T cell master cell bank

Abstract

To provide a T cell product delivery system, including a T cell master cell bank and / or a T cell working cell bank.SOLUTION: A T cell master cell bank can be constructed by dispensing T cells into multiple storage containers (e.g., sterile vials), freezing, and storing them. One container of T cells from this T cell master cell bank can be thawed and used for characterization analysis tests as appropriate. Alternatively, a T cell working cell bank can be constructed by dispensing T cells from the same container or from another thawed container into multiple appropriate containers, freezing, and storing them. T cell products can be manufactured by thawing one or more containers of T cells from the T cell master cell bank, introducing nucleic acid containing an exogenous gene into the T cells, and expanding them.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a T cell master cell bank and a T cell working cell bank, as well as a system for providing T cell products comprising the banks.

[0002] BACKGROUND OF THE INVENTION In recent years, immune cell therapy has been attracting attention as a cancer treatment. Immune cell therapy is a treatment in which immune cells that have been expanded and activated outside the patient's body are administered to the patient, allowing the immune cells to attack cancer cells. Immune cell therapy has the advantage of having almost no side effects compared to the three major conventional therapies of surgery, radiation therapy, and chemotherapy. There are various types of immune cell therapy, but off-the-shelf allogeneic (allogenic) T cell products are particularly promising.

[0003] As a system for providing the above-mentioned allogeneic T cell product, a system has been reported in which iPS cell banking technology (e.g., Patent Document 1) or technology for introducing a CAR gene into iPS cells (e.g., Patent Document 2) is used to create iPS cells by first introducing a CAR gene into iPS cells to produce them, which are then expanded and cultured, and the cells are stocked to create a master cell bank of iPS cells carrying a CAR gene, and iPS cells derived from the cell bank are differentiated into T cells to obtain T cells, which are then used as an allogeneic T cell product (e.g., Non-Patent Document 1).

[0004] The above system requires the introduction of exogenous genes, such as CAR genes, into pluripotent stem cells (e.g., iPS cells) and the differentiation of these cells into T cells. Therefore, the maintenance, differentiation, and culture processes of cells (e.g., iPS cells, T cells, and CAR-T cells) at each stage of the process must be carried out while meeting the Good Manufacturing Practice (GMP) standards set by the relevant national authorities. For example, because a master cell bank of iPS cells is used, iPS cells must be differentiated into T cells to prepare T cell products. However, because iPS cells are pluripotent, high-level process and quality control is required each time to induce uniform differentiated cells for each lot, resulting in extremely high personnel, time, and financial costs. These costs significantly impact the stable supply of T cell products. When attempting to provide multiple types of T cell products, the cumulative costs and time required for each step add up, making the impact even more severe. Therefore, a system that can provide products at low cost while still meeting GMP standards, and even when multiple types of T cell products are used, is desired. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Publication No. 2009-0299763 [Patent Document 2] International Publication No. 2017 / 088012 [Non-patent literature]

[0006] [Non-Patent Document 1] Bob Valamehr (Vice President of Fate Therapeutics) “Generation of off-the-shelf TCR-less CAR T cells from renewable pluripotent cells”, April 14-18, 2018 (AACR Annual Meeting 2018 Press Program) Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, an objective of the present invention is to provide a system for providing high-quality off-the-shelf allogenic T cell products that can reduce personnel, time, and financial costs and has minimal lot-to-lot variation, as well as a T cell master cell bank, a T cell working cell bank, and a collection of these that can be used in providing the system. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems. They found that conventional techniques, such as those described in Non-Patent Document 1, focus exclusively on methods for constructing a master cell bank of iPS cells. They hypothesized that this is due to the superior proliferation capacity of iPS cells compared to T cells. They then came up with the idea that if T cells could be sufficiently expanded by expansion culture, T cells or the expanded T cells could be used as a master cell bank. That is, rather than constructing a master cell bank of iPS cells carrying an exogenous gene such as a CAR gene, they came up with the idea that by constructing a master cell bank of T cells containing the exogenous gene, high-quality T cell products could be rapidly provided from the cell bank. To the best of their knowledge, the idea of ​​constructing a master cell bank of T cells was completely unknown and novel. Based on these findings, the present inventors conducted further research and ultimately completed the present invention.

[0009] That is, the present invention provides the following. [1] A T cell product delivery system, including a T cell master cell bank and / or a T cell working cell bank. [2] The system described in [1], wherein the T cell master cell bank and / or the T cell working cell bank contain T cells derived from induced pluripotent stem cells. [3] The system described in [1] or [2], wherein the T cell master cell bank and / or the T cell working cell bank contain T cells in which the expression of at least one type of HLA gene is suppressed. [3a] The system described in any one of [1] to [3], wherein the T cell master cell bank and / or the T cell working cell bank contain T cells into which an exogenous gene has been introduced. [3b] The system described in any one of [1] to [3a], further comprising a step of collecting a T cell master cell bank and / or a T cell working cell bank to construct a T cell master cell bank collection and / or a T cell working cell bank collection including two or more types of T cell master cell banks and / or T cell working cell banks. [3c] The system described in any one of [1] to [3b], wherein the T cell master cell bank and / or the T cell working cell bank are of two or more types. [3d] The system described in any one of [1] to [3c], further comprising a step of selecting a T cell master cell bank and / or a T cell working cell bank. [4] The system described in any one of [1] to [3d], which includes a step of introducing a nucleic acid containing an exogenous gene into T cells prepared from the T cell master cell bank and / or the T cell working cell bank. [5] The system described in [4], wherein the exogenous gene is a CAR gene or an exogenous TCR gene. [6] The system described in any one of [1] to [5], wherein the types of T cell products are two or more. [6a] A system described in any one of [1] to [6], further comprising a step of selecting a T cell product. [7] The system described in any one of [1] to [6a], further comprising a step of expanding T cells prepared from the T cell master cell bank and / or the T cell working cell bank. [8] The system described in [7], wherein the step of expanding the T cells includes a step of stimulating the cells with a CD30 agonist. [8a] The system described in [7], wherein the step of expanding the T cells comprises culturing the cells in the presence of a CD30 agonist. [9] The system described in any one of [7] to [8a], further comprising a step of producing a frozen T cell product containing the expanded T cells.

[10] The system described in any one of [6] to [9], further comprising a step of collecting T cell products to construct a T cell product collection containing two or more types of T cell products. [10a] The system according to any one of [1] to

[10] , further comprising a step of acquiring information about the subject. [10b] The system described in [10a], further comprising a step of selecting an appropriate T cell master cell bank and / or an appropriate T cell working cell bank based on information about the subject. [10c] The system described in [10a], further comprising a step of selecting an appropriate T cell product based on information about the subject.

[11] The system according to any one of [1] to [10c], further comprising a step of identifying a tumor-specific antigen or a tumor-associated antigen expressed in the subject's tumor.

[12] The system described in

[11] , further comprising a step of selecting a T cell product expressing a CAR or an exogenous TCR that recognizes and binds to the identified antigen from a T cell product collection containing two or more types of T cell products.

[13] A method for producing a T cell product, comprising the step of establishing a T cell master cell bank and / or a T cell working cell bank.

[14] The method for producing a T cell product described in

[13] , wherein the T cell master cell bank and / or the T cell working cell bank contain T cells derived from induced pluripotent stem cells.

[15] The method for producing a T cell product described in

[13] or

[14] , wherein the T cell master cell bank and / or the T cell working cell bank contain T cells in which the expression of at least one type of HLA gene is suppressed. [15a] A method for producing a T cell product according to any one of

[13] to

[15] , wherein the T cell master cell bank and / or the T cell working cell bank contain T cells into which an exogenous gene has been introduced.

[16] The method for producing a T cell product according to any one of

[13] to [15a], further comprising the step of introducing a nucleic acid containing an exogenous gene into T cells prepared from the T cell master cell bank and / or the T cell working cell bank.

[17] The method for producing a T cell product described in

[16] , wherein the exogenous gene is a CAR gene or an exogenous TCR gene.

[18] The method for producing a T cell product according to any one of

[13] to

[17] , further comprising a step of expanding the T cells.

[19] The method for producing a T cell product according to

[18] , further comprising stimulating the T cells with a CD30 agonist during the expansion and culture of the T cells. [19a] The method for producing a T cell product according to

[18] , wherein the step of expanding the T cells comprises culturing the cells in the presence of a CD30 agonist.

[20] T cell master cell bank and / or T cell working cell bank.

[21] The T cell master cell bank and / or T cell working cell bank according to

[20] , which contains T cells derived from induced pluripotent stem cells.

[22] A T cell master cell bank and / or a T cell working cell bank according to

[20] or

[21] , which comprises T cells in which the expression of at least one type of HLA gene is suppressed. [22a] A T cell master cell bank and / or a T cell working cell bank according to any one of

[20] to

[22] , which comprises T cells into which an exogenous gene has been introduced. A T cell master cell bank collection and / or a T cell working cell bank collection comprising two or more types of T cell master cell banks and / or T cell working cell banks described in any one of

[23]

[20] to [22a].

[24] A method for constructing a T cell master cell bank and / or a T cell working cell bank, comprising the steps of: (I) differentiating induced pluripotent stem cells that do not have a chimeric antigen receptor (CAR) gene into T cells for CAR-T therapy; (II) stocking the differentiated T cells; and (III) characterizing the differentiated T cells

[25] A method for constructing a T cell master cell bank and / or a T cell working cell bank, comprising the following steps: (i) differentiating induced pluripotent stem cells that do not have exogenous T cell receptor (TCR) genes into T cells for TCR-T therapy; (ii) stocking the differentiated T cells; and (iii) characterizing the differentiated T cells [25a] The method according to

[24] or

[25] , wherein the induced pluripotent stem cells have an exogenous gene.

[26] The method described in

[24] or [25a], wherein the induced pluripotent stem cells have an exogenous T cell receptor (TCR) gene.

[27] The method according to any one of

[24] to

[26] , wherein the induced pluripotent stem cells lack at least one type of HLA gene. [27a] The method according to any one of

[24] to

[26] , wherein the induced pluripotent stem cells have suppressed expression of at least one type of HLA gene.

[28] The method according to any one of

[24] to [27a], wherein the T cells express CD8αβ.

[29] A T cell master cell bank and / or a T cell working cell bank constructed by the method described in any one of

[24] to [28a].

[30] A method for producing a T cell product expressing a CAR or an exogenous TCR, comprising the steps of: (A) preparing T cells from the T cell master cell bank and / or the T cell working cell bank described in

[29] ; (B) introducing a CAR gene or an exogenous TCR gene into the prepared T cells; and (C) Expanding and culturing the T cells into which the CAR gene or exogenous TCR gene has been introduced. [30a] The method according to

[30] , further comprising the step (D) of freezing the expanded T cells.

[31] The method described in

[30] or [30a], wherein the CAR or exogenous TCR recognizes and binds to a tumor-specific antigen or a tumor-associated antigen.

[32] The method according to any one of

[30] to

[31] , wherein the step (C) comprises stimulating T cells with a CD30 agonist. [32a] The method for producing a T cell product according to any one of

[30] to

[31] , wherein step (C) comprises culturing the T cells in the presence of a CD30 agonist.

[33] A T cell product produced by the method described in any one of

[30] to [32a].

[34] A method for constructing a T cell product collection comprising two or more types of T cell products, the method comprising the step of collecting the T cell products described in

[33] .

[35]

[34] T cell product collection constructed by the method described.

[36] A method for providing a T cell product suitable for a subject, comprising the steps of: (x) identifying tumor-specific or tumor-associated antigens expressed in the subject's tumor; and (y) selecting a T cell product expressing a CAR or an exogenous TCR that recognizes and binds to the identified antigen from the T cell product collection described in

[35] .

[37] A method for providing a T cell product suitable for a subject, comprising the following steps. (p) A step of obtaining information of a subject, and (q) A step of selecting an appropriate T cell master cell bank and / or an appropriate T cell working cell bank based on the obtained subject information

Effect of the Invention

[0010] According to the present invention, a system and a method for providing an off-the-shelf allogeneic T cell product with reduced human, time, and monetary costs, small lot-to-lot differences, and high quality are provided. Such a system and method for providing are particularly excellent when providing multiple types of T cell products. Also provided are a T cell master cell bank and a T cell working cell bank that can be used for providing the above system or method, and collections thereof.

[0011] Various cells used in this specification may be referred to as follows respectively. An overview of each cell is described together. <Induced pluripotent stem cell (iPSC)> αβ-iPSC: An iPS cell into which a TCR-α chain gene (TRA gene) and a TCR-β chain gene (TRB gene) have been introduced Vγ9Vδ2-iPSC: An iPS cell into which a TCR-γ chain gene (TRG gene) encoding Vγ9Vδ2 TCR G115 and a TCR-δ chain gene (TRD gene) have been introduced <Hematopoietic progenitor cell (HPC)> Vγ9Vδ2-iHPC: An HPC into which a TCR-γ chain gene (TRG gene) encoding Vγ9Vδ2 TCR G115 and a TCR-δ chain gene (TRD gene) have been introduced <T cell> iγδTC: A T cell differentiated from an iPS cell into which an exogenous TCR has not been introduced iαβTC: A T cell differentiated from αβ-iPSC Vγ9Vδ2-iTC: A T cell differentiated from Vγ9Vδ2-iPSC Vγ9Vδ2-iHTC: T cells differentiated from Vγ9Vδ2-iHPC <T cells transfected with the CAR gene> CD19iαβCARTC: T cells prepared by introducing the gene encoding anti-CD19-CAR into iαβTC BCMAiαβCARTC: T cells prepared by introducing the gene encoding anti-BCMA-CAR into iαβTC CD19-CD30-iαβCARTC: T cells prepared by introducing the gene encoding anti-CD19-CAR containing the intracellular domain derived from CD30 into iαβTC <T cells transfected with the CAR gene and the IL-15Rα / IL-15 gene> CD19 / IL15iαβCARTC: T cells prepared by introducing the gene encoding anti-CD19-CAR and the gene encoding the IL-15Rα / IL-15 chimeric protein into iαβTC CD19 / IL15iVγ9Vδ2CARTC: T cells prepared by introducing the gene encoding anti-CD19-CAR and the gene encoding the IL-15Rα / IL-15 chimeric protein into Vγ9Vδ2-iTC CD19 / IL15iγδCARTC: T cells prepared by introducing the gene encoding anti-CD19-CAR and the gene encoding the IL-15Rα / IL-15 chimeric protein into iγδTC

Brief Description of the Drawings

[0012] [Figure 1] An example of a system for providing a T cell product containing a T cell bank is shown. [Figure 2] An example of a system in which a plurality of T cell products are provided from a T cell bank collection is shown. "Armored" in Fig. 2 means that an exogenous gene related to the secretion of cytokines and / or chemokines has been introduced. [Figure 3] The expression of CD3, γδTCR, and αβTCR on the cell membrane surface of iγδTC is shown. The filled peak indicates the result of the non-antibody staining group, and the blank peak indicates the staining results using each antigen-specific antibody. [Figure 4] This shows the expression of TCR-Vδ1 chain and TCR-V2δ chain on the cell membrane surface of iγδTC. The horizontal and vertical axes represent the expression of TCR-Vδ1 chain (Vdelta1) and TCR-Vδ2 chain (Vdelta2), respectively. [Figure 5] This shows the expression of CD3 and γδTCR molecules on the cell membrane surface of Vγ9Vδ2-iTCs. The horizontal and vertical axes of the left panel represent CD3 and pan-γδTCR expression, respectively. The horizontal and vertical axes of the right panel represent TCR-Vδ2 chain and TCR-Vγ9 chain expression, respectively. [Figure 6] The figure shows the expression of CD3 and γδ TCR molecules on the cell membrane surface of Vγ9Vδ2-iHTCs. The horizontal and vertical axes of the left panel show the expression of CD3 and pan-γδ TCR, respectively. The horizontal and vertical axes of the right panel show the expression of CD3 and TCR-Vγ9 chain, respectively. [Figure 7]

[0039] Figure 1 shows the cell proliferation of iγδTC. The vertical axis indicates the cell number, and the horizontal axis indicates the number of days elapsed since the start of expansion culture. The arrow indicates the day on which stimulation with immobilized anti-CD3 agonist antibody / Retronectin (registered trademark) and anti-CD30 agonist antibody was initiated. [Figure 8]

[0033] Figure 1 shows Vγ9Vδ2-iTC cell proliferation. The vertical axis indicates the number of cells, and the horizontal axis indicates the number of days since the start of expansion culture. The arrow indicates the day when stimulation with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark) was started. [Figure 9] This figure shows the growth curve of CD19-iαβCARTC stimulated with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark). The vertical axis shows the cell number, and the horizontal axis shows the number of days since the start of the stimulation. The arrow indicates the day when stimulation with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark) was started. [Figure 10]These figures show the antigen-specific cytotoxic activity of CART cells (CD19iαβCARTC (A) and BCMAiαβCARTC (B) respectively) and iαβTCs produced by introducing a gene encoding an anti-CD19-CAR or a gene encoding an anti-BCMA-CAR into iαβTCs against CD19-positive Raji cells (A) or BCMA-positive H929 cells (B). [Figure 11] 1 shows the cytotoxic activity of CD19-CD30-iαβCARTC against CD19-positive Raji cancer cells. The vertical axis shows the percentage of cells killed after 2 hours, and the horizontal axis shows the mixture ratio of effector cells (CD19iαβCARTC) and target cells (CD19-positive Raji cancer cells). [Figure 12] This figure shows the antigen-specific cytotoxic activity of CD19 / IL15iγδCARTC. The filled and open circles represent the cytotoxic activity against CD19-positive Raji cancer cells and CD19-negative CCRF-CEM cancer cells, respectively. The vertical axis represents the percentage of remaining cells after 2 hours, and the horizontal axis represents the mixture ratio of effector cells (CD19 / IL15iγδCARTC) to target cells (CD19-positive Raji cancer cells or CD19-negative CCRF-CEM cancer cells). [Figure 13]

[0039] Figure 1 shows cell proliferation of CD19 / IL15iVγ9Vδ2CARTC. The vertical axis indicates the number of cells, and the horizontal axis indicates the number of days since the start of expansion culture. The arrow indicates the day when stimulation with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark) was started. [Figure 14] FIG. 10 shows the effect of CD19 / IL15iγδCARTC in extending the survival time of human CD19-expressing Nalm6 tumor xenograft mice. [Figure 15] FIG. 1 shows the effect of CD19 / IL15iVγ9δ2CARTC in extending the survival time of human CD19-expressing Nalm6 xenograft mice. [Figure 16] An example of a system for providing T cell products including a T cell bank is shown. [Figure 17] FIG. 10 is a diagram illustrating an example of a hardware configuration included in a T cell product selection unit.

