may be considered in formulating the administration schedule.

In the case where the pharmaceutical composition containing the regenerative T cells of the present invention is used for preventing or treating cancer in a cancer patient, when the TCR on the regenerative T cells is allogenic, the pharmaceutical composition containing the regenerative T cells into which the TCR that does not exhibit an allogenic response to normal cells of the cancer patient has been introduced is used to avoid an allogenic response in the cancer patient.

The pharmaceutical composition of the present invention can be used for preventing or treating cancer. Examples of the cancer include, but are not limited to, hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, malignant melanoma, and other skin cancers, bladder cancer, head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, brain tumor, sarcoma, and B-cell non-hodgkin lymphoma. The pharmaceutical composition of the present invention can be applied to cancers having high immunogenicity, such as hepatocellular carcinoma, hepatoblastoma, colorectal cancer, lung cancer and malignant melanoma. "highly immunogenic cancer" is a cancer having a strong ability to induce an immune response, and means a cancer in which a large number of gene mutations are found in cancer cells (1000 or more or 5000 or more per cell), which are recognized by immune cells including T cells, an immune checkpoint inhibitor shows an effect, and in which a large number of immune cells infiltrate into tumor tissue (a large number of immune cells are found by pathological tissue staining).

The present invention is illustrated with reference to the following examples, however, the present invention is not limited to the following examples.

Example 1

[ production of regenerative T cells from peripheral blood non-T non-B cells or monocyte initialized iPS cells ]

FIG. 1 shows the steps for producing regenerative T cells of the present invention. Monocytes are taken from the tumor tissue of the cancer patient (subject) being treated, T cells specifically responsive to the tumor antigen are isolated as CD106 positive T cells, and the TCR gene is isolated from the CD106 positive T cells (fig. 1- (2')). TCR genes expected to have higher therapeutic effects were screened for their ability to bind to tumors based on TCR encoded by the TCR gene (fig. 1- (3')). When tumor recurrence and tumor cell mutation occur in the patient undergoing treatment, the effective TCR gene for treatment can be screened again. In order to obtain a sufficient therapeutic effect, it is important to obtain a plurality of types of TCRs (TCR pools).

In addition, iPS cells were produced from non-T non-B cells or monocytes of the patient peripheral blood (fig. 1- (1) and (2)). Next, iPS cell clones with good differentiation efficiency into T cells were selected from the obtained iPS cells (fig. 1- (3)). It has been demonstrated that by this screening, the deviation in induction efficiency to T cells due to the difference in iPS cell clones is significantly reduced, and the robustness and efficiency of regenerative T cell production are improved. The selected TCR gene group, which specifically reacted to the tumor antigen, was expressed in iPS cell clones with good differentiation efficiency to the T cells (fig. 1- (4)), and each selected TCR gene was differentiated into mature T cells and proliferated (fig. 1- (5)). In addition, the mature T cells are tumor antigen specific cytotoxic T cells and are regenerative T cells. These regenerative T cells were administered to cancer patients (fig. 1- (6)).

In the method of the present invention, since the step of producing iPS cells (fig. 1- (1) to (3)) and the step of obtaining TCR genes responsive to tumor antigens (fig. 1- (2 ') and (3')) can be performed in parallel, these steps can be completed in a shorter period of time (about 55 days) than in the case where these steps are performed sequentially.