[0013] (Detailed Description of the Invention) 1. T cell master cell bank, T cell working cell bank, and T cell product distribution system The present invention provides a T cell master cell bank and / or a T cell working cell bank. Hereinafter, the term "T cell cell bank" may be used to encompass both a T cell master cell bank and a T cell working cell bank. In another aspect, the present invention provides a T cell product delivery system (hereinafter, sometimes referred to as the "delivery system of the present invention") that includes a T cell master cell bank and / or a T cell working cell bank.

[0014] As used herein, the term "cell bank" refers to a set of identical, characterized starting materials that meet the quality standards established by drug authorities around the world in accordance with the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) guidelines (ICH guideline Q5D). Generally, cell banks are constructed by freezing cells packed in appropriate containers. This distinguishes them from frozen stocks, which are obtained simply by freezing cells. If necessary, cell banks are subjected to quality assessment tests in addition to characterization. The cell bank construction method and the test items for characterization and quality assessment of the constructed cell bank vary depending on the biological properties (e.g., auxotrophy), culture history, and feasibility of the cells to be banked, but are appropriately determined by those skilled in the art or based on discussions between the artisan and regulatory authorities. Information about the construction method and the results of the characterization and quality assessment are submitted to drug authorities in each country's marketing authorization application. A "master cell bank" refers to a seed cell line from which all manufacturing cell seeds are derived, grown under specific culture conditions through a minimum number of passages and dispensed into multiple ampoules. A "T cell master cell bank" refers to a cell line containing T cells, which is pooled from one or more master cell banks, further cultured under conditions confirmed to be sufficiently stable, and dispensed into multiple ampoules. A "T cell working cell bank" refers to a cell line containing T cells (herein, both the T cell master cell bank and the T cell working cell bank are sometimes referred to as "T cell cell banks"). According to ICH Q5D, characterization testing is generally performed on the master cell bank, and typically, some characterization testing is also performed on each working cell bank.

[0015] Characterization studies primarily assess the absence of adventitious infectious agents, particularly those contaminated with adventitious viruses, which can have serious consequences in clinical use, and are therefore assessed according to ICH Q5A(R1). Other tests include identity testing to detect cross-contamination with other cell lines, and may also include karyotyping and tumorigenicity testing. Test items for quality assessment include confirmation tests using appropriate cell phenotypes as T cells, purity tests, tests for impurities derived from the manufacturing process, and content tests such as cell count and cell viability. T cell banks can be constructed by the manufacturer and / or provider of T cell products themselves, or they can be introduced from others and used to produce T cell products. According to ICH Q5D, the two-tiered cell banking concept, where a working cell bank is constructed from a master cell bank, is generally accepted as the most practical method for providing a cell substrate for continuous drug manufacturing.

[0016] A working cell bank is derived from one or more master cell banks. The working cell bank can be updated from the master cell bank as needed. ICH Q5D states that newly established working cell banks should be appropriately qualified through characterization and other procedures.

[0017] In one embodiment of the present invention, a T cell master cell bank can be constructed by dispensing T cells into multiple storage containers (e.g., sterile vials), freezing them, and storing them. One container of T cells from this T cell master cell bank can be thawed and subjected to appropriate characterization tests. Furthermore, a T cell working cell bank can be constructed by dispensing T cells from the same container or from a separate thawed container into multiple appropriate containers, freezing them, and storing them. In one embodiment of the present invention, a T cell product can be produced by thawing one or more containers of T cells from a T cell bank, introducing a nucleic acid containing an exogenous gene into the T cells, and expanding the cells.

[0018] In the present invention, "stocking" means properly storing multiple containers into which cells such as T cells have been allocated, and managing storage conditions such as temperature, receipt and dispensing, etc. The containers may be stored in one location or in multiple locations.

[0019] In the present invention, "T cells" refers to CD3-positive cells. Examples of T cells that can be used in the present invention include CD8-positive cytotoxic T cells (CTLs), CD4-positive helper T cells, regulatory T cells, effector T cells, and γδ T cells, which are T cell receptor (TCR) γ chain and TCR δ chain-positive cells. CD4 / CD8-positive cells are also included in T cells. The TCR in T cells may be a TCR resulting from expression of an endogenous TCR gene, or a TCR resulting from expression of an exogenous TCR gene. The exogenous TCR may be a modified TCR, as described below. The T cells in the T cell bank provided by the present invention are preferably cytotoxic T cells, more preferably CD8αβ-positive cytotoxic T cells that express CD8αβ. Furthermore, in this specification, the term "T cell product" refers to T cells packed in a storage container (e.g., a sterile vial or a transfusion bag), or to T cells that have been appropriately packaged, labeled, or otherwise treated. T cell products may be frozen or unfrozen, but are preferably frozen for long-term storage in terms of T cell stability. Herein, a frozen T cell product may be referred to as a "frozen T cell product." Unless otherwise specified, the term "T cell product" herein encompasses both unfrozen and frozen T cell products (frozen T cell products). Herein, the term "T cell product" encompasses both clinical trial products administered to mammalian subjects, including humans, and commercially available products.

[0020] As described above, conventional methods for providing T cell products using master cell banks or working cell banks of iPS cells involve extremely high personnel, time, and financial costs. In particular, when attempting to provide two or more types of T cell products, the costs and time required for each are cumulative, resulting in even more serious impacts. On the other hand, the delivery system of the present invention uses a master cell bank or working cell bank of T cells, which significantly reduces personnel, time, and financial costs compared to conventional techniques, particularly when providing two or more types of T cell products. Therefore, while the delivery system of the present invention may provide only one type of T cell product, it is particularly advantageous when providing two or more types.

[0021] As used herein, "positive" means that the protein or gene is expressed in a detectable amount by techniques known in the art. Protein detection can be carried out using immunological assays using antibodies, such as ELISA, immunostaining, and flow cytometry. In addition, in the case of proteins that are expressed intracellularly and not present on the cell surface (e.g., transcription factors or their subunits), the target protein can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Gene detection can be carried out using nucleic acid amplification and / or nucleic acid detection methods, such as RT-PCR, biochips (e.g., microarrays), and RNAseq.

[0022] As used herein, "negative" means that the expression level of a protein or gene is below the lower limit of detection by all or any of the above-mentioned known techniques. The lower limit of detection of protein or gene expression may vary depending on the technique.

[0023] As used herein, "gene expression" includes both the synthesis of mRNA from a specific nucleotide sequence of the gene (also referred to as transcription or mRNA expression) and the synthesis of a protein based on the information in the mRNA (also referred to as translation or protein expression). However, unless otherwise specified, "gene expression" or simply "expression" refers to protein expression.

[0024] As used herein, "culturing" refers to maintaining, propagating (growing), and / or differentiating cells in an in vitro environment. "Culturing" refers to maintaining, propagating (growing), and / or differentiating cells outside a tissue or body, for example, in a cell culture plate, dish, or flask.

[0025] T cells constituting a T cell bank can be produced by inducing the differentiation of stem cells capable of differentiating into T cells. Examples of such stem cells include pluripotent stem cells and multipotent stem cells. As used herein, "pluripotent stem cells" refer to stem cells that can differentiate into tissues and cells with various different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (also referred to herein as "iPS cells"), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer embryonic stem cells: ntES cells), pluripotent germ stem cells, and embryonic germ stem cells (EG cells). As used herein, "pluripotent stem cells" refer to stem cells that have the ability to differentiate into cells of a limited number of lineages. Examples of "pluripotent stem cells" include hematopoietic stem cells, dental pulp stem cells, stem cells derived from oral mucosa, hair follicle stem cells, cultured fibroblasts, and somatic stem cells derived from bone marrow stem cells. Preferred pluripotent stem cells are ES cells and iPS cells, with iPS cells being particularly preferred. When the pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be produced by destroying the embryo or without destroying the embryo, but preferably are produced without destroying the embryo. The stem cells are preferably derived from mammals (e.g., mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans), and more preferably from humans. Therefore, human iPS cells are the most preferred stem cells for use in the present invention.

[0026] "Induced pluripotent stem cells (iPS cells)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells (iPS cells)." These include iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872), Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells created using a method that does not include c-Myc (Nakagawa M, Alternatively, iPS cells established by introducing six factors using a virus-free method (Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106) or iPS cells established by introducing six factors using a virus-free method (Okita K et al., Nat. Methods 2011 May;8(5):409-12, Okita K et al., Stem Cells. 31(3):458-66.) can also be used. Other examples that can be used include induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, as developed by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells developed by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells developed by Sakurada et al. (JP Patent Publication No. 2008-307007). In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, Any of the induced pluripotent stem cells known in the art and described in the Japanese Patent Laid-Open Publication Nos. 2008-307007, 2008-283972, US2008-2336610, US2009-047263, WO2007 / 069666, WO2008 / 118220, WO2008 / 124133, WO2008 / 151058, WO2009 / 006930, WO2009 / 006997, and WO2009 / 007852 can be used. Various iPS cell lines established by the NIH, RIKEN, Kyoto University, and other institutions can be used as induced pluripotent stem cell lines. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain; Kyoto University's 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain; and iPS cell stocks for regenerative medicine (e.g., Ff-I01s04 strain, QHJI strain, etc.).

[0027] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (for example, blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al., (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al., (1998), Science. 282:1145-1147; Suemori H. et al., (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al., (2006), Proc. Natl. Acad. Sci. USA 103:9554-9559; Suemori H. et al., (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al., (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al., (2006), Nature. 444:481-485, etc.Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage before the blastocyst stage (Chung Y. et al., (2008), Cell Stem Cell 2: 113-117), or can be established using developmentally arrested embryos (Zhang X. et al., (2006), Stem Cells 24: 2669-2676). Regarding "ES cells," various mouse ES cell lines established by inGenious targeting laboratory, Inc., RIKEN (Riken), and other institutions are available, while various human ES cell lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, and other institutions are available. For example, human ES cell lines that can be used include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0028] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al., (2001), Science, 292:740-743; S. Wakayama et al., (2005), Biol. Reprod., 72:932-936; Byrne J. et al., (2007), Nature, 450:497-502). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al., (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (mentioned above) (Wakayama Sayaka et al., (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg can be reprogrammed by culturing it for several hours.

[0029] Pluripotent germline stem cells (GS cells) are pluripotent stem cells derived from germline stem cells (GS cells). Similar to embryonic stem cells, these cells can be induced to differentiate into various cell lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be produced (Kanatsu-Shinohara M. et al., (2003) Biol. Reprod., 69:612-616; Shinohara K. et al., (2004), Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for embryonic stem cells (Takebayashi M. et al., (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0030] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al., (1992), Cell, 70:841-847; JL Resnick et al., (1992), Nature, 359:550-551).

[0031] 1-1. Induction of T cell differentiation The differentiation of stem cells into T cells can be induced by any known method, without particular limitation, as long as it can differentiate into T cells. When pluripotent stem cells are used as stem cells, the differentiation into T cells can include, for example, (1) a process of differentiating the pluripotent stem cells into hematopoietic progenitor cells, and (2) a process of differentiating the hematopoietic progenitor cells into T cells.

[0032] (1) A process for differentiating pluripotent stem cells into hematopoietic progenitor cells As used herein, "hematopoietic progenitor cells (HPCs)" refer to CD34-positive cells, preferably CD34 / CD43 dual-positive (DP) cells. In the present invention, hematopoietic progenitor cells and hematopoietic stem cells are not distinguished from each other and refer to the same cells unless otherwise specified.

[0033] The method for differentiating pluripotent stem cells into hematopoietic progenitor cells is not particularly limited as long as it allows differentiation into hematopoietic progenitor cells. For example, methods include culturing pluripotent stem cells in a medium for inducing hematopoietic progenitor cells, as described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, and Liu S. et al., Cytotherapy, 17 (2015); 344-358.

[0034] In the present invention, the medium for inducing hematopoietic progenitor cells is not particularly limited, and a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include Dulbecco's medium (e.g., IMDM), Eagle's medium (e.g., DMEM, EMEM, BME, MEM, αMEM), Ham's medium (e.g., F10 medium, F12 medium), RPMI medium (e.g., RPMI-1640 medium, RPMI-1630 medium), MCDB medium (e.g., MCDB104, 107, 131, 151, 153 medium), Fisher's medium, 199 medium, primate ES cell medium (primate ES / iPS cell culture medium, ReproCell), mouse ES cell medium (TX-WES culture medium, ThromboX), serum-free medium (mTeSR, Stemcell Technology), ReproFF, StemSpan (registered trademark) SFEM, StemSpan (registered trademark) H3000, Stemline II, ESF-B medium, ESF-C medium, CSTI-7 medium, and Neurobasal medium (Thermo Fisher Examples of suitable media include, but are not limited to, StemPro-34 medium, StemFit (registered trademark) (e.g., StemFit AK03N, StemFit AK02N), etc. Furthermore, these media can be mixed and used as needed, for example, to produce DMEM / F12 medium.

[0035] If necessary, the basal medium may contain medium additives, such as serum, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax (registered trademark)), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines.

[0036] In the present invention, vitamin C refers to L-ascorbic acid and its derivatives, and L-ascorbic acid derivatives refer to those that become vitamin C through an enzymatic reaction in vivo. Examples of ascorbic acid derivatives used in the present invention include vitamin C phosphate (e.g., ascorbic acid 2-phosphate), ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6 palmitate. Vitamin C phosphate (e.g., ascorbic acid 2-phosphate) is preferred, including L-ascorbate phosphates such as sodium L-ascorbate phosphate and magnesium L-ascorbate phosphate.

[0037] When vitamin C is used, it is preferably added (supplemented) separately every 4 days, every 3 days, every 2 days, or every day, and more preferably added every day. In one embodiment, the vitamin C is added in an amount equivalent to 5 ng / ml to 500 ng / ml in the culture medium (e.g., an amount equivalent to 5 ng / ml, 10 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml). In another embodiment, the vitamin C compound is added in an amount equivalent to 5 μg / ml to 500 μg / ml in the culture medium (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0038] The medium used in step (1) may further contain at least one cytokine selected from the group consisting of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), Flt-3 Ligand (FLT-3L), and basic fibroblast growth factor (bFGF). Cultures supplemented with BMP4, VEGF, and bFGF are more preferred, and cultures supplemented with BMP4, VEGF, SCF, and bFGF are even more preferred.

[0039] When cytokines are used, the concentrations in the medium may be, for example, 5 ng / ml to 500 ng / ml for BMP4, 5 ng / ml to 500 ng / ml for VEGF, 5 ng / ml to 100 ng / ml for SCF, 1 ng / ml to 100 ng / ml for TPO, 1 ng / ml to 100 ng / ml for FLT-3L, and 5 ng / ml to 500 ng / ml for bFGF.

[0040] The medium may also contain a TGFβ inhibitor. TGFβ inhibitors are small molecule inhibitors that interfere with signal transduction of the TGFβ family, and include, for example, SB431542, SB202190 (RK Lindemann et al., Mol. Cancer 2:20(2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories). For example, when the TGFβ inhibitor is SB431542, its concentration in the medium is preferably 0.5 μM to 100 μM.

[0041] Pluripotent stem cells may be cultured in either adherent or suspension culture. Adherent culture may be performed using a culture vessel coated with an extracellular matrix component, or co-culture with feeder cells. Examples of feeder cells include, but are not limited to, fibroblasts (mouse embryonic fibroblasts (MEF) and mouse fibroblasts (STO)). Feeder cells are preferably inactivated by known methods, such as irradiation (e.g., gamma rays) or treatment with an anticancer drug (e.g., mitomycin C). Examples of extracellular matrix components include Matrigel (Niwa A, et al., PLoS One. 6(7):e22261, 2011), fibrous proteins such as gelatin, collagen, and elastin, glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesive proteins such as fibronectin, vitronectin, and laminin.

[0042] Suspension culture is the cultivation of cells in a non-adherent state to a culture vessel, and can be carried out using, but is not limited to, a culture vessel that has not been artificially treated to improve adhesion to the cells (e.g., coated with an extracellular matrix, etc.), or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or a nonionic surface-active polyol (e.g., Pluronic F-127)). When performing suspension culture, it is preferable to form and cultivate embryoid bodies (EBs).

[0043] In the present invention, hematopoietic progenitor cells can also be prepared from a net-like structure (also called ES-sac or iPS-sac) obtained by culturing pluripotent stem cells. Here, the term "net-like structure" refers to a three-dimensional sac-like structure (with an internal space) derived from pluripotent stem cells, formed from an endothelial cell population or the like, and containing hematopoietic progenitor cells.