The above is explained in more detail. The isolation of the tumor antigen specific TCR genes and antigen specific reactivity was validated as described below (see upper panel of fig. 2). Tumors (specimens) isolated from cancer patients (subjects) were co-cultured with CD 8-positive cytotoxic T cells, and then single cell sorting was performed on the activated CD 106-positive cytotoxic T cells (CD 8-positive and CD 106-positive cells) by a cell sorter (fig. 2- (2)). TCR gene pairs (TCR alpha chain genes and TCR beta chain genes) were isolated from the isolated single cells by single cell PCR, the isolated TCR gene pairs were sequence analyzed, and the types of tumor-reactive T cells (TCR repertoires) and their frequency of occurrence were analyzed (fig. 2- (3)). The isolated TCR gene pairs were expressed in TCR gene-deficient T cell lines that had bound the reporter gene, and the TCR gene complex was reconstituted (fig. 2- (4)). Subsequently, the reactivity of the reconstituted TCR gene complex with tumors (antigen-specific reactivity) was verified (fig. 2- (5)). That is, when a T cell line expressing the TCR gene pair is co-cultured with a tumor cell of interest, but the expressed TCR specifically binds to a tumor antigen, the TCR gene signal is activated and a reporter gene integrated downstream of the target gene promoter is expressed. Thereafter, antigen-specific reactivity was verified by measuring reporter activity and performing binding evaluation of tumor antigen-specific TCRs. Through the above steps, a plurality of genes having a high frequency of occurrence are selected from TCR gene pairs exhibiting specific reactivity to tumors, and used as the introduced genes of iPS cells (M-iPS cells) derived from non-T non-B cells or monocytes, which will be described later.

Production of iPS cells and regenerative T cells into which the TCR gene was introduced was performed as follows (see lower panel of fig. 2). iPS cells were prepared from peripheral blood mononuclear cells of cancer patients (fig. 2- (2')). The M-iPS cells depicted in the figures are iPS cells not derived from T cells, and are cells that have not undergone gene reconstruction of TCR genes. Next, an M-iPS cell line having excellent differentiation efficiency into T cells was selected from the produced M-iPS cell line group (fig. 2- (3')). For the selected M-iPS cell lines, tumor-specific TCR genes (two genes of TCR. Alpha. Chain gene and TCR. Beta. Chain gene, see FIG. 2- (3)) were subjected to gene transfer using transposon vectors (see FIG. 2- (6) and FIG. 3), and M-iPS cell lines (TCR-iPS cell lines) expressing the TCR genes were selected by a cell sorter (MACQUANT Tyto (registered trademark) cell sorter, miltenyi Biotec Co.) using the expression of the marker CD19 as an index. Next, hematopoietic stem cell precursors are induced by embryoid body-like methods and induced into mature T cells by differentiation of immature T cells by stepwise differentiation induction methods mimicking T cell production in vivo.

The phenotype of regenerative T cells (tumor antigen specific CD8 positive cytotoxic T cells) induced by iPS cells as described above was analyzed. The results of analysis of regenerative T cells based on antigen markers on the cell surface using a flow cytometer are shown in fig. 4. Confirmation of expression of CD45 by regenerative T cells induced by iPS cells ⁺ TCRαβ ⁺ CD3 ⁺ CD4 ^- CD8αβ ⁺ The CD45 ⁺ TCRαβ ⁺ CD3 ⁺ CD4 ^- CD8αβ ⁺ Expression was found in mature cytotoxic T cells in vivo.

The analysis results of telomeres (index of rejuvenation) as markers of cell aging for the regenerative T cells via iPS cells of the present invention are shown in fig. 5. The telomere length recovery was confirmed in (C) and (D) by the tumor antigen-specific regenerative T cells (D) of the present invention, which were about 3 times as long as the telomere length of iPS cells (C) produced from tumor antigen-specific T cells (a) and pre-regenerative tumor antigen-specific T cells (B) of the patient, compared to the telomere length of peripheral blood mononuclear cells (a) of the patient. That is, an improvement in cell aging is demonstrated in the regenerative T cells of the present invention.

For the regenerative T cells via iPS cells of the present invention, the expression of PD-1 and TIGIT molecules, one of the cell failure markers associated with immune checkpoints, was analyzed by staining with anti TIGIT antibodies and anti PD-1 antibodies and flow cytometry. For comparison, tumor antigen specific T cells prior to regeneration were also analyzed. The results are shown in FIG. 6. It was confirmed that the expression of PD-1 and TIGIT molecules was significantly reduced in the regenerative T cells of the present invention compared to the tumor antigen specific T cells before regeneration. Therefore, it was shown that the regenerative T cells of the present invention have high cytotoxic activity.