[0044] The culture temperature is not particularly limited, but is, for example, approximately 37°C to 42°C, preferably approximately 37°C to 39°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of hematopoietic progenitor cells, etc. The number of days is not particularly limited as long as hematopoietic progenitor cells are obtained, but is, for example, at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, preferably 14 days. Long culture periods are not usually problematic in the production of hematopoietic progenitor cells, but a period of, for example, 35 days or less is preferred, and 21 days or less is more preferred. Culture may also be performed under hypoxic conditions. In the present invention, hypoxic conditions include oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0045] (2) Differentiating hematopoietic progenitor cells into T cells The method for differentiating hematopoietic progenitor cells into T cells is not particularly limited as long as it allows hematopoietic progenitor cells to be differentiated into T cells. For example, a method of culturing hematopoietic progenitor cells under culture conditions similar to those used for inducing T cells from hematopoietic progenitor cells, such as those described in International Publication No. 2016 / 076415 or International Publication No. 2017 / 221975, can be used.

[0046] In the present invention, the medium for inducing differentiation into T cells is not particularly limited, but a medium used for culturing animal cells can be prepared as the basal medium. Furthermore, the basal medium may contain medium additives, etc., as needed. Examples of the basal medium and medium additives include those used in step (1) above.

[0047] When vitamin C is used in step (2), the vitamin C may be the same as those described in step (1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0048] In step (2), it is preferable to use a p38 inhibitor and / or SDF-1 (stromal cell-derived factor 1). In the present invention, the term "p38 inhibitor" refers to a substance that inhibits the function of p38 protein (p38 MAP kinase), and examples thereof include, but are not limited to, chemical inhibitors of p38, dominant-negative mutants of p38, and nucleic acids encoding the same.

[0049] Examples of chemical inhibitors of p38 used in the present invention include, but are not limited to, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and derivatives thereof, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol) and derivatives thereof, SB220025 and derivatives thereof, PD169316, RPR200765A, AMG-548, BIRB-796, SCIO-469, SCIO-323, VX-702, and FR167653. These compounds are commercially available, for example, SB203580, SB202190, SC239063, SB220025, and PD169316 are available from Calbiochem, and SCIO-469 and SCIO-323 are available from Scios. Preferred chemical inhibitors of P38 include SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives.

[0050] Dominant-negative mutants of p38 used in the present invention include p38T180A, in which threonine at position 180 in the DNA-binding domain of p38 is mutated to alanine, and p38Y182F, in which tyrosine at position 182 of human and mouse p38 is mutated to phenylalanine. The p38 inhibitor is contained in the medium at a concentration ranging from about 1 μM to about 50 μM. When SB203580 is used as the p38 inhibitor, it can be contained in the medium at a concentration ranging from 1 μM to 50 μM, 5 μM to 30 μM, or 10 μM to 20 μM.

[0051] The SDF-1 used in the present invention may be not only SDF-1α or its mature form, but also isoforms such as SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, or SDF-1φ, or their mature forms, or a mixture of these in any proportion. Preferably, SDF-1α is used. SDF-1 is also referred to as CXCL-12 or PBSF.

[0052] In the present invention, SDF-1 may have one or more amino acids deleted, substituted, inserted, and / or added in its amino acid sequence, so long as it retains its chemokine activity (SDF-1 with such amino acid deletions, substitutions, insertions, and / or additions is also referred to as an "SDF-1 mutant"). Similarly, SDF-1 or an SDF-1 mutant may have a sugar chain deleted, substituted, inserted, and / or added. Examples of the SDF-1 mutant include those that retain at least four cysteine ​​residues (Cys30, Cys32, Cys55, and Cys71 in the case of human SDF-1α) and have 90% or more identity to the native amino acid sequence, but are not limited to these amino acid mutations. SDF-1 may be from mammals, such as humans, or non-human mammals, such as monkeys, sheep, cows, horses, pigs, dogs, cats, rabbits, rats, and mice. For example, the protein registered under GenBank accession number NP_954637 can be used as human SDF-1α, and the protein registered under GenBank accession number NP_000600 can be used as SDF-1β.

[0053] SDF-1 may be commercially available, purified from nature, or produced by peptide synthesis or genetic engineering techniques. SDF-1 is contained in the medium, for example, in a range of about 10 ng / ml to about 100 ng / ml. Alternatively, SDF-1 substitutes having SDF-1-like activity may be used instead of SDF-1. Examples of such SDF-1 substitutes include CXCR4 agonists, and low-molecular-weight compounds having CXCR4 agonistic activity may be added to the medium instead of SDF-1.

[0054] The medium used in step (2) may further contain at least one, preferably all, of cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT-3L, and IL-7. The concentrations of these cytokines are, for example, 10 ng / ml to 100 ng / ml for SCF, 10 ng / ml to 200 ng / ml for TPO, 1 ng / ml to 100 ng / ml for IL-7, and 1 ng / ml to 100 ng / ml for FLT-3L.

[0055] In step (2), hematopoietic progenitor cells may be cultured in an adherent or suspension culture. In the case of adherent culture, the culture vessel may be coated, or the cells may be co-cultured with feeder cells or the like. An example of a feeder cell to be co-cultured is the bone marrow stromal cell line OP9 cells (available from the RIKEN BioResource Center). The OP9 cells are preferably OP9-DL4 cells or OP9-DL1 cells, which constitutively express DLL4 or DLL1 (e.g., Holmes R1 and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009(2)). When OP9 cells are used as feeder cells in the present invention, they may be appropriately added to the medium with separately prepared DLL4 or DLL1, or a fusion protein of DLL4 or DLL1 with Fc or the like. When feeder cells are used, it is preferable to appropriately replace the feeder cells during culture. Feeder cell replacement can be performed by transferring the target cells during culture onto previously seeded feeder cells. The replacement can be performed every 5 days, 4 days, 3 days, or 2 days. Furthermore, when hematopoietic progenitor cells are obtained by suspension culture of embryoid bodies, they are preferably dissociated into single cells and then cultured in an adherent manner. Co-culture with feeder cells is also possible, but preferably, the culture is performed without feeder cells. In the case of adherent culture, examples of coating agents for coating culture vessels include Matrigel (Niwa A, et al., PLos One, 6(7):e22261, 2011)), collagen, gelatin, laminin, heparan sulfate proteoglycan, RetroNectin (registered trademark), DLL4 or DLL1, or fusion proteins of DLL4 or DLL1 with the Fc region of an antibody (hereinafter sometimes referred to as Fc) (e.g., DLL4 / Fc chimera), entactin, and / or combinations thereof, with a combination of RetroNectin and a fusion protein of DLL4 with Fc or the like being preferred.

[0056] In step (2), the culture temperature conditions are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of T cells, etc. The number of days is not particularly limited as long as T cells are obtained, but is typically at least 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, or 20 days or more, and preferably 21 days. Furthermore, 90 days or less is preferred, and 42 days or less is more preferred.

[0057] The cell population obtained by the above steps contains T cells, and step (2) may further include the following step (3).

[0058] (3) A process for enriching T cells The method for enriching T cells is not particularly limited as long as the T cells are enriched. For example, a method of culturing T cells under culture conditions similar to those used in the process of inducing CD8-positive T cells from CD4CD8-positive T cells, as described in WO 2016 / 076415 and WO 2017 / 221975, can be used.

[0059] As used herein, "enriching" refers to increasing the proportion of a particular component in a composition, such as a composition of cells, and "enriched," when used to describe a composition of cells, e.g., a cell population, refers to a cell population in which the amount of a particular component is increased compared to the proportion of such component in the cell population prior to enrichment. For example, a composition, such as a cell population, can be enriched for a target cell type, thus increasing the proportion of the target cell type compared to the proportion of target cells present in the cell population prior to enrichment. Cell populations can also be enriched for a target cell type by cell selection and sorting methods known in the art. Cell populations can also be enriched by certain culture methods, sorting, or selection processes described herein. In certain embodiments of the present invention, a method of enriching a target cell population results in a cell population that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% enrichment for the target cell population.

[0060] As used herein, a "cell population" refers to two or more cells of the same or different types. A "cell population" also refers to a mass of cells of the same or different types.

[0061] In the present invention, the medium used for enriching T cells is not particularly limited, but a medium used for culturing animal cells can be prepared as the basal medium. If necessary, the basal medium may contain medium additives. Examples of the basal medium and medium additives include those used in step (1) above.

[0062] When vitamin C is used in step (3), the vitamin C may be the same as those described in step (1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0063] When a hormone is used in step (3), the hormone may be an adrenocortical hormone. The adrenocortical hormone is a glucocorticoid or a derivative thereof, and examples thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. Dexamethasone is preferred. When the adrenocortical hormone is dexamethasone, its concentration in the medium is 1 nM to 100 nM.

[0064] In step (3), the medium may contain a CD3 / TCR complex agonist. The CD3 / TCR complex agonist is not particularly limited, as long as it is a molecule capable of specifically binding to the CD3 / TCR complex and transmitting a signal from the CD3 / TCR complex into T cells. Examples of CD3 / TCR complex agonists include CD3 agonists and / or TCR agonists. Examples of CD3 agonists include anti-CD3 agonist antibodies (also simply referred to as "anti-CD3 antibodies") or binding fragments thereof. Examples of TCR agonists include at least one selected from the group consisting of anti-TCR agonist antibodies (also simply referred to as "anti-TCR antibodies") or binding fragments thereof, MHC / antigen peptide complexes or multimers thereof, and MHC / superantigen complexes or multimers thereof. When an anti-CD3 antibody is used, the anti-CD3 antibody encompasses both polyclonal and monoclonal antibodies, with monoclonal antibodies being preferred. The antibody may belong to any immunoglobulin class, including IgG, IgA, IgM, IgD, or IgE, with IgG being preferred. Examples of anti-CD3 antibodies include the antibody produced from the OKT3 clone (OKT3) and the antibody produced from the UCHT1 clone (UCHT1), with UCHT1 being preferred. The concentration of the anti-CD3 antibody in the culture medium is, for example, 10 ng / ml to 1000 ng / ml, preferably 50 ng / ml to 800 ng / ml, and more preferably 250 ng / ml to 600 ng / ml. The CD3 / TCR complex agonist may be commercially available, purified from nature, or produced by peptide synthesis, genetic engineering, or chemical synthesis. For example, OKT3 and UCHT1 can be purchased from ThermoFisher, GeneTex, or other companies.

[0065] When a cytokine is used in step (3), examples of the cytokine include IL-2 and IL-7. When the cytokine is IL-2, its concentration in the medium is 10 U / ml to 1000 U / mL, and when the cytokine is IL-7, its concentration in the medium is 1 ng / ml to 1000 ng / mL.

[0066] In step (3), the culture temperature conditions are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, those skilled in the art can appropriately determine the culture period while monitoring the number of T cells, etc. As long as the cells are concentrated, the number of days is not particularly limited, but is, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, or more, and preferably 6 days. Furthermore, 28 days or less is preferred, and 14 days or less is more preferred.

[0067] The expression of endogenous genes of the T cells constituting the T cell bank may be appropriately adjusted. Specifically, from the viewpoint of reducing rejection in allogeneic transplantation, it is preferable that the expression of at least one (e.g., one, two, three, six, or nine) HLA (human leukocyte antigen) gene be suppressed or that the HLA gene be deleted in the T cells constituting the T cell bank. Therefore, in one embodiment of the present invention, the donation system of the present invention comprises T cells in which the expression of at least one HLA gene is suppressed. As used herein, suppression of HLA gene expression also encompasses deletion of the gene.

[0068] Specific examples of the HLA genes include one or more HLA genes selected from the group consisting of class I HLA (i.e., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G) genes and class II HLA (i.e., HLA-DR, HLA-DQ, and HLA-DP) genes. Alternatively, the expression of genes important for the expression of the HLA genes or their presentation on the cell surface may be suppressed. Examples of such genes include the B2M gene, which encodes B2M, a protein important for presenting HLA class 1 HLA on the cell surface, and the CIITA gene, which encodes CIITA, a protein important for the expression of class II HLA genes. On the other hand, since suppression of HLA gene expression makes cells in which HLA gene expression is suppressed more likely to become targets of natural killer (NK) cells, it is also preferable to overexpress the CD47 gene or to suppress the expression of only two genes, the HLA-A gene and the HLA-B gene, while maintaining the expression of the HLA-C gene, which is important for avoiding attack by NK cells, as well as the HLA-E gene, HLA-F gene, and HLA-G gene (in this case, the expression of class II HLA genes may or may not be suppressed, but from the standpoint of immune compatibility, it is preferable to suppress the expression of these genes). Thus, in a preferred embodiment, combinations of HLA genes whose expression is to be inhibited include (i) a combination of HLA-A genes and HLA-B genes, (ii) a combination of HLA-A genes, HLA-B genes, HLA-DR genes, HLA-DQ genes, and HLA-DP genes, and (iii) a combination of HLA-A genes, HLA-B genes, HLA-C genes, HLA-E genes, HLA-F genes, HLA-G genes, HLA-DR genes, HLA-DQ genes, and HLA-DP genes. While the above description has been given using HLA, a human major histocompatibility complex (MHC), as an example, it is also preferable to inhibit the expression of at least one MCH gene in a similar manner when using non-human MHC.

[0069] Methods for suppressing HLA gene expression can be appropriately used, including known methods. For example, HLA gene expression can be knocked down by introducing siRNA, shRNA, miRNA, antisense oligonucleotides, or ribozymes targeting HLA mRNA, B2M mRNA, and / or CIITA mRNA into cells at the stage of differentiation from pluripotent cells to T cells (e.g., pluripotent stem cells, hematopoietic progenitor cells (CD34+ / CD43+), ProT cells (CD4- / CD8-), CD3+ / CD4+ / CD8+ T cells, CD3+ / CD4- / CD8+ T cells, or other cells (e.g., CD3- / CD4+ / CD8+ cells, etc.)). Alternatively, nucleic acids encoding such molecules can be introduced into such cells using the methods described below. Alternatively, the HLA gene, B2M gene, and / or CIITA gene in cells can be knocked out by genome editing (e.g., CRISPR system, TALEN, ZFN, etc.).

[0070] Furthermore, it is preferable that the expression of endogenous TCRs in the T cells constituting the T cell bank be suppressed by silencing the expression of the TCR chain using siRNA or by introducing an exogenous TCR known to be harmless to the subject receiving the T cells. When applying the siRNA-based method, it is preferable to use a nucleic acid encoding the TCR with a different nucleotide sequence (codon-altered sequence) from the nucleotide sequence corresponding to the RNA on which the siRNA acts to suppress the expression of the endogenous TCR chain, in order to avoid the effect of the siRNA on the exogenous TCR. These methods are described, for example, in WO 2008 / 153029. The nucleotide sequence can be prepared by introducing silent mutations into a nucleic acid encoding a naturally occurring TCR or by chemically synthesizing an artificially designed nucleic acid. Alternatively, to avoid mispairing with the endogenous TCR chain, part or all of the constant region of the nucleic acid encoding the introduced TCR may be replaced with a constant region derived from an animal other than the subject.

[0071] Furthermore, the T cells constituting the T cell bank may be introduced with a nucleic acid containing an exogenous gene, and the exogenous gene product may be expressed.

[0072] The exogenous gene is not particularly limited, and can be an exogenous gene encoding a protein, cytokine, chemokine, or the like that can contribute to the regulation of T cell function. Examples of the exogenous gene include a gene encoding a chimeric antigen receptor (CAR) (hereinafter also referred to as a "CAR gene") and a gene encoding an exogenous T cell receptor (TCR) (hereinafter also referred to as a "TCR gene"). The CAR and TCR expressed from the gene introduced into a cell can recognize and bind to an antigen and / or the antigen-HLA complex. As used herein, a nucleic acid encoding a TCR refers to a nucleic acid containing a base sequence encoding one chain that forms a TCR and a base sequence encoding the other chain.

[0073] As used herein, the term "capable of binding" means "having an ability to bind" and refers to the ability to form a non-covalent complex with one or more other molecules. Various methods and assays for determining binding ability are known in the art. Binding is typically high affinity, with binding affinity, measured by KD values, preferably less than 1 μM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, and even more preferably less than 1 pM. The term "KD" or "KD value" is related to the equilibrium dissociation constant, as known in the art, and represents the binding affinity between molecules. A smaller KD value indicates a higher binding affinity.

[0074] The TCRs used in the present invention include not only heterodimers formed by the α and β chains of TCR (i.e., αβTCR) or the γ and δ chains of TCR (i.e., γδTCR), but also homodimers. Furthermore, TCRs lacking part or all of the constant region or with modified amino acid sequences may also be used.

[0075] Furthermore, the constant regions of the above-mentioned TCR chains may be modified in a specific manner in the constant regions of the TCR chains of the cytotoxic T cell (CTL) clones from which they are derived, such that, for example, specific amino acid residues in the TCR constant regions of the CTL clones are substituted with cysteine ​​residues to enhance the efficiency of dimer formation through disulfide bonds between the TCR chains, but this modification is not limited to this.

[0076] Furthermore, the TCR may be a variant, and hereinafter, unless otherwise specified, the term "TCR" encompasses such variants. Examples of such variants include a variant of a T cell receptor developed by the present inventors, which combines two polypeptides containing the constant regions of TCR chains selected from the group consisting of the α chain, β chain, γ chain, and δ chain. A variant of a TCR is characterized by not containing the complementarity-determining regions (CDRs) of the TCR α chain and β chain, or the complementarity-determining regions (CDRs) of the same chain (preferably, a part or all of the variable region containing the CDRs), and by not containing the complementarity-determining regions (CDRs) of the T cell receptor chain from which the constant region is derived, or the complementarity-determining regions (CDRs) of the same chain (preferably, a part or all of the variable region containing the CDRs). Therefore, the variants do not include natural or artificial (e.g., the constant and variable regions are derived from different animal species) αβ TCRs or γδ TCRs, or TCR variants to which additional amino acids have been added to these TCRs. For example, when a polypeptide corresponding to at least one chain of a variant of the present invention contains a constant region of the T cell receptor α chain, the polypeptide does not contain the complementarity-determining region of the T cell receptor α chain, preferably a part or all of the variable region containing the complementarity-determining region, but may contain the CDRs and variable region of a different TCR chain other than the α chain and the β chain, such as the γ chain or the δ chain. Similarly, when a polypeptide corresponding to at least one chain of the above variant contains a constant region of the T cell receptor β chain, the polypeptide does not contain the complementarity-determining region of the T cell receptor β chain, preferably a part or all of the variable region containing the complementarity-determining region, but may contain the CDRs and variable region of a different TCR chain other than the α chain and the β chain, such as the γ chain or the δ chain. The same applies to the γ chain and the δ chain.

[0077] The TCR may have a membrane-translocating signal peptide (hereinafter referred to as "signal peptide") added thereto. The signal peptide may be a membrane-translocating signal peptide derived from a gene encoding various peptides having a transmembrane domain, such as CD8, immunoglobulin-H (IGH), or CD4, and / or an amino acid sequence in which one or several (e.g., two, three, four, or five) amino acids have been deleted, substituted, inserted, and / or added within the amino acid sequence of the signal peptide, or a signal peptide consisting of an amino acid sequence identical to the amino acid sequence of the signal peptide. When a signal peptide is added, the binding position and the number of signal peptides are not particularly limited.

[0078] In the present invention, the term "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain of the CAR comprises a single-chain fragment (scFv) consisting of the light chain (VL) and heavy chain (VH) of the variable region of an antibody linked in tandem via a spacer such as a linker (e.g., a linker consisting of G and S (GS linker)). After recognizing an antigen via the scFv domain, T cells expressing the CAR transmit the recognition signal into the T cell via the intracellular signaling domain. Introducing a CAR into T cells can confer specificity for a target antigen. Furthermore, because CARs can directly recognize antigen molecules independent of HLA class I or class II, they can elicit a strong immune response even against cells with reduced expression of HLA class I or class II genes. Antigens targeted by the CAR include the same antigens targeted by the TCR.

[0079] Examples of the transmembrane domain of the CAR include, but are not limited to, transmembrane domains derived from one or more proteins selected from the group consisting of the α chain, β chain, or ζ chain of TCR, CD28, CD3ε chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB (CD137), and CD154. The transmembrane domain of the molecule from which the first intracellular signaling domain linked to the antigen-binding domain is derived may be used. For example, if the molecule from which the first intracellular signaling domain linked to the antigen-binding domain is derived is CD28, the transmembrane domain may also be derived from CD28. Alternatively, an artificially designed transmembrane domain may be used.

[0080] Examples of the intracellular signaling domain of a CAR include, but are not limited to, intracellular domains derived from one or more proteins selected from the group consisting of CD3 ζ chain (TCR ζ chain), FcR γ chain, FcR β chain, CD3 γ chain, CD3 δ chain, CD3 ε chain, CD5, CD22, CD79a, CD79b, and CD66d. Among these, an intracellular signaling domain derived from the CD3 ζ chain is preferred. The intracellular signaling domain may further include the intracellular domain of a costimulatory molecule, such as the intracellular domain of one or more proteins selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. The strength and duration of CAR activity can be controlled by selecting the type and number of costimulatory molecules to be bound (e.g., Mol Ther. 2009;17:1453-1464.).

[0081] A spacer may be inserted between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular signaling domain and the transmembrane domain of the CAR. The spacer may be a peptide typically consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. Specific examples of the spacer include, but are not limited to, a hinge region derived from IgG1, or a peptide containing the CH2CH3 region of an immunoglobulin and a portion of CD3.

[0082] Specific examples of CARs include, but are not limited to, first-generation CARs in which an scFV and a CD3ζ chain are linked via a spacer; second-generation CARs in which a transmembrane domain and an intracellular domain derived from CD28 are incorporated between the scFV and CD3ζ chain of the first-generation CAR to enhance its ability to activate T cells; and third-generation CARs in which an intracellular domain of a costimulatory molecule other than CD28 (4-1BB or OX40) is incorporated between the CD28 intracellular domain and the CD3ζ chain of the second-generation CAR.

[0083] More specifically, the CAR used in the present invention includes a chimeric antigen receptor comprising an scFv that recognizes CD19 as the antigen-binding domain, a CD8 transmembrane domain as the transmembrane domain, and an intracellular signaling domain derived from CD28, CD30, 4-1BB, or CD3ζ chain. The order of the intracellular domains contained in the intracellular signaling domain is not particularly limited, but may be, for example, the order of CD28-derived intracellular domain, CD30-derived intracellular domain or 4-1BB-derived intracellular domain, and CD3ζ chain-derived intracellular domain. More specifically, the chimeric antigen receptor of the present invention comprises, for example, the amino acid sequence represented by SEQ ID NO: 4 or 5, or an amino acid sequence in which one or more amino acids (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to several (2, 3, 4, or 5)) have been deleted, substituted, inserted, and / or added from the amino acid sequence represented by SEQ ID NO: 4 or 5.

[0084] Furthermore, examples of the intracellular domain derived from CD30 include amino acid sequences in which one or more amino acids (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to several (2, 3, 4, or 5)) have been deleted, substituted, inserted, and / or added from the amino acid sequence shown in SEQ ID NO: 7. When an amino acid sequence has been deleted, substituted, inserted, and / or added as described above, the position of the deletion, substitution, insertion, and / or addition is not particularly limited, as long as the function of the intracellular domain of CD30 is maintained.

[0085] Antigens targeted by the TCRs and CARs include, but are not limited to, tumor antigens, which may be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). Specific examples of such tumor antigens include one or more antigens selected from the group consisting of differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2; tumor-specific multilineage antigens such as WT1, Glypican-3, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; fetal antigens such as CEA; overexpressed oncogenes or mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA These include, but are not limited to, 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0086] Whether the above-mentioned TCR or CAR (hereinafter sometimes abbreviated as "TCR, etc.") can specifically recognize and bind to an antigen can be confirmed by known methods, and suitable methods include, for example, a dextramer assay or an ELISPOT assay. By performing an ELISPOT assay, it can be confirmed that a T cell expressing a TCR, etc. on its cell surface recognizes a target antigen via the TCR, etc., and that the signal is transmitted into the cell.

[0087] Furthermore, the present inventors have previously found that cells expressing a fusion protein containing IL-15 and IL-15Rα together with the above-mentioned CAR (hereinafter, sometimes abbreviated as "IL-15 / IL-15Rα") have increased cytotoxic activity compared to cells expressing only the CAR. Therefore, from the viewpoint of cytotoxic activity, it is preferable that the T cells constituting the T cell bank express IL-15 / IL-15Rα, and more preferably that they also express the above-mentioned CAR. In order to obtain T cells expressing IL-15 / IL-15Rα, the IL-15 / IL-15Rα gene is expressed in cells at the stage of differentiation from pluripotent cells to T cells (e.g., pluripotent stem cells, hematopoietic progenitor cells (CD34 + / CD43 + ), ProT cells (CD4 - / CD8 - ), CD3 + / CD4 + / CD8 + T cells, CD3 + / CD4 - / CD8 + T cells or other cells (e.g., CD3 - / CD4 + / CD8 + IL-15 / IL-15Rα is preferably introduced into cells in the form of nucleic acids encoding them, and such introduction can be carried out in the same manner as in the nucleic acid introduction step described above.

[0088] In the IL-15 signal transduction system, IL-15Rα expressed on antigen-presenting cells typically binds to IL-15 and presents IL-15 to the IL-15 receptor, consisting of IL-15Rβ and the common gamma chain (γc), on CD8+CD4-negative cells (trans-presentation), thereby maintaining the cytotoxic activity of CD8+CD4-negative cells. Therefore, when T cells expressing IL-15 / IL-15Rα are CD8+CD4-negative, they can transduce the IL-15 signal into their own cells via the IL-15 receptor. Alternatively, T cells expressing IL-15 / IL-15Rα can transduce the IL-15 signal into other CD8+CD4-negative cells via the IL-15 receptor. As described above, IL-15 / IL-15Rα can maintain the cytotoxic activity of CD8+CD4-negative cells, and thus a continuous cytotoxic effect against cells targeted by CAR can be expected.

[0089] IL-15 / IL-15Rα may be a transmembrane protein or a secreted protein. It is known that the IL-15-binding domain, consisting of amino acids 1-65 from the N-terminus of the mature IL-15Rα protein, is responsible for binding to IL-15 (Wei X. et al., J. Immunol., 167: 277-282, 2001). Therefore, a transmembrane protein may be any protein that retains the IL-15-binding domain and the transmembrane domain of IL-15Rα. On the other hand, a secreted protein may be any protein that retains the IL-15-binding domain but lacks the transmembrane domain of IL-15Rα (e.g., a protein consisting of amino acid residues 1-65, 1-85, or 1-182 of IL-15Rα, or a peptide containing an amino acid sequence 85% or more (e.g., 90%, 95%, 97%, 98%, or 99%) identical to the amino acid sequence).

[0090] IL-15 / IL-15Rα may incorporate a spacer between IL-15 and IL-15Rα, and the spacer may be a peptide generally consisting of 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 20 to 50 amino acids. Specific examples of the spacer include, but are not limited to, the GS linker described above.

[0091] IL-15 / IL-15Rα is not particularly limited as long as it is a fusion protein of IL-15 and IL-15Rα, and a specific example is the peptide consisting of SEQ ID NO: 7. Alternatively, IL-15 / IL-15R is not particularly limited as long as it is capable of binding to the IL-15 receptor and transducing the IL-15 signal into cells, and examples include peptides comprising an amino acid sequence that has about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 7. Here, "homology" or "identity" refers to the percentage (%) of identical and similar amino acid residues (in the case of identity, identical amino acid residues) relative to the total overlapping amino acid residues in the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). "Similar amino acids" refer to amino acids similar in physicochemical properties, and include, for example, amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitution with such similar amino acids is expected to not change the phenotype of the protein (i.e., conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).The homology or identity of amino acid sequences herein can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0092] Introduction of an exogenous gene can be carried out by known methods. When the nucleic acid to be introduced is in the form of DNA, it can be carried out by, for example, calcium phosphate coprecipitation, PEG, electroporation, microinjection, lipofection, etc. For example, methods described in Cell Engineering, Special Issue 8, New Cell Engineering Experimental Protocols, pp. 263-267 (1995) (Shujunsha), Virology, Vol. 52, p. 456 (1973), and Folia Pharmacol. Jpn., Vol. 119 (No. 6), pp. 345-351 (2002) can be used. When a viral vector is used, the nucleic acid, together with a packaging vector if necessary, can be introduced into appropriate packaging cells (e.g., Plat-E cells) or complementation cell lines (e.g., 293 cells), and the viral vector produced in the culture supernatant can be collected and introduced into cells by infecting the cells with the vector using a method appropriate for the viral vector. Examples of such viral vectors include retroviral vectors (including lentiviral vectors and pseudotyped vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and episomal vectors. Transposon expression systems (PiggyBac systems) may also be used. Examples of plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo). Specific methods for using retroviral vectors as vectors are disclosed in International Publication No. 2007 / 69666, Cell, 126, 663-676 (2006), and Cell, 131, 861-872 (2007). In particular, when using retroviral vectors, the use of the recombinant fibronectin fragment CH-296 (Takara Bio Inc.) enables highly efficient gene transfer into various cells. When the nucleic acid to be introduced is in the form of DNA and a viral vector is used, the type of viral vector can be appropriately selected depending on the type of cell to be introduced.When the target cells are T cells, a retroviral vector is preferably used, and a gamma retroviral vector is more preferably used, and when the target cells are iPS cells, a lentiviral vector is preferably used.

[0093] The nucleic acid to be introduced may be directly introduced into cells in the form of RNA, and the introduced gene may be expressed in the cells. A known method for introducing RNA can be used, and for example, lipofection or electroporation can be suitably used. Furthermore, when the reprogramming factor is in the form of a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (e.g., HIV-derived TAT and polyarginine), or microinjection.

[0094] The above-mentioned TCRs and the like are introduced into cells in the form of nucleic acids encoding the TCRs and the like. Fusion proteins containing IL-15 and IL-15Rα are also introduced into cells in the form of nucleic acids encoding the fusion proteins. The nucleic acids and the nucleic acids for suppressing expression of the above-mentioned HLA genes may be DNA, RNA, or DNA / RNA chimeras, but are preferably DNA. The nucleic acids may be double-stranded or single-stranded. If double-stranded, they may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. When the nucleic acid is RNA, T in the RNA sequence is to be read as U. The nucleic acid may contain natural nucleotides, modified nucleotides, nucleotide analogs, or mixtures thereof, as long as it is capable of expressing a polypeptide in vitro or in cells.

[0095] The above nucleic acids can be constructed by methods known per se. For example, DNA encoding the full length or a portion of a TCR or CAR can be constructed by chemically synthesizing a DNA strand based on the amino acid sequence or nucleic acid sequence of a known TCR or CAR, or by connecting synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson Assembly. Nucleic acids for suppressing HLA gene expression and nucleic acids encoding fusion proteins containing IL-15 and IL-15Rα can also be constructed in a similar manner.

[0096] The above-mentioned nucleic acid can be incorporated into an expression vector. The vector may or may not be integrated into the genome of the target cell. In one embodiment, the vector that is not integrated into the genome can replicate outside the genome of the target cell. The vector may exist in multiple copies outside the genome of the target cell. In another embodiment of the present invention, the vector is integrated into the genome of the target cell. Alternatively, the introduced nucleic acid can be integrated into the genome by homologous recombination using genome editing (e.g., CRISPR system, TALEN, ZFN, etc.). In a preferred embodiment, the nucleic acid, such as a vector, is integrated into a predetermined location in the genome of the target cell.

[0097] Examples of promoters used in the above vectors include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR Vα gene promoter, and TCR Vβ gene promoter. Of these, the EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, and SRα promoter are preferred.

[0098] In addition to the promoter, the vector may optionally contain transcriptional and translational regulatory sequences, a ribosome binding site, an enhancer, a replication origin, a poly(A) addition signal, a selection marker gene, etc. Examples of selection marker genes include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.

[0099] In one embodiment of the present invention, an expression vector containing a nucleic acid encoding a TCR α chain and a nucleic acid encoding a β chain can be introduced into a target cell to form a heterodimer of the TCR α chain and β chain within the target cell or on the cell surface. In this case, the nucleic acid encoding the TCR α chain and the nucleic acid encoding the β chain may be incorporated into separate expression vectors or may be incorporated into a single expression vector. When incorporated into a single expression vector, these two types of nucleic acids are preferably incorporated via a sequence that enables polycistronic expression. The use of a sequence that enables polycistronic expression enables more efficient expression of multiple genes incorporated into a single expression vector. Examples of sequences that enable polycistronic expression include 2A sequences (e.g., 2A sequences (F2A) derived from foot-and-mouth disease virus (FMDV), 2A sequences (E2A) derived from equine rhinitis A virus (ERAV), 2A sequences (P2A) derived from porcine teschovirus (PTV-1), and 2A sequences (T2A) derived from Thosea asigna virus (TaV) (PLoS ONE 3, e2532, 2008; Stem Cells 25, 1707, 2007), and internal ribosome entry sites (IRES) (US Patent No. 4,937,190). From the viewpoint of uniform expression levels, however, P2A and T2A sequences are preferred. The same applies when using an expression vector containing a nucleic acid encoding a TCR gamma chain and a nucleic acid encoding a TCR delta chain.

[0100] The exogenous gene to be introduced into the T cells constituting the T cell bank can be introduced into cells generated during the process of differentiating stem cells (e.g., iPS cells or ES cells) into T cells. Here, the cells generated during the process of differentiating stem cells (e.g., iPS cells or ES cells) into T cells include stem cells (e.g., iPS cells or ES cells) and T cells.

[0101] In one embodiment of the present invention, the exogenous gene to be introduced into the T cells constituting the T cell bank is introduced into iPS cells, hematopoietic progenitor cells and / or T cells. Furthermore, in one embodiment of the present invention, before constructing a T cell bank, the T cells obtained above can be expanded as needed by the method described below in "1-5. Expansion culture." A T cell master cell bank can be constructed by dispensing and storing the T cells obtained by the above method into multiple storage containers (such as sterile vials). The stored T cells are preferably frozen. One container of T cells from this T cell master cell bank can be thawed as needed and subjected to appropriate characterization tests. Alternatively, T cells prepared from the same container or from a different container can be expanded as needed by the method described below in "1-5. Expansion Culture," and the resulting T cells can be dispensed into multiple containers for expansion and storage to construct a T cell working cell bank. The stored T cells are preferably frozen. Therefore, the provision system of the present invention may include a step of constructing a T cell master cell bank and / or a T cell working cell bank (hereinafter also referred to as a "T cell cell bank construction step"). Hereinafter, the part configured to execute the T cell cell bank construction step may be referred to as a "T cell cell bank construction unit."

[0102] 1-2.Freezing of T cells As described above, the T cells in the T cell bank are preferably frozen. Freezing of T cells can be performed by known methods. Examples of such methods include, but are not limited to, placing a container containing the T cells in a freezer (e.g., an ultra-low temperature freezer) or contacting the cells with a low-temperature medium (e.g., liquid nitrogen) and storing them in a freezer or cryopreservation system (e.g., a locator). The freezing temperature is typically 0°C or below, preferably −20°C or below, more preferably −40°C or below, and even more preferably −80°C or below. The cooling rate during the freezing procedure typically requires 1 to 5 hours, preferably 2 to 4 hours, and particularly about 3 hours from 4°C to −80°C. This cooling rate can be achieved by subjecting the container containing the T cells directly or in a freezing container to a freezing means set at the desired temperature. The freezing container may have a function to control the rate at which the temperature inside the container drops to a predetermined rate. Such a freezing container can be, for example, BICELL (registered trademark) (Nippon Freezer). The above cooling rate can be achieved by using a freezer that can control the cooling rate by programming. Any known freezer can be used, such as a programmable freezer (e.g., CryoMed (Thermo Fisher), PDF-2000G (Strex), or KRYO-560-16 (Asahi Life Sciences)).