Cytokine production was analyzed, which is an important indicator of cytotoxicity. Cytotoxic T cells (CD 8 alpha/beta double positive cells) and regenerative T cells of the invention in peripheral blood mononuclear cells collected from healthy persons were stimulated with PMA (phorbol 12-myristate 13-acetate) and Ionomycin (Ionomycin), and the amounts of IL-2 and IFN- γ produced were compared. The results are shown in FIG. 7. The results show a significant increase in cells producing IL-2 and IFN-gamma in the regenerative T cells of the invention compared to cytotoxic T cells obtained from healthy humans.

Example 2

[ preparation of iPS cell clone ]

Mononuclear cells were isolated from peripheral blood collected from patients with hepatocellular carcinoma or hepatoblastoma using a mononuclear cell isolation solution Lymphoprep (registered trademark). From the obtained monocytes, CD19/CD20 positive B cells and CD3/CD4/CD8 positive T cells were removed using FACS or MACS beads to obtain non-T non-B cells or monocytes. Infection of the Sendai virus (CytoTune (registered trademark) 2.0) loaded with factor 4 (Oct 3/4, sox2, klf4 and c-Myc) in mountain and Sendai virus encoding SV40Tag with the obtained non-T non-B cell or monocyte populations had MOI (multiplicity of infection) of 5 to 20. In addition SV40 may be removed.

The obtained iPS cells consisted of a plurality of iPS cell clones. Thus, colonies were picked and cloned. All cloned iPS cells were cryopreserved. The cloned iPS cells were cultured in a differentiation medium for about 10 days to induce hematopoietic stem cells, and CD34/CD43 double positive hematopoietic stem cells were isolated. Isolated hematopoietic stem cells were cultured on FcDLL4 (which is a fusion protein of DLL4 protein and Fc region of immunoglobulin) coated plates for about 21 days and induced to differentiate into T cells.

The frequency of the immature cytotoxic T cells obtained after the above-mentioned 21 days of culture was verified by the double positive rate of CD8 alpha chain/beta chain, and the clone with the highest frequency of occurrence of the double positive cells of CD8 alpha chain/beta chain was selected.

Several iPS cell clones with good differentiation efficiency into T cells were expanded and cultured for two weeks in an iPS cell maintenance medium, and then they were dispensed into a cell storage container for cryopreservation, thereby constructing a master cell bank.

Example 3

[ introduction of TCR Gene into iPS cell clone ]

For iPS cell clones obtained in example 2, which had good differentiation efficiency into T cells and were derived from non-T non-B cells or monocytes, piggyBac (registered trademark) transposon vectors having genes encoding T cell receptor α and β chains, which have confirmed tumor antigen specificity, were introduced using electroporation. Then, iPS cells expressing the target T cell receptor α chain and β chain were isolated by a cell sorter using the expression of the marker molecule CD19 as an index. The isolated iPS cells were cultured in differentiation medium for about 10 days, CD34/CD43 biscationic hematopoietic stem cells were induced and isolated by a cell sorter. The isolated hematopoietic stem cells were cultured on FcDLL 4-coated plates for about 21 days and induced to differentiate into T cells.

The CD8 alpha chain/beta chain double-positive immature T cells obtained after the above-mentioned 21 days of culture were isolated and purified using a cell sorter. Then, the immature T cells are co-cultured in the presence of PHA (phytohemagglutinin) and peripheral blood mononuclear cells as feeder cells, in the presence of RetroNectin (registered trademark) and an anti-CD 3 antibody, or in the presence of an anti-CD 3 antibody and an anti-CD 28 antibody to be induced into mature cytotoxic T cells. These stimuli are performed more than once. The properties of the obtained T cells were confirmed by the cytotoxic activity of GPC 3-specific target cells, production of IFN- γ, and antigen binding ability.