[0103] The freezing procedure may be performed after adding a cryoprotectant to a culture medium or physiological buffer solution in which the colonies have been immersed, or by replacing the culture medium with a cryoprotectant-containing cryoprotectant-containing cryoprotectant. When replacing the culture medium with a cryoprotection medium, the culture medium may be substantially completely removed before adding the cryoprotection medium, or the culture medium may be partially left behind before adding the cryoprotection medium. Commercially available cryoprotection mediums may be used, such as CryoStor (registered trademark) CS10 and STEM-CELLBANKER (registered trademark) (ZENOAQ).

[0104] Cryoprotectants are not particularly limited and include, for example, dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), and glycerin. Cryoprotectants may be used alone or in combination of two or more. Cryoprotectants may also be used in combination with extracellular cryoprotectants. Examples of extracellular cryoprotectants include polyethylene glycol, sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxyethyl starch (HES), dextran, and albumin.

[0105] The concentration of the cryoprotectant added to the culture medium or the cryopreservation solution is typically 2 to 20% (v / v), preferably 5 to 15%, and more preferably 8 to 13%, of the total culture medium or cryopreservation solution. This concentration can be adjusted appropriately depending on the type of cryoprotectant. For example, when DMSO is used as the cryoprotectant, the concentration of DMSO is typically 2 to 20% (v / v), preferably 2.5 to 12.5%, and more preferably 5 to 10%, of the total culture medium or cryopreservation solution. Specific examples of cryopreservatives and freezers are as described above. A cryopreservation container can be used as the container for containing T cells, and specific examples of cryopreservation containers include sterile vials (e.g., glass ampoules, polymer vials (AT-closed vials), etc.).

[0106] 1-3.Preparation of T cells In the present invention, since a T cell bank typically contains one or more containers into which T cells have been dispensed and frozen, preparation of T cells also includes steps of collecting one or more containers from the T cell bank and thawing the T cells, as well as, if necessary, washing the thawed cells, pre-culturing the cells to bring them to sleep, maintaining the cells in a culture, etc. Each of the steps above may be performed by the same entity or different entities.

[0107] The T cells can be thawed by known methods, including, but not limited to, contacting a container containing frozen T cells collected from a T cell bank with a solid, liquid, or gaseous medium (e.g., water, culture medium) at a temperature higher than the freezing point using a water bath, incubator, or the like. The temperature of the medium is typically 4°C to 50°C, preferably 30°C to 40°C, and more preferably 36°C to 38°C. The thawing time is typically within 2 minutes, and a reduction in cell viability can be significantly suppressed by keeping it within 20 seconds. The thawing time can be adjusted, for example, by changing the temperature of the thawing method or immersion medium, or the volume or composition of the culture medium or cryopreservation solution used at the time of freezing.

[0108] Cell washing can be performed by known methods, for example, by suspending the cells in a cell washing solution (e.g., culture medium or physiological buffer solution that may contain serum or serum components (e.g., serum albumin)), centrifuging, discarding the supernatant, and recovering the precipitated cells, but is not limited to this. In the cell washing step, the cycle of suspension, centrifugation, and recovery may be performed one or more times (e.g., 2, 3, 4, 5 or more times). In one embodiment of the present invention, the cell washing step is performed immediately after the step of thawing T cells collected from a T cell bank. An example of a commercially available cell washing solution that can be used in the present invention is Cell Lotion (Nippon Zenyaku Kogyo Co., Ltd.).

[0109] In the present invention, the medium for the pre-culture and maintenance culture is not particularly limited, but a medium used for culturing animal cells can be prepared as the basal medium. If necessary, the basal medium may contain medium additives. Examples of the basal medium and medium additives include those listed in step (1) of 1-1 above.

[0110] The culture period for pre-culture and maintenance culture can be appropriately determined depending on the purpose, but is preferably 1 day or more (e.g., 5, 6, or 7 days), and preferably 10 days or less (e.g., 7, 8, 9, or 10 days). The culture temperature is not particularly limited, but is 30°C to 40°C, preferably 37°C, and culture is performed in the presence of CO2-containing air, with a CO2 concentration of preferably 2 to 5%.

[0111] 1-4.Transfection of nucleic acids into T cells prepared from a T cell bank The delivery system of the present invention may include a step of introducing a nucleic acid containing an exogenous gene into T cells prepared from a T cell bank (hereinafter also referred to as a "nucleic acid introduction step"). As used herein, "including a step" or "including a process" means including a step or a part configured to perform a process. In addition, hereinafter, the part configured to perform the nucleic acid introduction step may be referred to as a "nucleic acid introduction unit." The method for introducing the nucleic acid in the nucleic acid introduction step, as well as the exogenous gene and the nucleic acid containing the same, are the same as those described above. The exogenous gene introduced in the nucleic acid introduction step may be the same as or different from the exogenous gene already introduced into the T cells that constitute the T cell bank.

[0112] The nucleic acid introduction unit may comprise a basal medium, a culture vessel, and a sterile space, and may be configured to introduce a nucleic acid containing an exogenous gene into the prepared T cells. As used herein, the term "sterile space" refers to a sterile internal space defined by a clean chamber, clean bench, sterile room, or the like, and includes the sterile internal space defined by the entire nucleic acid introduction unit, as well as a sterile portion of the internal space of the nucleic acid introduction unit. The device, medium, and other components provided in the nucleic acid introduction unit are appropriately selected depending on the characteristics of the device, the nucleic acid introduction method, and the like. Furthermore, the nucleic acid introduction section may, as necessary, be equipped with one or more of the following: medium additives, clean bench, incubator, cell culture bioreactor, microscope, centrifuge, aspirator, nucleic acid for introduction, nucleic acid encoding a protein for genome editing, etc., pipette, tube, buffer (e.g., acetate buffer, phosphate buffer, citrate buffer, citrate phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, McIlvaine buffer, etc.), gene introduction aid, etc., and may further be equipped with additional reagents and devices depending on the type of nucleic acid introduction method.

[0113] Specific examples of the basal medium and medium additives provided in the nucleic acid introduction unit are as described above. Examples of the culture vessel include those commonly used for cell culture, such as petri dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, cell culture flasks, spinner flasks, tubes, trays, culture bags, and roller bottles. The material of these culture vessels is not particularly limited, and examples include glass, polyvinyl chloride, cellulose-based polymers such as ethyl cellulose and acetyl cellulose, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymer, poly(butadiene-styrene) copolymer, poly(butadiene-acrylonitrile) copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, polychloroprene, styrene resin, chlorosulfonated polyethylene, ethylene vinyl acetate, acrylic block copolymer, and other plastics.

[0114] For example, when nucleic acid introduction is performed by calcium phosphate co-precipitation, the nucleic acid introduction unit may include one or more of various reagents and solutions (e.g., CaCl2 and its solution, HCl and its solution, NaCl and its solution, MgCl2 and its solution, Na2HPO4 and its solution, KH2PO4 and its solution, NP-40 and its solution, HEPES and its solution, DMSO and its solution, etc.) as the other reagents and devices. For example, when electroporation is used, the nucleic acid introduction unit may include one or more of an electroporator, electrodes (e.g., platinum electrodes, cuvette electrodes, petri dish platinum plate electrodes, etc.), electroporation plates, chambers (e.g., electrode chambers, plate chambers, etc.), etc. For example, when using lipofection, the system may be equipped with one or more transfection reagents (e.g., Lipofectamine (Invitrogen), jetPEI (Polyplus-transfection), Xfect™ (Clontech TaKaRa Cellartis), GenomONE™ (Ishihara Sangyo Kaisha, Ltd.)), Trans IT (Mirus Bio LLC), RmesFect / RmesFect Stem (OZ BIOSCIENCES), etc.). For example, when nucleic acid transfer is performed using a viral vector, the nucleic acid transfer unit may be equipped with one or more of the various viral vectors listed above, packaging vector plasmids, packaging cells (e.g., Plat-E cells), complementation cell lines, biosafety level 2 laboratories, etc.

[0115] 1-5. Expansion culture The provision system of the present invention may include a step of expanding T cells prepared from a T cell master cell bank or T cells into which a nucleic acid containing an exogenous gene has been introduced (hereinafter also referred to as an "expansion step"). Hereinafter, the part configured to perform the expansion step may be referred to as an "expansion section."

[0116] As used herein, "expansion culture" refers to culturing a desired cell population for the purpose of expanding the cell population and increasing the cell number. The increase in cell number is achieved by the increase in cell number due to cell proliferation exceeding the decrease in cell number due to cell death, and does not necessarily mean that all cells in the cell population proliferate. The increase in cell number can be 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1,000-fold, 3,000-fold, 5,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, or 1,000,000-fold or more compared to before the start of expansion culture.

[0117] The expansion step can be performed by known methods, but from the viewpoint of cell proliferation efficiency, it is preferable to perform it by a method discovered by the present inventors, which includes a step of stimulating T cells with a CD30 agonist. Because culturing T cells in the presence of a CD30 agonist enables stimulation of T cells by the CD30 agonist, it is preferable to perform it by a method including a step of culturing T cells in the presence of a CD30 agonist. Therefore, in one embodiment of the present invention, the expansion step can include a step of culturing T cells in the presence of a CD30 agonist. Furthermore, when expansion is performed in the presence of a CD30 agonist, it is preferable that a CD3 / TCR complex agonist and fibronectin or a modified form thereof are also present in the culture medium. The CD3 / TCR complex agonist and CD30 agonist can stimulate the CD3 / TCR complex and CD30, respectively. Because the proliferation potential of T cells differentiated from iPS cells is lower than that of iPS cells, constructing a master cell bank and / or working cell bank at the T cell stage has made it difficult to supply T cells and T cell products in quantities sufficient to achieve the expected therapeutic effect when administered to a human or multiple humans. The present invention overcomes these difficulties. Furthermore, by using a method that includes a step of stimulating T cells with the above-mentioned CD30 agonist in the expansion culture step, it is possible to construct a master cell bank and / or working cell bank at the T cell stage and to more stably supply T cell products.

[0118] As used herein, the term "stimulation" means that a substance binds to various receptors and activates downstream signal pathways.

[0119] In the present invention, the medium used in the expansion step of T cells is not particularly limited, but a medium used for culturing animal cells can be prepared as the basal medium. If necessary, the basal medium may contain medium additives. Examples of the basal medium and medium additives include those listed in step (1) of section 1-1 above. The culture can be performed, for example, in a CO2 incubator under an atmosphere with a CO2 concentration of about 1 to about 10%, preferably about 2 to about 5%, at about 30 to about 40°C, preferably about 37°C. Those skilled in the art can appropriately determine the culture period while monitoring the number of T cells, etc. The number of days is not particularly limited as long as T cells can be obtained, but is, for example, at least 3 days, 5 days, 7 days, 10 days, 14 days, or 21 days or more, and preferably 7 to 15 days. Furthermore, 30 days or less is preferred, and 21 days or less is more preferred. The culture period is, for example, 3 to 30 days, 5 to 30 days, 7 to 30 days, 10 to 30 days, 14 to 30 days, 21 to 30 days, 3 to 21 days, 5 to 21 days, 7 to 21 days, 10 to 21 days, 14 to 21 days, 3 to 15 days, 5 to 15 days, 7 to 15 days, 10 to 15 days, 14 to 15 days, etc.

[0120] When vitamin C is used in the expansion culture step, the vitamin C may be the same as those described in step (2-1), and may be added in the same manner. In one embodiment, the concentration of vitamin C in the medium or culture solution is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added in an amount equivalent to 5 μg / ml to 500 μg / ml of the culture solution (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, or 500 μg / ml).

[0121] When a CD3 / TCR complex agonist is used in the expansion step, examples of the CD3 / TCR complex agonist used in this step include those described in step (3) of 1-1 above. The concentration of the CD3 / TCR complex agonist in the medium is also the same as the concentration described in step (3) of 1-1 above.

[0122] In the expansion culture step, it is preferable that fibronectin or a variant thereof is present in the medium. Such fibronectin is not particularly limited as long as it is a molecule capable of binding to T cells. The fibronectin variant is not particularly limited as long as it is a molecule capable of binding to VLA-5 and VLA-4 on the surface of T cells, and examples thereof include retronectin. Fibronectin or a variant thereof may be present in any form in the medium. For example, it may be contained in the medium during culture or may be immobilized on the culture vessel, but is preferably immobilized on the culture vessel.

[0123] When fibronectin or a variant thereof is contained in a medium, the medium may be the same as the medium containing a CD3 / TCR complex agonist. Furthermore, the presence or absence of serum, additives, etc. may also be the same as the medium containing a CD3 / TCR complex agonist. When fibronectin or a variant thereof is contained in a medium, the concentration of the fibronectin or variant thereof may be, at the lower limit, 10 ng / ml or more, preferably 100 ng / ml or more, and at the upper limit, 10,000 μg / ml or less, preferably 1,000 μg / ml or less.

[0124] In the expansion step, it is also preferable that a CD30 agonist be present in the medium. Such a CD30 agonist is not particularly limited as long as it is a molecule that can specifically bind to CD30 and thereby transmit a signal from CD30 into the cell. Examples of the CD30 agonist include at least one selected from the group consisting of an anti-CD30 agonist antibody (also simply referred to as "anti-CD30 antibody") or a binding fragment thereof, and a CD30 ligand or a binding fragment thereof.

[0125] As with the CD3 / TCR complex agonist, the CD30 agonist used in the expansion step may be present in any form as long as it is capable of contacting CD30 during culture. For example, it may be contained in the culture medium during culture or may be immobilized on a culture vessel, but is preferably contained in the culture medium.

[0126] When a CD30 agonist is contained in the medium, the medium may be the same as the medium containing a CD3 / TCR complex agonist. Furthermore, the presence or absence of serum, additives, and the like may be the same as the medium containing a CD3 / TCR complex agonist. When a CD30 agonist is contained in the medium, the concentration of the CD30 agonist in the medium may be appropriately determined by those skilled in the art depending on the CD30 agonist. For example, when the CD30 agonist is an anti-CD30 agonist antibody or a binding fragment thereof, the concentration of the anti-CD30 agonist antibody or a binding fragment thereof in the medium is usually 1 ng / ml to 10,000 ng / ml, and preferably 30 ng / ml to 300 ng / ml.

[0127] Furthermore, when a CD30 agonist is immobilized on a culture vessel, the culture vessel may be the same as the culture vessel on which the CD3 / TCR complex agonist is immobilized. Furthermore, the method for immobilizing the CD30 agonist on a culture vessel may be the same as the method for immobilizing the CD30 agonist on a culture vessel. The concentration of the CD30 agonist solution when immobilizing the CD30 agonist on a culture vessel may be, at the lower limit, 0.1 ng / ml or more, preferably 1 ng / ml or more, and at the upper limit, 10,000 ng / ml or less, preferably 1,000 ng / ml or less. Such concentrations are, for example, 0.1 to 10,000 ng / ml, 1 to 10,000 ng / ml, 0.1 to 1,000 ng / ml, 1 to 1,000 ng / ml, 30 ng / ml to 300 ng / ml, etc.

[0128] When cytokines are used in the expansion culture step, examples of the cytokines include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, etc., and these may be used alone or in combination with multiple types (preferably all types). When the cytokine is IL-2, its concentration in the medium may be 10 U / ml to 1000 U / ml or 0.01 ng / ml to 1,000,000 ng / ml. When the cytokine is IL-7, its concentration in the medium may be 0.1 ng / ml to 1,000 ng / ml, 1 ng / ml to 500 ng / ml, or 5 ng / ml to 100 ng / ml (e.g., 10 ng / ml). The concentration of IL-12 in the medium may be 0.1 ng / ml to 1000 ng / ml, 1 ng / ml to 500 ng / ml, or 5 ng / ml to 100 ng / ml (e.g., 50 ng / ml), the concentration of IL-15 in the medium may be 0.1 ng / ml to 1000 ng / ml, 1 ng / ml to 500 ng / ml, or 5 ng / ml to 100 ng / ml (e.g., 10 ng / ml), the concentration of IL-18 in the medium may be 0.1 ng / ml to 1000 ng / ml, 1 ng / ml to 500 ng / ml, or 5 ng / ml to 100 ng / ml (e.g., 50 ng / ml), and the concentration of IL-21 in the medium may be 0.1 ng / ml to 1000 ng / ml, 1 ng / ml to 500 ng / ml, or 5 It may be ng / ml to 100 ng / ml (for example, 20 ng / ml).

[0129] In the expansion step, the medium may further contain a TNF family cytokine as a cytokine. Examples of TNF family cytokines include TNF-α, TNF-β, lymphotoxin α, Fas ligand, TRAIL, TWEAK, TL1A, RANK ligand, OX40 ligand, APRIL, AITRL, BAFF, 4-1BBL, and CD40 ligand, with TL1A being preferred. When TL1A is used, its concentration in the medium may be 5 ng / ml to 500 ng / ml, preferably 10 ng / ml to 300 ng / ml, and more preferably 20 ng / ml to 200 ng / ml (e.g., 50 ng / ml).