Example 4

[ specificity of CD106 expression in tumor-toxic T cells ]

In melanoma patients with significant anti-PD-1 antibody efficacy, tumor tissue was obtained from surgical specimens prior to initiation of anti-PD-1 antibody therapy. The resulting tumor tissue was subjected to enzyme treatment by adding collagenase and DNase thereto and stirring at 37 ℃ for 30 minutes, to isolate cells. CD3 positive cells were sorted from a mixture of cells derived from tumor tissue and tumor infiltrating immune cells using a BD FACSAria III (registered trademark) cell sorter (BD Biosciences Co.) to obtain cells expressing TCR.

For each of the obtained T cells, single cell gene expression analysis was performed by sequencing using both the gene expression and TCR sequences of the marker candidate using the 5Prime kit and the V (D) J engineering kit of the chromosome system (10X Genomics). As a sequencer, hiSeq 3000 (Illumina corporation) was used.

And according to the obtained fastg file, using a UMAP library of Python to manufacture a UMAP graph. On the UMAP map obtained, the expression of each gene is mapped (mapping).

The results are shown in FIG. 8. In each combination (panel) of fig. 8, the cell staining described in the combination expressed positive for the marker is shown. The cells are clustered according to expression pattern, and the encircled portions correspond to tumor-toxic T cells. It is understood that CD106 (VCAM 1) positive cells have highly specific expression in a tumor-toxic T cell population. It was shown that CD106 can be used as a selection marker for tumor-toxic T cells, with a higher specificity than surface markers other than CD 106. In the case of TCR sequences, the β chain is obtained in cells that account for 9 or more adults, and the α chain is obtained in cells that account for 6 to 9 adults.

Example 5

[ tumor aggressiveness of CD 106-Positive cell population ]

Cells of tumor tissue were isolated as in example 5, and CD3 positive cells were sorted from the obtained cell mixture using BD FACSAria III to obtain T cells expressing TCR as tumor infiltrating T cells. The tumor infiltrating T cells were co-cultured with a cell line established from autologous tumors as a stimulator, and IFN-gamma production was compared to the non-stimulatory case. IFN-gamma production was determined by intracellular staining using anti-IFN-gamma antibodies. Cell staining was performed using anti-PD-1 antibodies, anti-TIGIT antibodies, anti-LAG 3 antibodies, and anti-CD 106 antibodies. As negative control, isotype control antibodies against anti-CD 106 antibodies were used.

The results are shown in FIG. 9. In only the CD106 positive cell subsets, stimulation of autologous tumor cell lines produced IFN-gamma, whereas in the marker positive cell subsets other than CD106, although autologous tumor stimulation produced IFN-gamma, IFN-gamma was also produced in the marker negative cell subsets, thus demonstrating inferior specificity to CD106.

Example 6

[ introduction of TCR Gene into mature T cells clonally differentiated from iPS cells ]

For mature T cells differentiated from iPS cell clones obtained in example 2, which had good differentiation efficiency into T cells and were derived from non-T non-B cells or monocytes, through hematopoietic stem cells and immature T cells, genes (cdnas) encoding GPC3 antigen-specific T cell receptor alpha and beta chains were introduced using piggyBac (registered trademark) system in the same manner as in example 3.

The results of analyzing the phenotype of mature T cells derived from the gene-introduced iPS cells by flow cytometry are shown in fig. 10. In fig. 10, "no EP" shows the analysis result of the mature T cell expressing WT1 antigen-specific T cell receptor alpha chain and beta chain as the mature T cell for gene transfer; "EGFP" shows the result of analysis of the mature T cell into which the expression vector into which the tracer gene (EGFP (enhanced green fluorescent protein) gene) as a gene transfer manipulation marker has been introduced; "Empty-CD19" shows the result of analysis of the mature T cell into which the piggyBac (registered trademark) transposon vector having integrated therein only the intracellular defective human CD19 gene as a tracer gene is introduced; "TCR-CD19" shows the result of analysis of the mature T cells into which the piggyBac (registered trademark) transposon vector having the GPC3 antigen-specific T cell receptor alpha chain beta chain gene and the intracellular defective human CD19 gene integrated in series has been introduced.

Of the mature T cells derived from iPS cells, denoted by "no EP", only the T cell marker CD3 was detected. That is, the cells used for gene transfer were confirmed to be T cells.

In mature T cells derived from iPS cells, represented by "EGFP", expression of CD3 gene and EGFP gene was detected. That is, it can be seen that the gene transfer operation has been properly performed.

In mature T cells derived from iPS cells, indicated by "Empty-CD19", the expression of the CD3 gene and the intracellular defective human CD19 gene as a tracer gene integrated into piggyBac (registered trademark) transposon vector was detected. That is, it can be seen that the gene transfer operation has been properly performed.

In mature T cells derived from iPS cells represented by "TCR-CD19", binding to GPC3 peptide/HLA complex (GPC 3-Dex) recognized by GPC3 antigen-specific T cell receptor alpha chain beta chain was detected in addition to expression of CD3 gene and intracellular defective human CD19 gene as a tracer gene integrated into piggyBac (registered trademark) transposon vector. That is, it has been shown that by introducing a GPC3 antigen-specific T cell receptor α chain β chain gene into mature T cells derived from iPS cells using piggyBac (registered trademark) system, the GPC3 antigen-specific T cell receptor α chain β chain expressed on the cells functions as a molecule recognizing GPC3 peptide/HLA complex (GPC 3-Dex). Thus, it has been shown that by using novel tumor-associated antigens (neoantigens) and other tumor-associated antigen-specific T cell receptor alpha chain beta chain genes as T cell receptor alpha chain beta chain genes introduced into iPS cell-derived mature T cells, iPS cell-derived mature T cells recognizing these antigens can be produced.

Example 7

[ production of regenerative T cells from iPS cells initialized by peripheral blood T cells ]

Fig. 11 shows a method of screening iPS cell clones having high differentiation efficiency into T cells in the step of producing regenerative T cells from iPS cells in which peripheral blood T cells have been initialized. Monocytes were isolated from peripheral blood of EBV (Epstein-Barr virus) infected subjects, the isolated monocytes were stimulated with EBV antigen in vitro, and a population of CD8 positive T cells recognizing the EBV antigen was isolated. Mountain factor 4 (Oct 3/4, sox2, klf4, and c-Myc) and SV40T antigens were introduced into an isolated CD8 positive T cell population using sendai virus vectors to obtain an iPS cell population. iPS cell clones were isolated from the obtained iPS cell population, and for each clone, the ability of differentiation into T cells was examined, and iPS cell clones having high differentiation efficiency into T cells were selected. T cells recognizing EBV antigen are cells that hardly cause an allogeneic reaction even in the case of allogeneic transplantation.