[0130] Furthermore, in the expansion culture step, the medium may further contain an apoptosis inhibitor. Examples of apoptosis inhibitors include protease inhibitors, such as caspase inhibitors. A preferred caspase inhibitor is the Pan Caspase FMK inhibitor Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone) (hereinafter, sometimes referred to as "Z-VAD-FMK"). The concentration of the inhibitor in the medium may be 1 μM to 1000 μM, preferably 1 μM to 500 μM, more preferably 1 μM to 200 μM, and particularly preferably 1 μM to 50 μM (e.g., 10 μM).

[0131] In the present invention, the obtained T cells may be isolated and used, or they may be used as is (i.e., as a cell population that may contain other cell types). When isolating, the cells can be isolated using at least one molecule selected from the group consisting of αTCR, βTCR, and CD3 as an indicator, and methods well known to those skilled in the art can be used for the isolation method. Examples of isolation methods include, but are not limited to, isolation by flow cytometry or magnetic cell separation using antibodies to αTCR, βTCR, and CD3 (optionally bound to magnetic beads, etc.), and purification methods using an affinity column on which a desired antigen is immobilized. When used as is, the proportion of T cells in the cell population may be increased using methods well known to those skilled in the art, including, but not limited to, those described in Front. Immunol., 5:636 (2014), JP 2017-537625, and JP 2003-529363.

[0132] Therefore, the expansion culture section may comprise a basal medium, a culture vessel, and a sterile space, preferably also comprising a CD3 agonist, and may be configured to expand T cells. The device, culture medium, etc. provided in the nucleic acid introduction section are appropriately selected depending on the characteristics of the device, the expansion culture method, etc. When a CD3 agonist is used, the culture vessel and CD30 agonist provided in the expansion culture section may be in a form in which CD30 is immobilized on the culture vessel. Furthermore, the expansion culture section may comprise one or more of the following, as needed: a CD3 / TCR complex agonist, fibronectin or its modified form, an apoptosis inhibitor, a medium additive, a clean bench, an incubator, a cell culture bioreactor, a microscope, a centrifuge, an aspirator, a pipette, a tube, a buffer (e.g., acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, McIlvaine buffer, etc.), and may further comprise additional reagents and devices depending on the type of culture method. In particular, when cytokines are used as medium additives, suitable cytokines include, for example, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and TNF family cytokines.

[0133] Specific examples of the basal medium, medium additives, CD3 agonist, CD3 / TCR complex agonist, fibronectin or its modified form, and apoptosis inhibitor provided in the expansion culture section are as described above. Specific examples of the culture vessel include those described in 1-4. The expanded T cells are filled into an appropriate storage container (such as a sterile vial or a transfusion bag), and if necessary, the container is labeled and packaged to produce a T cell product.

[0134] 1-6. Manufacturing of frozen T cell products The provision system of the present invention may include a step of producing a frozen T cell product from the expanded T cells (hereinafter also referred to as a "frozen T cell product production step"). Hereinafter, the part configured to carry out the frozen T cell product production step may be referred to as a "frozen T cell product production unit."

[0135] The frozen T cell product production step can be carried out in the same manner as in 1-2.

[0136] Therefore, the frozen T cell product manufacturing unit may be configured to produce frozen stocks of expanded T cells, and may include at least a cryopreservative, a cryopreservation container, and a freezer and / or cryopreservation system (e.g., a locator, etc.). The frozen stock manufacturing unit may also include one or more low-temperature media (e.g., liquid nitrogen, etc.), pipettes, tubes, buffers (e.g., acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, McIlvaine's buffer, etc.), as needed.

[0137] Specific examples of cryopreservatives and freezers provided by the Frozen T Cell Product Manufacturing Department are as described above. Specific examples of cryopreservation containers include cryovials (e.g., glass ampoules, plastic tubes, etc.).

[0138] 1-7. Building a T cell product collection The provision system of the present invention may include a step of collecting T cell products and constructing a T cell product collection containing two or more types of T cell products (hereinafter also referred to as the "T cell product collection construction step"). Hereinafter, the part configured to execute the T cell product collection construction step may be referred to as the "T cell product collection construction unit."

[0139] The T cell products constituting the T cell product collection may be frozen T cell products, and from the viewpoint of T cell stability, frozen T cell products are preferable for long-term storage.

[0140] The T cell product collection construction step can also be performed by known methods. For example, two or more types of T cell products can be produced by the above-described method, and the T cell products can be stored with labels or other distinguishable labels. The types of collection can be expanded by adding different types of T cell products to the constructed T cell product collection. For ease of storage, it is preferable to store the products in the same space (e.g., in the same freezer, in the same room, in the same building, on the same site, etc.), but they can also be stored in physically separate spaces. Any method that allows for distinguishing the types of T cell products can be used to distinguish the types. For example, the types can be distinguished by attaching labels or other distinguishing labels to the containers, or, if the products are stored in separate freezers or cryopreservation systems, by attaching labels or other distinguishing labels to the freezers or cryopreservation systems, or by using management software or other distinguishing methods.

[0141] Therefore, the T cell product collection construction unit may be configured to include at least the T cell products to be collected and to collect the T cell products constructed as described above to construct a T cell product collection containing two or more types of T cell products. Furthermore, the T cell product collection construction unit may include one or more of the following, as needed: a label, a freezer, a cryopreservation system, and management software. Specific examples of freezers are as described above.

[0142] 1-8. Antigen Identification The delivery system of the present invention may include a step of identifying a tumor-specific antigen or tumor-associated antigen (hereinafter simply referred to as "antigen") expressed in a tumor of a subject (hereinafter also referred to as an "antigen identification step"). Hereinafter, the part configured to perform the antigen identification step may be referred to as an "antigen identification unit."

[0143] The antigen identification step can also be performed by known methods. For example, a biological sample such as a tumor tissue fragment or a body fluid (e.g., blood, serum, plasma, etc.) that may contain tumor cells is collected from a subject, mRNA is extracted from the sample, cDNA is synthesized from the mRNA as needed, and the nucleic acid is used for identification using a nucleic acid amplification method and / or a nucleic acid detection method such as digital PCR (e.g., ddPCR), RT-PCR, a biochip (e.g., microarray), or RNAseq. PCR can be performed, for example, using a primer set capable of amplifying mRNA of a tumor-specific antigen or tumor-associated antigen and a PCR device. Furthermore, when RNAseq is used, it can be performed using a next-generation sequencer.

[0144] Therefore, the antigen identification unit may be configured to identify tumor-specific or tumor-associated antigens expressed in the tumor of a subject. For example, the antigen identification unit may include at least the primer set and a PCR device, the biochip, or the next-generation sequencer.

[0145] Examples of the next-generation sequencer include, but are not limited to, devices manufactured by Illumina (e.g., MiSeq, HiSeq2500), devices manufactured by Thermo Fisher Scientific (e.g., Ion Proton, Ion PGM), and devices manufactured by Roche Diagnostics (e.g., GS FLX+, GS Junior).

[0146] 1-9.Selection of T cell products The provision system of the present invention can acquire subject information and select an appropriate T cell product based on that information. Therefore, the provision system of the present invention may include a step of acquiring subject information and / or a step of selecting a T cell product appropriate for the subject. Hereinafter, the part configured to execute the subject information acquisition step may be referred to as the "subject information acquisition unit." The subject information is not particularly limited, and examples include the subject's genetic information (e.g., HLA gene-related information, abnormal gene information, etc.), diagnostic results, medical history, medication history, age, sex, blood type, height, weight, and, if the subject has a tumor, information on tumor-specific antigens or tumor-associated antigens expressed in the tumor.

[0147] In one embodiment of the present invention, the method may include a step of selecting a T cell product expressing a CAR or an exogenous TCR that recognizes and binds to an antigen identified from information about the subject from a T cell product collection containing T cell products (hereinafter also referred to as a "T cell product selection step"). Hereinafter, the part configured to perform the T cell product selection step may be referred to as a "T cell product selection unit." The T cell product in the T cell product selection step may be a frozen T cell product, and from the viewpoint of T cell stability, a frozen T cell product is preferable.

[0148] The step of selecting a T cell product expressing a CAR or exogenous TCR that recognizes and binds to the identified antigen from a T cell product collection containing T cell products is conceptually included in the step of selecting a T cell product appropriate for the subject based on the acquired subject information. The subject information acquisition unit may include an antigen identification unit. In one aspect of the present invention, the T cell product selection step can select a T cell bank containing T cells expressing an appropriate CAR, exogenous TCR, cytokine, chemokine, etc., based on, for example, the contents of the manufacturing plan for the T cell product to be provided.

[0149] The T cell product selection step can be performed by a known method. For example, based on the document (paper or electronic data medium) showing the identification results from the antigen identification step, a T cell product containing T cells expressing a CAR or exogenous TCR that recognizes and binds to the identified antigen can be selected from a pre-created list of T cell products. If the expression of multiple tumor-specific or tumor-associated antigens is confirmed in the antigen identification step, a T cell product containing T cells can be selected based on evaluation criteria such as the highest expression level or the T cell product that is expected to be most effective. Only one type of T cell product may be selected, or two or more types may be selected. These steps may also be performed using software designed for the above-mentioned T cell product selection. T cell products can be selected according to instructions (such as a prescription) from a doctor or medical institution.

[0150] Therefore, the T cell product selection unit may be configured to at least include a document showing the identification result from the antigen identification step, and to select a T cell product expressing a CAR or exogenous TCR that recognizes and binds to the identified antigen from a T cell product collection containing T cell products. Furthermore, the T cell product selection unit may include, as necessary, one or more of a list of T cell products, software designed for the above-mentioned T cell product selection, etc.

[0151] The T Cell Bank Construction Department, T Cell Bank Collection Construction Department (described below), T Cell Bank Selection Department (described below), Frozen T Cell Product Manufacturing Department, T Cell Product Collection Construction Department, and T Cell Product Selection Department manage information about the T cells that make up the T cell bank and T cell products (hereinafter referred to as "T cell-related information"). T cell-related information includes, for example, production records, characterization results, quality assessment results, storage temperature control records, and production plans for the T cells. Furthermore, if the T cells are iPS cell-derived T cells, the information also includes production records, characterization results, quality assessment results, storage temperature records, and specific genetic information for the iPS cells. Furthermore, if the T cells are T cells into which a nucleic acid encoding an exogenous gene has been introduced, the information also includes information about the exogenous gene (e.g., the type of protein, cytokine, etc. encoded by the exogenous gene). The T Cell Bank Selection Department and T Cell Product Selection Department select T cell banks and T cell products, respectively, based on the details of the production plans for the T cell products to be provided. The T cell bank selection unit and the T cell product selection unit may further select an appropriate T cell bank and T cell product, respectively, based on the subject information acquired by the subject information acquisition unit. Thus, the T cell bank selection unit and the T cell product selection unit can be used even when there is only one type of T cell bank and / or T cell product.

[0152] One embodiment of the present invention will be described with reference to Fig. 16. In this specification and drawings, components having substantially the same functional configurations are denoted by the same reference numerals, and redundant description will be omitted. 16, the T cell product provision system 100 prepares T cells from a T cell bank 102 to provide a T cell product 131. The T cell bank 102 may be constructed by a T cell bank construction unit 101. Furthermore, when two or more types of T cell banks are constructed, these may be collected by a T cell cell bank collection construction unit 111 to construct a T cell cell bank collection 112. A desired exogenous gene may be introduced into the prepared T cells in nucleic acid introduction unit 103, as needed. The T cells may then be expanded in expansion culture unit 104, as needed. The T cells may then be frozen in frozen T cell product production unit 105, as needed, to form frozen T cell product 106. When two or more types of frozen T cell products are produced, they may be collected by T cell product collection construction unit 121 to construct T cell product collection 122. Furthermore, the T cell cell bank selection unit 154 and the T cell product selection unit 155 can select appropriate T cell cell banks and T cell products as the T cell products 131 to be provided, respectively, based on the T cell-related information 171. Such T cell-related information is managed in the T cell cell bank construction unit 101, the T cell cell bank collection construction unit 111, the T cell cell bank selection unit 154, the T cell product collection construction unit 121, and the T cell product selection unit 155. Furthermore, to provide a T cell product more suitable for treatment and / or prevention of a subject, subject information 151 can be acquired by a subject information acquisition unit 152, and an appropriate T cell product 131 can be selected by a T cell product selection unit 155 based on the subject information. The T cell product 131 may be the same as the T cell product 106 if there is only one type of T cell product, or may be one or more types of T cell products included in the T cell product collection 122 if selected from the T cell product collection 122. The subject information acquisition unit may include an antigen identification unit 153. An appropriate T cell cell bank 161 may be selected by a T cell cell bank selection unit 154 based on the information acquired by the subject information acquisition unit 152. The T cell cell bank 161 may be the same as the T cell bank 102 if there is only one type of T cell bank, or may be one or more types of T cell banks included in the T cell cell bank collection 112 if selected from the T cell cell bank collection 112. T cells can be prepared from the T cell bank 161 and provided as described above.

[0153] Next, we will explain the hardware configuration that may be included in the subject information acquisition unit, T cell cell bank construction unit, T cell cell bank selection unit, and / or T cell product selection unit. Since the T cell cell bank selection unit and the T cell product selection unit have similar hardware configurations, we will explain the hardware configuration that may be included in the T cell product selection unit. Figure 17 is a diagram showing an example of a hardware configuration that may be included in the T cell product selection unit 200. As shown in Figure 17, the T cell product selection unit 200 has a processor 201, memory 202, auxiliary storage device 203, I / F (Interface) device 204, communication device 205, and drive device 206. The hardware components of the T cell product selection unit 200 are connected to each other via a bus 207. The processor 201 has various arithmetic devices such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), etc. The processor 201 reads out programs such as various software programs installed in the auxiliary storage device 203 onto the memory 202 and executes them. The memory 202 has a main storage device such as a ROM (Read Only Memory), a RAM (Random Access Memory), etc. The processor 201 and the memory 202 form a so-called computer, and the processor 201 executes programs such as various software loaded onto the memory 202, thereby enabling the computer to realize various functions. The auxiliary storage device 203 stores various software programs and various information (for example, T cell-related information, subject information, etc.) and data used when the processor 201 executes the various software programs. The I / F device 204 is a connection device that connects the operation device 210 and display device 211 with the T cell product selection unit 200. The I / F device 204 accepts various instructions for the T cell product selection unit 200 via the operation device 210. The I / F device 204 also outputs the processing results by the T cell product selection unit 200 via the display device 211. The communication device 205 is a communication device for communicating with other devices via a network. The drive device 206 is a device for loading a recording medium 212. The recording medium 212 here includes media that record information optically, electrically, or magnetically, such as a CD-ROM, a flexible disk, a magneto-optical disk, etc. The recording medium 212 may also include semiconductor memories that record information electrically, such as ROMs, flash memories, etc. The various software programs and the like to be installed in the auxiliary storage device 203 are installed, for example, by setting the distributed recording medium 212 in the drive device 206 and reading the various software programs and the like recorded on the recording medium 212 by the drive device 206. Alternatively, the various software programs and the like to be installed in the auxiliary storage device 203 may be installed by being downloaded from a network via the communication device 205.

[0154] 1-10. Uses of T cell products The T cell products provided by the delivery system of the present invention can exhibit cytotoxic activity against, for example, cancer cells, cancer stem cells, tumor cells, etc., and can therefore be used to prevent or treat cancer or tumors, and can be administered to subjects. As used herein, the term "subject" refers to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans) that receive T cell products in clinical trials, etc., with humans being the preferred subject.

[0155] Cancers and tumors that can be prevented or treated by the above-mentioned T cell products are described, for example, in "Daniel Baumhoer et al., Am J. Clin Pathol, 2008, 129, 899-906." Specific examples include liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., ovarian clear cell adenocarcinoma), childhood cancer, lung cancer (e.g., squamous cell carcinoma, small cell lung carcinoma), testicular cancer (e.g., non-seminomatous germ cell tumor), soft tissue tumor (e.g., liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g., cervical intraepithelial neoplasia, cervical squamous cell carcinoma), melanoma, adrenal gland tumor (e.g., adrenal adenoma), neural tumor (e.g., schwannoma), gastric cancer (e.g., gastric adenocarcinoma), kidney cancer (e.g., Grawitz tumor), breast cancer (e.g., invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g., medullary carcinoma), laryngeal cancer (e.g., squamous cell carcinoma), and bladder cancer (e.g., invasive transitional cell carcinoma).

[0156] The T cells contained in the T cell product may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before administration to a subject. Stimulatory molecules include, but are not limited to, cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, with IL-2 being preferred. The concentration of IL-2 in the medium is not particularly limited, but is preferably 0.01 to 1 × 10. 5 U / mL, more preferably 1 to 1 × 10 4 U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, anti-CD3 antibody, anti-CD30 antibody, and anti-IL-4 antibody. Other lymphocyte-stimulating factors, such as lectin, can also be added. Furthermore, serum or plasma may be added to the medium. The amount of these to be added to the medium is not particularly limited, but examples include 0% to 20% by volume. The amount of serum or plasma used can be varied depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources, but from the viewpoint of safety, autologous sources are preferred.

[0157] The T cells are preferably administered parenterally to a subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but typically, the number of cells is 1 x 10 per administration for a subject weighing 60 kg. 6 ~1×10 10 Preferably 1 x 10 7 ~1×10 9 5×10 7 ~5×10 8 The dose may be administered once or multiple times.

[0158] 2. Methods for constructing a T cell master cell bank and / or a T cell working cell bank The present invention also provides a method for constructing the above-mentioned T cell master cell bank and / or T cell working cell bank (hereinafter, these may be referred to as the "cell bank construction method of the present invention"). In one embodiment of the present invention, when the exogenous gene is a CAR gene, the cell bank construction method of the present invention comprises the steps of (I) differentiating induced pluripotent stem cells that do not have a CAR gene into T cells for CAR-T therapy, (II) stocking the differentiated T cells, and (III) characterizing the differentiated T cells. The above steps (II) and (III) are usually performed in this order, but step (II) may be performed after step (III). In another embodiment, when the exogenous gene is a TCR gene, the cell bank construction method of the present invention comprises the steps of (i) differentiating induced pluripotent stem cells that do not have an exogenous TCR gene into T cells for TCR-T therapy, (ii) stocking the differentiated T cells, and (iii) characterizing the differentiated T cells. The above steps (ii) and (iii) are usually performed in this order, but step (ii) may be performed after step (iii). The above steps may be performed by the same entity or different entities.

[0159] In another aspect of the present invention, there is provided a T cell master cell bank and / or a T cell working cell bank (hereinafter sometimes referred to as the "T cell cell bank of the present invention") constructed by the cell bank construction method of the present invention.

[0160] 2-1. Induction of T cell differentiation from induced pluripotent stem cells without CAR genes or exogenous TCR genes Steps (I) and (i) in 2. above can be performed by the method described in steps (1) and (2) in 1-1. above, using induced pluripotent stem cells that do not have a CAR gene and / or an exogenous TCR gene. "CAR-T therapy" in step (I) refers to therapeutic or prophylactic use using a CAR expressed in T cells, and excludes therapeutic or prophylactic use using an exogenous TCR or an endogenous TCR expressed in T cells. However, the T cells may contain exogenous TCR genes introduced for purposes other than therapeutic or prophylactic purposes, such as suppressing the expression of endogenous TCR genes and efficiently producing homogeneous T cells, as described in 1-1. above. Similarly, in step (i), "for TCR-T therapy" refers to therapeutic or prophylactic use using an exogenous TCR expressed in T cells, and excludes therapeutic or prophylactic use using a CAR expressed in T cells and an endogenous TCR. However, the T cells may contain an exogenous TCR gene introduced for purposes other than therapeutic or prophylactic purposes, such as suppressing the expression of an endogenous TCR gene, as described in 1-1 above.

[0161] The CAR and TCR in steps (I) and (i), specific examples of antigens targeted by the TCR and CAR, and definitions of terms such as T cells are as described in 1. and 1-1. above. Specific examples of T cells obtained by differentiation induction in the above steps are as described in 1. above. Such T cells are preferably cytotoxic T cells, more preferably CD8αβ-positive cytotoxic T cells that express CD8αβ.

[0162] 2-2. Stock of differentiated T cells Steps (III) and (iii) of 2. above can be carried out by the methods described in 1-1. and 1-2. above using the T cells obtained in 2-1. above.

[0163] The induced pluripotent stem cells used in the cell bank construction method of the present invention and the T cell bank of the present invention can be established by the method described in 1-1 above. Furthermore, the induced pluripotent stem cells or the T cells used in steps (II) and (ii) of 2 above are preferably those in which the expression of at least one HLA gene is suppressed, similar to the cells described in 1-1 above. Specific HLA genes and combinations, as well as the definition and method of suppressing the expression of the genes, are as described in 1-1 above.

[0164] 2-3. Characterization of T cells The test items for characterization in steps (III) and (iii) of 2 above will vary depending on the biological properties (e.g., auxotrophy), culture history, and feasibility of the cells to be banked, but will be appropriately selected by those skilled in the art or based on discussions between those skilled in the art and regulatory authorities. Information on the construction method and the results of characterization and quality assessment will be submitted to the drug authorities in each country's marketing authorization application. Characterization of T cell banks primarily involves assessing the absence of adventitious infectious agents. Contamination with adventitious viruses, in particular, can have serious consequences in clinical use, so thorough evaluations are performed in accordance with ICH Q5A(R1). Other tests include identity testing to detect cross-contamination with other cell lines. Karyotyping and tumorigenicity testing may also be performed. Test items for quality assessment include confirmation tests using appropriate cell phenotypes as T cells, purity tests, tests for impurities derived from the manufacturing process, and content tests such as cell count and cell viability.

[0165] 2-4. Construction of a T cell bank collection The provision system of the present invention may include a step (hereinafter also referred to as a "T cell cell bank collection construction step") of collecting a T cell master cell bank and / or a T cell working cell bank and constructing a T cell master cell bank collection and / or a T cell working cell bank collection (hereinafter also referred to as a "T cell cell bank collection") including two or more types of T cell master cell banks and / or T cell working cell banks. In this specification, a part configured to perform the T cell cell bank collection construction step may be referred to as a "T cell cell bank collection construction unit."

[0166] When there is a possibility that a T cell bank for providing one or more T cell products may be insufficient, or when it is deemed appropriate to use a T cell product from a different type of T cell bank depending on the characteristics of the patient to be administered, a T cell bank constructed separately can be added to an existing T cell bank. In this way, by collecting multiple T cell banks and constructing a T cell bank collection, a more stable and robust T cell product delivery system can be provided.

[0167] The T cell bank collection construction step can also be performed by known methods. For example, two or more types of T cell banks can be produced by the cell bank construction method of the present invention, and the T cell banks can be stored with labels or other distinguishable labels. The types of collections can be expanded by adding different types of T cell banks to the constructed T cell bank collection. For ease of storage, it is preferable to store the T cell banks in the same space (e.g., in the same freezer, in the same room, in the same building, on the same site, etc.), but they can also be stored in physically separate spaces. Any method that allows distinguishing between types of T cell banks can be used. For example, labels can be attached to cryopreservation containers, or, if cells are stored in separate freezers or cryopreservation systems, labels can be attached to the freezers or cryopreservation systems, or management software can be used to distinguish between the types.

[0168] Therefore, the T cell cell bank collection construction unit may be configured to include at least a T cell bank to be collected, collect the T cell bank constructed as described above, and construct a T cell cell bank collection including two or more types of T cell banks. Furthermore, the T cell cell bank collection construction unit may include one or more of a label, a freezer, a cryopreservation system, management software, etc., as needed. Specific examples of freezers are as described above. The construction of the T cell cell bank collection may be performed by the same entity as the entity constructing the T cell bank, or by a different entity. The T cell cell bank collection may include T cell banks constructed by different entities.

[0169] 2-4-1. Selection of T cell bank The provision system of the present invention may include a step of selecting a T cell bank for producing the provided T cell product from a T cell bank collection containing T cell banks (hereinafter also referred to as a "T cell bank selection step"). In this specification, the part configured to perform the T cell bank selection step may be referred to as a "T cell bank selection unit." The T cell bank selection step can be performed by known methods, such as selecting a T cell bank containing T cells expressing an appropriate T cell receptor based on the details of the manufacturing plan for the T cell product to be provided. In one embodiment of the present invention, information about a subject can be acquired and an appropriate T cell bank can be selected based on the acquired information. Therefore, the provision system of the present invention may include a step of acquiring information about the subject (hereinafter also referred to as a "subject information acquisition step") and / or a step of selecting a T cell bank appropriate for the subject. Hereinafter, the part configured to execute the step of acquiring subject information may be referred to as a "subject information acquisition unit." The subject information is not particularly limited, and examples include the subject's genetic information (e.g., HLA gene-related information, abnormal gene information, etc.), diagnostic results, medical history, medication history, age, sex, blood type, height, weight, and, if the subject has a tumor, information on tumor-specific antigens or tumor-associated antigens expressed in the tumor.

[0170] 3. Methods for producing T cell products In another aspect, the present invention provides a method for producing a T cell product (hereinafter, sometimes referred to as the "method for producing a T cell product of the present invention"), which comprises the steps of: (A) preparing T cells from the T cell bank of the present invention; (B) introducing a CAR gene or an exogenous TCR gene into the prepared T cells; and (C) expanding the T cells into which the CAR gene or exogenous TCR gene has been introduced. The method for producing a T cell product of the present invention may further comprise the step of (D) freezing the expanded T cells. Also provided is a T cell product (hereinafter, sometimes referred to as the "T cell product of the present invention") produced by the method for producing a T cell product of the present invention. Each of the above steps may be performed by the same or different entities.

[0171] 3-1.Preparation of T cells Step (A) of 3 above can be carried out by the method described in 1-3 above using the T cell bank of the present invention.

[0172] 3-2. Nucleic acid introduction Step (B) in 3. above can be performed by the method described in 1-4. above using T cells prepared by the method described in 3-1. above. Furthermore, it is preferable that the T cells used in step (B) express IL-15 / IL-15Rα. The types of nucleic acids, explanations of CAR, TCR, and IL-15 / IL-15Rα, definitions of each term, and specific examples of antigens targeted by TCR and CAR are as described in 1-1. above. Thus, in one aspect, the T cells used in the method for producing a frozen stock of the present invention or the T cells contained in the frozen T cell stock of the present invention can be characterized in that the CAR or exogenous TCR recognizes and binds to the tumor-specific antigen or tumor-associated antigen described in 1-1. above.

[0173] 3-3. Expansion culture Step (C) in 3. above can be carried out by the method described in 1-5. above using T cells into which nucleic acid has been introduced by the method described in 3-2. above. The method for expansion, specific examples of compounds used in expansion, the concentrations of the compounds in the medium, and definitions of each term are as described in 1-5. above. Thus, in one embodiment, the method for producing a frozen stock of the present invention can include a step of culturing the T cells in the presence of a CD30 agonist.

[0174] 3-4.Freezing of T cells Step (D) of 3 above can be carried out by the method described in 1-2 above using T cells expanded by the method described in 3-3 above.

[0175] 4. How to build a T cell product collection Furthermore, in another aspect, the present invention provides a method for constructing a T cell product collection comprising two or more types of T cell products (hereinafter, this may be referred to as the "T cell product collection construction method of the present invention"), which may comprise the step of collecting the T cell products of the present invention. Also provided is a T cell product collection constructed by the T cell product collection construction method of the present invention (hereinafter, this may be referred to as the "T cell product collection of the present invention"). The T cell products constituting the T cell product collection of the present invention may be frozen T cell products, and frozen T cell products are preferred from the viewpoint of T cell stability.

[0176] The step of collecting T cell products of the present invention can be carried out using the T cell products of the present invention by the methods described in 1-7 above. The T cell products included in the T cell product collection may be produced by the same entity as the entity constructing the T cell product collection, or by a different entity. Thus, the T cell product collection may include T cell products produced by different entities.

[0177] 5. Methods for manufacturing T cell products The present invention also provides a method for producing a T cell product (hereinafter sometimes referred to as the "method for producing a T cell product of the present invention"), which may include the step of constructing a T cell master cell bank and / or a T cell working cell bank.

[0178] Specific examples and definitions of terms such as T cells, T cell products, master cell banks, and working cell banks are as described in 1-1 above. The pluripotent stem cells, T cells, T cell products, master cell banks, and working cell banks can be produced or constructed, for example, by the methods described in 1 and 2 above. The method for producing a T cell product of the present invention may include (a) differentiating pluripotent stem cells into T cells, (b) stocking the differentiated T cells, (c) characterizing the differentiated T cells, (d) preparing T cells from the stock of cells, (e) introducing a nucleic acid containing an exogenous gene into T cells, and / or (f) expanding and culturing the T cells. The T cells used in the method for producing a T cell product of the present invention may be T cells in which the expression of at least one HLA gene is suppressed. Specific HLA genes and combinations, as well as the definition and method of suppressing the expression of the genes, are as described in 1-1 above. Each of the above steps may be performed by the same or different entities.

[0179] Step (a) of 5. above can be carried out by the method described in steps (1) and (2) of 1-1. above using the pluripotent stem cells described in 1-1. above. The pluripotent stem cells are preferably induced pluripotent stem cells, more preferably human induced pluripotent stem cells. The induced pluripotent stem cells used in the cell bank construction method of the present invention and the T cell cell bank of the present invention can be established by the method described in 1-1. above. Step (b) of 5. above can be carried out by the methods described in 1-1. and 1-2. above using T cells differentiated in step (1). Step (c) of 5. above can be carried out by the method described in 2-2. above using T cells differentiated in step (1). Step (d) above can be carried out by the method described in 1-3. above using the cell stock produced in step (c).

[0180] Step (e) in 5. above can be performed by the method described in 1-4. above using T cells differentiated in step (a), cells stocked in step (b), T cells characterized in step (c), or T cells prepared in step (d). The T cells used in step (e) preferably express IL-15 / IL-15Rα. The types of nucleic acids, explanations of CAR, TCR, and IL-15 / IL-15Rα, definitions of each term, and specific examples of antigens targeted by TCR and CAR are as described in 1-1. Thus, in one aspect, the T cells used in the method for producing a frozen stock of the present invention or the T cells contained in the frozen stock of T cells of the present invention can be characterized in that the CAR or exogenous TCR recognizes and binds to the tumor-specific antigen or tumor-associated antigen described in 1-1.

[0181] Step (f) in 5. above can be carried out by the method described in 1-5. above using T cells into which nucleic acid has been introduced in step (e). The expansion method, specific examples of compounds used in expansion, the concentrations of the compounds in the medium, and definitions of each term are as described in 1-5. above. Thus, in one embodiment, the method for producing a T cell product of the present invention can include a step of stimulating the T cells by culturing the T cells in the presence of a CD30 agonist.

[0182] 6. How T cell products are provided Furthermore, the present invention provides a method for providing a T cell product suitable for a subject (hereinafter, sometimes referred to as the "providing method of the present invention"). To provide a T cell product suitable for a subject, information about the subject can be acquired, and an appropriate T cell bank and / or appropriate T cell product can be selected based on the acquired information. Thus, the providing method of the present invention comprises (p) acquiring information about the subject, and (q) selecting an appropriate T cell bank and / or appropriate T cell product based on the acquired information about the subject. Using the selected T cell bank, a T cell product can be produced and provided to the subject by performing step (e) and / or step (f) in 5 above. Furthermore, when a subject has a tumor, the providing method of the present invention can acquire information about tumor-specific or tumor-associated antigens expressed in the tumor, and select an appropriate T cell product based on the information. Thus, the providing method of the present invention comprises (x) identifying tumor-specific or tumor-associated antigens expressed in the subject's tumor, and (y) selecting a T cell product from the T cell product collection of the present invention that expresses a CAR or exogenous TCR that recognizes and binds to the identified antigen. (x) identifying a tumor-specific or tumor-associated antigen expressed in a subject's tumor and (y) selecting a T cell product expressing a CAR or exogenous TCR that recognizes and binds to the identified antigen from the T cell product collection of the present invention are conceptually encompassed within (p) acquiring subject information and (q) selecting an appropriate T cell product based on the acquired subject information. The terms T cell, T cell product, subject, and other terms and their definitions are as described above in 1-1 to 1-10. Each of the above steps may be performed by the same or different entities.

[0183] Step (x) in 6 above can be carried out using a biological sample derived from a subject by the method described in 1-8 above. Specific examples of the biological sample derived from a subject are as described in 1-8 above.

[0184] Step (y) in 6 above can be performed by the method described in 1-9 above, based on the identification results of step (x). Explanations and definitions of CAR and TCR, specific examples of tumor-specific antigens and tumor-associated antigens, specific examples of evaluation criteria, etc. are as described in 1-1 and 1-9 above.

[0185] Specific examples of cancers and tumors that can be prevented or treated by the T cell product provided by the method of the present invention are as described above in 1-10. In addition, the method for culturing and / or stimulating the T cells contained in the T cell product, the route of administration, the dosage, the type of subject, etc. are also as described above in 1-1-10.

[0186] The present invention will be explained in more detail in the following examples, but the scope of the present invention is not limited to these examples. [Example]

[0187] [Example 1] 1. iPS Cell Preparation The iPS cells used were Ff-I01s04 iPS cells (derived from peripheral blood mononuclear cells of a healthy individual) provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University. iPS cell culture was performed according to the protocol "Feeder-free culture of human iPS cells" provided by CiRA.

[0188] 2.Transfer of T cell receptor (TCR) genes into iPS cells 2-1.Transfection of WT1-TCR gene A gene (WT1-TCR) consisting of the TRB and TRA genes encoding the HLA-A*24:02-restricted WT1-specific TCR derived from TAK1, provided by Professor Masaki Yasukawa of the Ehime University Graduate School of Medicine, linked by a P2A sequence, was introduced into iPS cells. The gene was introduced into iPS cells by incorporating it into the CS-UbC-RfA-IRES2-hKO1 lentiviral vector provided by RIKEN and infecting the iPS cells. Hereinafter, iPS cells transfected with the WT1-TCR gene will be referred to as "WT1αβ-iPSCs."

[0189] 2-2.Transduction of Vγ9Vδ2-TCR gene The Vγ9Vδ2 T cell receptor (Vγ9Vδ2TCR G115) was derived from the G115 γδ T cell clone and encoded by the TRG and TRD genes linked by a P2A sequence. Vγ9Vδ2TCR G115 is an artificially synthesized oligonucleotide that encodes a polypeptide (SEQ ID NO: 1) designed to be arranged in the order shown in Table 1 from the N-terminus.

[0190] [Table 1]

[0191] A lentiviral vector was constructed using pLVSIN-Ub, which was derived by removing the neomycin resistance gene from pLVSIN-CMV Neo (Clontech) and replacing the CMV promoter with a human ubiquitin promoter. The artificial oligonucleotide encoding the Vγ9Vδ2 TCR G115 synthesized above was inserted into the multicloning site of the pLVSIN-Ub lentiviral vector. This plasmid was then used with Clontech's Lenti-X TM 293T cell line and Lenti-X TM A lentiviral vector was produced using Packaging Single Shots (VSV-G). The iPS cells prepared in Example 1, section 1, were infected with the produced lentiviral vector to introduce the Vγ9Vδ2-TCR gene into the iPS cells. Hereinafter, iPS cells into which the Vγ9Vδ2TCR G115 gene has been introduced may be referred to as "Vγ9Vδ2-iPSCs."