Fig. 12 shows a method of genome editing iPS cell clones selected as cells having high differentiation efficiency into T cells and producing regenerative T cells in the step of producing regenerative T cells from iPS cells in which peripheral blood T cells have been initialized. Regarding iPS cell clones derived from CD8 positive T cells recognizing EBV antigen and having high differentiation efficiency into T cells, CRISPR/Cas9 was used to delete β2m and CIITA genes related to MHC class I and MHC class II expression, PVR genes related to activated natural killer cells (NK), and Rag2 genes related to T cell receptor rearrangement, on the other hand, fusion genes (HLA-E) of β2m/binding peptide/HLA-E, which are inhibitory ligands of NK cells, were expressed, thereby protecting iPS cells from attack by T cells and NK cells of the host. In the genome editing, the cells can be removed after administration into a living body by expressing suicide genes such as drug-induced caspase 9 and/or specific marker genes such as EGFR (epidermal growth factor receptor), CD19 and CD 20. After genome editing, the cells are re-cloned, and when it is confirmed that the differentiation efficiency into T cells is high, clones of regenerative T cells derived from iPS cells can be stored to construct a master cell bank. The regenerative T cells differentiated and proliferated by iPS cells are host T cells for producing regenerative T cells for treating cancer, and a master cell bank may be constructed from the host T cells. The host T cells can be used as a material for producing regenerative T cells into which TCR genes or CAR (chimeric antigen receptor) genes have been introduced. Since the host T cells are T cells recognizing EBV antigens, even if transferred into a living body, an allogeneic reaction is not easily caused. "host T cells" means T cells which are not used per se for the treatment of a patient, but are used as starting materials for the production of a cancer prophylactic or therapeutic agent comprising regenerative T cells as an active ingredient.

FIG. 13 illustrates a method for producing regenerative T cells from the host T cells that recognize cancer antigens. In host T cells recognizing EBV antigens, the T cell receptor β chain recognizing the rearranged EBV antigen is replaced by a conjugate mediating T2a recognizing the T cell receptor β chain and T cell receptor α chain of the cancer antigen, respectively, by gene replacement using genome editing of CRISPR/Cas 9. On the other hand, by genome editing using CRISPR/Cas9, T cell receptor alpha chains recognizing EBV antigens are removed. According to the method described in FIG. 13, regenerative T cells recognizing cancer antigens can be produced.

Fig. 14 summarizes the methods shown in fig. 11 through 13. The regenerative T cells (iPS-T cells) produced from the same general iPS cells into which the peripheral blood T cells have been initialized can be used as starting materials for producing T cells into which the TCR gene or CAR gene has been introduced. "Universal iPS cells" refers to iPS cells that can be used without causing rejection even in patients with any kind of MHC (Major Histocompatibility Complex; major histocompatibility Complex) due to low immunogenicity. That is, they are iPS cells that can be given to a patient without regard to MHC matching. The universal iPS cells may be produced by knocking out MHC class I or MHC class II molecules and expressing a ligand that inhibits NK cells.

For introducing the TCR gene or CAR gene, a viral vector used for production of T cells used in conventional T cell replacement therapy or regenerative therapy may be used. By using iPS-T cells as a starting material, T cells required as an active ingredient of a cancer preventive or therapeutic agent can be produced in a short time. In addition, it is also easy to introduce TCRs recognizing a neoantigen, and iPS-T cells can be produced while maintaining polyclonal properties by introducing a plurality of TCR genes recognizing different neoantigens.

Claims

1. A method of producing regenerative T cells by iPS cells, comprising:

(1) A step of preparing cdnas encoding T cell receptor alpha and beta chains, respectively, in each individual cell, in a population of T cells which are T cells obtained from a subject, are reactive to a tumor-associated antigen and are CD 106-positive;

(2) Initializing peripheral blood mononuclear cells or T cells of the subject from which the B cells and T cells have been removed into iPS cells, and selecting iPS cell clones having high differentiation efficiency into T cells from the iPS cells thus obtained;

(4) A step of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell obtained in step (3) and into which the cDNA has been introduced into the mature T cell and proliferating the mature T cell.

2. The method of claim 1, wherein step (2) is performed prior to step (1) or in parallel with step (1).

3. The method of claim 1 or 2, wherein the subjects in steps (1) and (2) are the same individual.

4. The method of claim 1 or 2, wherein the subjects in steps (1) and (2) are different individuals, and the subject in step (2) is a subject for cancer prevention or treatment.

5. The method of claim 1 or 2, wherein the subjects in steps (1) and (2) are different individuals, and the subject in step (1) is a subject for cancer prevention or treatment.

6. The method of claim 1 or 2, wherein the subjects in steps (1) and (2) are the same individual or different individuals, and the subjects different from the subjects in steps (1) and (2) are subjects for cancer prevention or treatment.

7. The method according to claim 1 or 2, further comprising the step of: selecting T cells which do not exhibit an allogeneic response to cells from a subject who is a subject to cancer prevention or treatment from among T cells into which the cDNA obtained in step (4) has been introduced.

8. The method according to claim 1 or 2, further comprising the step of: screening the cDNA prepared in the step (1) for cDNA encoding a T cell receptor that does not induce an allogeneic response to cells from a subject being a subject for cancer prevention or treatment.

9. The method of claim 7 or 8, wherein the cells from a subject as a subject for cancer prevention or treatment are peripheral blood mononuclear cells.

10. The method according to any one of claims 1 to 9, wherein the cDNA is introduced into the iPS cell clone, the hematopoietic stem cells differentiated from the iPS cell clone, the immature T cells differentiated from the hematopoietic stem cells, or the mature T cells differentiated from the immature T cells in step (3) using a viral vector, a non-viral vector, or a genome editing technique.

11. The method of claim 10, wherein the genome editing technique is CRISPR/Cas9 or TALEN.

12. The method of claim 10, wherein the non-viral vector is a transposon vector.

13. The method of claim 12, wherein the transposon vector is a piggyBac (registered trademark) vector.

14. The method according to any one of claims 1 to 13, wherein in step (4), the steps of differentiating the iPS cell clone, the hematopoietic stem cell, or the immature T cell into a mature T cell, into which the cDNA is introduced, and proliferating the mature T cell are performed in the presence of feeder cells and PHA (phytohemagglutinin), in the presence of RetroNectin (registered trademark) and an anti-CD 3 antibody, or in the presence of an anti-CD 3 antibody and an anti-CD 28 antibody.

15. The method of claim 14, wherein the feeder cells are autologous or allogeneic peripheral blood mononuclear cells.

16. The method of any one of claims 1 to 15, wherein the subject in step (1) or (2) is a patient having skin cancer, bladder cancer, head and neck cancer, uterine cancer, cervical cancer, glioblastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, large intestine cancer, brain tumor, sarcoma, or B-cell non-hodgkin's lymphoma, such as hepatocellular carcinoma, hepatoblastoma, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, ovarian cancer, malignant melanoma.

17. The method of any one of claims 1 to 15, wherein the subject in step (1) or (2) is a highly immunogenic cancer patient.

18. The method of claim 17, wherein the highly immunogenic cancer is hepatocellular carcinoma, hepatoblastoma, colorectal cancer, lung cancer, or malignant melanoma.

19. The method of any one of claims 1 to 18, wherein the population of T cells in step (1) is double positive for CD3/CD 106.

20. The method of any one of claims 1-19, wherein the hematopoietic stem cells are double positive for CD34/CD 43.

21. The method of any one of claims 1 to 20, wherein the immature T cells are CD8 a chain/β chain double positive.

22. The method according to any one of claims 1 to 21, wherein the iPS cell clone selected in step (2) that has high differentiation efficiency into T cells, or the hematopoietic stem cell differentiated from the iPS cell clone in step (3), the immature T cells differentiated from the hematopoietic stem cells, or the mature T cells differentiated from the immature T cells are stored, and a master cell bank is constructed.

23. The method of claim 22, wherein the preservation is cryopreservation.

24. The method of claim 22, wherein steps (3) and (4) are performed on the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell stored in the master cell bank.

25. The master cell bank of claim 22, comprising the iPS cell clone, the hematopoietic stem cell, the immature T cell, or the mature T cell.

26. A regenerative T cell produced by the method of any one of claims 1 to 24.

27. A pharmaceutical composition comprising the regenerative T cell of claim 26.

28. A method of preventing or treating cancer using the pharmaceutical composition of claim 27.