[0192] [Example 2] 1. Differentiation of iPS cells into hematopoietic progenitor cells (HPCs) For differentiation of iPS cells into hematopoietic progenitor cells (HPCs), a suspension cell population differentiated according to known methods (e.g., the methods described in Cell Reports 2 (2012) pp. 1722-1735 and WO 2017 / 221975) was used. Specifically, the iPS cells obtained in Example 1-1, Example 1-2-1, and Example 1-2-2, WT1αβ-iPSCs, and Vγ9Vδ2-iPSCs were plated at 3 x 10 in a 6-well plate treated with ultra-low adhesion. 5 Cells were seeded at 1000 cells / well and cultured in EB medium (StemPro34 supplemented with 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 2 mM L-glutamine, 45 mM α-monothioglycerol, and 50 μg / ml ascorbic acid 2-phosphate) containing 10 ng / ml BMP4, 50 ng / ml bFGF, 15 ng / ml VEGF, and 2 μM SB431542 under hypoxic conditions (5% O2) for 5 days. Subsequently, 50 ng / ml SCF, 30 ng / ml TPO, and 10 ng / ml Flt3L were added, and the culture was continued for an additional 5–9 days to obtain a suspension cell population. The medium was changed every 2–3 days during the culture period. The suspension cell population, including HPCs, was stained with the antibody set listed in Table 2. The cell population stained as described above was subjected to sorting by FACSAria.

[0193] [Table 2]

[0194] 2. Transduction of Vγ9Vδ2-TCR gene into HPCs A gene (Vγ9Vδ2TCR G115) in which the TRG gene and TRD gene encoding the Vγ9Vδ2 T cell receptor derived from the G115 γδ T cell clone were linked by a P2A sequence was introduced into the cell fraction obtained in 1. of [Example 2]. The gene introduction was carried out in the same manner as in 2-2. of [Example 1]. Hereinafter, HPCs into which the Vγ9Vδ2TCR G115 gene has been introduced may be referred to as "Vγ9Vδ2-iHPCs."

[0195] [Example 3] 1. Differentiation of HPCs into T cells The cell fraction obtained in [Example 2] 1. and the Vγ9Vδ2-iHPC obtained in [Example 2] 2. were differentiated into lymphoid cells according to known methods (e.g., the methods described in Journal of Leukocyte Biology 96 (2016) 1165-1175 and WO 2017 / 221975). Specifically, the hematopoietic progenitor cell population was seeded at 2000 cells / well onto a 48-well plate coated with recombinant h-DLL4 / Fc chimera (Sino Biological) and Retronectin (Takara Bio), and cultured at 5% CO2 and 37°C. The medium was changed every 2 or 3 days during the culture period. The culture medium was αMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 55 μM 2-mercaptoethanol, 50 μg / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 50 ng / ml SCF, 50 ng / ml IL-7, 50 ng / ml Flt3L, 100 ng / ml TPO, 15 μM SB203580, and 30 ng / ml SDF-1α. On days 7 and 14, the cells were passaged onto similarly coated 48-well plates. On day 21, all cells were harvested and confirmed by flow cytometry for the presence of CD45(+) and CD3(+) subpopulations. The obtained cells were seeded onto a 24-well plate and cultured under conditions of 5% CO2 and 37°C.The cells were cultured in αMEM medium containing 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 ng / ml streptomycin, 50 ng / ml ascorbic acid 2-phosphate, 10 μg / ml human insulin, 5.5 μg / ml human transferrin, 5 ng / ml sodium selenite, 500 ng / ml anti-CD3 antibody (UCHT1 or OKT3 clone), 10 nM dexamethasone (Fuji Pharma Co., Ltd.: 10171-H02H), 100 U / ml IL-2, and 10 ng / ml IL-7. All cells were harvested on day 41 of culture. Hereinafter, T cells differentiated from iPS cells obtained in 1. of Example 1 may be referred to as "iγδTC," T cells differentiated from WT1αβ-iPSCs obtained in 2-1. of Example 1 may be referred to as "iWT1αβTC," T cells differentiated from Vγ9Vδ2-iPSCs obtained in 2-2. of Example 1 may be referred to as "Vγ9Vδ2-iTC," and T cells differentiated from Vγ9Vδ2-iHPCs obtained in 2. of Example may be referred to as "Vγ9Vδ2-iHTC." iγδTC was stained using the antibody set in Table 3 (Figs. 3 and 4).

[0196] [Table 3]

[0197] For iγδTCs, the expression of CD3, VδTCR, αβTCR, TCR-Vδ1 chain, and TCR-Vδ2 chain on the cell membrane surface was measured using a flow cytometer (Figures 3 and 4).

[0198] For Vγ9Vδ2-iTCs (Fig. 5) and Vγ9Vδ2-iHTCs (Fig. 6), the expression of CD3, VδTCR, TCR-Vδ1 chain, and TCR-Vδ2 chain on the cell membrane surface was measured using a flow cytometer.

[0199] [Example 4] T cell expansion 1. Expansion of γδTCs 1-1. Expansion culture The iγδTC obtained in [Example 3] were suspended at 2,000,000 cells / mL in α-MEM medium containing 15% FBS and the cytokines listed in Table 4. The suspension was seeded onto plates coated with anti-CD3 antibody (UCHT1) and retronectin and cultured for 3 days at 5% CO₂ and 37°C. On day 3 of culture, the cells were harvested from the plates and counted using a NucleoCounter® NC-200 (ChemoMetec). The cells were then suspended in an appropriate volume of α-MEM medium containing 15% FBS and the cytokines listed in Table 5. The cells were then added to non-coated G-Rex® 6-well plates (WILSONWOLF) and cultured at 5% CO₂ and 37°C. Subsequently, 4 to 6 times on days 5, 6, 7, 8, 9, 10, 11, and 14 of culture, a portion of the cells was harvested from the plates and counted using a NucleoCounter® NC-200. Anti-CD3 antibody and Retronectin were immobilized on culture plates as follows: Anti-CD3 antibody (UCHT1, final concentration 3000 ng / mL) and Retronectin (final concentration 150 μg / mL) dissolved in PBS at the required concentrations were added to the plate and then left to stand overnight at 4°C.

[0200] 1-2.Growth rate The proliferation rate of iγδTC cells when the expansion culture in 1-1 of [Example 4] was repeated five times is shown in Figure 7. iγδTC cells were at least 10 11 These cells can be expanded more than twice in culture and can be used as T cells to form a T cell master cell bank and / or a T cell working cell bank.

[0201] [Table 4]

[0202] [Table 5]

[0203] 2. Expansion of Vγ9Vδ2-iTCs 2-1. Expansion culture Vγ9Vδ2-iTC obtained in [Example 3] were suspended at 2,000,000 cells / mL in α-MEM medium containing 15% FBS and the cytokines listed in Table 6. They were then seeded onto plates coated with anti-CD3 antibody (OKT3) and retronectin and cultured for 3 days at 5% CO₂ / 37°C. On day 3 of culture, cells were harvested from the plates and counted using a NucleoCounter® NC-200 (ChemoMetec). The cells were then suspended in an appropriate volume in α-MEM medium containing 15% FBS and the cytokines listed in Table 5. The cells were then added to non-coated G-Rex® 6-well plates (WILSONWOLF) and cultured at 5% CO₂ / 37°C. Subsequently, 4 to 6 times on days 5, 6, 7, 8, 9, 10, 11, and 14 of culture, some cells were harvested from the plates and counted using a NucleoCounter® NC-200. Immobilization of anti-CD3 antibody and retronectin on the culture plate was carried out by the method described in 1-1 of [Example 4].

[0204] 2-2.Growth rate The proliferation rate of Vγ9Vδ2-iTC cells when step 2-1 of [Example 4] was repeated five times is shown in Figure 8. Vγ9Vδ2-iTC cells were at least 10 12 These cells can be expanded more than twice in culture and can be used as T cells to form a T cell master cell bank and / or a T cell working cell bank.

[0205] [Table 6]

[0206] 3. Expansion of WT1αβ-iTCs 3-1. Expansion culture The iWT1αβTC obtained in Example 3 was suspended at 2,000,000 cells / mL in α-MEM medium containing 15% FBS and the cytokines listed in Table 6. The suspension was seeded onto plates coated with anti-CD3 antibody (OKT3) and retronectin and cultured for 3 days at 5% CO₂ and 37°C. On day 3 of culture, the cells were harvested from the plates and counted using a NucleoCounter® NC-200 (ChemoMetec). The cells were then suspended in an appropriate volume in α-MEM medium containing 15% FBS and the cytokines listed in Table 5, added to non-coated G-Rex® 6-well plates (WILSONWOLF), and cultured at 5% CO₂ and 37°C. Subsequently, aliquots of cells were harvested from the plates 4 to 6 times on days 5, 6, 7, 8, 9, 10, 11, 14, and 17 of culture, and cell counts were counted using a NucleoCounter® NC-200. Anti-CD3 antibody and Retronectin were immobilized on culture plates as follows: Anti-CD3 antibody (OKT3, final concentration 3000 ng / mL) and Retronectin (final concentration 150 μg / mL) dissolved in PBS at the required concentrations were added to the plates and then left to stand overnight at 4°C.

[0207] 3-2.Growth rate The proliferation rate of iWT1αβTC cells after repeating the expansion culture in 3-1 of [Example 4] four times is shown in Figure 9. iWT1αβTC cells were at least 10 10 These cells can be expanded more than twice in culture and can be used as T cells to form a T cell master cell bank and / or a T cell working cell bank.

[0208] [Example 5] 1. Construction of a T cell master cell bank The T cell culture medium obtained in Example 4 is replaced with a cryopreservation medium (CryoStor CS10) containing a cryoprotectant, and dispensed into multiple storage containers (sterile vials (polymer vials (AT-closed vials))). The containers containing the T cells are placed in a freezer to freeze the T cells. A programmable freezer, CryoMed (Thermo Fisher), is used as the freezer. A master cell bank of frozen T cells is constructed by stockpiling these.

[0209] 2. Construction of a T cell working cell bank A container containing T cells is collected from the frozen T cell master cell bank described in Example 5, step 1. The container is immersed in a water bath at approximately 37°C for approximately 15 seconds to thaw the T cells. The thawed T cells are suspended in an appropriate amount of medium containing the cytokines listed in Table 5, added to a 6-well plate, and cultured at 5% CO2 and 37°C. After culturing for one to three days, the T cells are expanded using the method described in Example 4, and then frozen and stored in the same manner as above to construct a T cell working cell bank.

[0210] 3. Characterization and Quality Assessment As in Example 5, 2, one container is collected from the T cell master cell bank and / or the T cell working cell bank, and the T cells are thawed. The resulting T cells are subjected to characterization and quality assessment.

[0211] 4. Construction of a T Cell Bank Collection A master cell bank collection of multiple types of T cells obtained in 1. of [Example 5] is constructed by collecting the master cell banks and storing them with labels or the like to distinguish between the types of T cell master cell bank.

[0212] [Example 6] 1. T Cell Preparation As in Example 5, section 2, one container was collected from the T cell master cell bank and / or T cell working cell bank, and T cells were thawed. The thawed T cells were suspended in an appropriate volume in medium supplemented with the cytokines listed in Table 5, added to a 6-well plate, and cultured at 5% CO₂ / 37°C. After culturing for 1 to 3 days, the cells were suspended at 2,000,000 cells / mL in α-MEM medium supplemented with 15% FBS and the cytokines listed in Table 4 or Table 6. The cells were then seeded onto plates coated with anti-CD3 antibodies (UCHT1 or OKT3) and retronectin and cultured for 3 days at 5% CO₂ / 37°C. On the third day of culture, the cells were recovered from the plate, counted using a NucleoCounter® NC-200 (ChemoMetec), and then suspended in an appropriate volume in α-MEM medium supplemented with 15% FBS and the cytokines listed in Table 5. The cells were then transferred to a non-coated cell culture flask (25 cm). 2 or 75 cm 2 ) and cultured at 5% CO2 / 37°C for 1 day to prepare T cells.

[0213] 2.Transfer of nucleic acids into T cells 2-1. Anti-CD19 CAR gene transduction As a gene encoding anti-CD19-CAR, an oligo DNA encoding a polypeptide (SEQ ID NO: 2) designed to be arranged in the order shown in Table 7 from the N-terminus was artificially synthesized.

[0214] [Table 7]

[0215] The artificial oligo DNA synthesized above was inserted into the multicloning site of the pMEI-5 retroviral vector. Unitech Corporation was commissioned to produce the viral vector. The retroviral vector carrying the gene encoding the anti-CD19-CAR thus produced was infected into the iWT1αβTC cells produced in Example 3 to produce iPS cell-derived anti-CD19-CAR T cells (hereinafter sometimes referred to as "CD19iWT1αβCARTC").

[0216] 2-2. Anti-BCMA CAR gene transduction As a gene encoding anti-BCMA-CAR, an oligo DNA encoding a polypeptide (SEQ ID NO: 3) designed to be arranged in the order shown in Table 8 from the N-terminus was artificially synthesized.

[0217] [Table 8]

[0218] The artificial oligo DNA synthesized above was inserted into the multicloning site of the pMEI-5 retroviral vector. Unitech Corporation was commissioned to produce the viral vector. The retroviral vector carrying the gene encoding the anti-BCMA-CAR thus produced was infected into the iWT1αβTC cells produced in Example 3 to produce iPS cell-derived anti-BCMA-CART cells (hereinafter sometimes referred to as "BMCAiWT1αβCARTC").

[0219] 2-3.Transfection of anti-CD19 CAR gene containing CD30-derived intracellular domain As an anti-CD19-CAR gene containing a CD30-derived intracellular domain, an oligoDNA encoding a polypeptide (sequence number 4) designed to be arranged in the order shown in Table 9 from the N-terminus was artificially synthesized.

[0220] [Table 9]

[0221] The artificial oligo DNA synthesized above was inserted into the multicloning site of the pMY retroviral vector. A viral vector was produced using FLYRD18 cells for producing retroviral vectors. WT1αβ-iTCs produced in Example 1, section 3, were infected with the produced retroviral vector carrying an anti-CD19-CAR gene containing a CD30-derived intracellular domain to produce iPS cell-derived anti-CD19-CART cells containing a CD30-derived intracellular domain (hereinafter sometimes referred to as "CD19-CD30-iWT1αβCARTCs").

[0222] 2-4. Anti-CD19-CAR gene transduction As a gene encoding anti-CD19-CAR, an oligo DNA encoding a polypeptide (SEQ ID NO: 5) designed to be arranged in the order shown in Table 10 from the N-terminus was artificially synthesized.

[0223] [Table 10]

[0224] The artificial oligo DNA synthesized above was inserted into the multicloning site of the pMY retroviral vector. A viral vector was produced using FLYRD18 cells for producing retroviral vectors. The retroviral vector carrying the gene encoding the anti-CD19-CAR thus produced was infected into the iγδTCs produced in Example 3 to produce iPS cell-derived anti-CD19-CART cells (hereinafter sometimes referred to as "CD19iγδCARTCs").

[0225] [Example 7] 1. Cytotoxic activity of T cells, etc. 1-1. Cytotoxic activity of CD19iWT1αβCARTC and BMCAiWT1αβCARTC The cytotoxic activity of CD19iWT1αβCARTC and BMCAiWT1αβCARTC obtained in 2-1 and 2-2 of [Example 6] against CD19-positive Raji cancer cells and BCMA-positive H929 cancer cells, respectively, was evaluated. CD19iWT1αβCARTC or iWT1αβTC was mixed with Raji cells at a ratio of 1:16, and the cytotoxic activity of CD19iWT1αβCARTC was evaluated based on target cell death after 2 hours. BCMAiWT1αβCARTC or iWT1αβTC was also mixed with H929 cells at a ratio of 1:16, and the cytotoxic activity of BCMAiWT1αβCARTC was evaluated based on target cell death after 2 hours. CD19iWT1αβCARTC and BMCAiWT1αβCARTC had cytotoxic activity against CD19-positive Raji cancer cells and BCMA-positive H929 cancer cells, respectively, demonstrating that two types of CARTs can be produced from the same iWT1αβTC (Figure 10).

[0226] 1-2.Cytotoxic activity of CD19-CD30-iWT1αβCARTC The cytotoxic activity of CD19-CD30-iWT1αβCARTC obtained in 2-3 of [Example 6] against CD19-positive Raji cancer cells was evaluated. CD19-CD30-iWT1αβCARTC was mixed with Raji cells at a ratio of 0.5, 1, 2, 4, 8, or 16 times, and the cytotoxic activity of CD19-CD30-iWT1αβCARTC was evaluated based on target cell death after 2 hours. CD19-CD30-iWT1αβCARTC had cytotoxic activity against CD19-positive Raji cancer cells, demonstrating that different CARs can be produced from the same iWT1αβTC (Figure 11).

[0227] 1-3.Cytotoxic activity of CD19iγδCARTC The cytotoxic activity of CD19iγδCARTC obtained in 2-4 of [Example 6] was evaluated. CD19iγδCARTC was mixed with CD19-positive Raji cells and CD19-negative CCRF-CEN cells at a ratio of 0.5, 1, 2, 4, 8, or 16 times the target cells, and the cytotoxic activity of CD19iγδCARTC was evaluated based on target cell death after 2 hours. CD19iγδCARTC had cytotoxic activity against CD19-positive Raji cells, but not against CD19-negative CCRF-CEN cells, demonstrating antigen-specific cytotoxic activity (Figure 12).

[0228] [Example 8] 1.Transfection of anti-CD19-CAR gene and IL-15Rα / IL-15 gene As the IL-15Rα / IL-15 gene, an oligoDNA encoding a polypeptide (SEQ ID NO: 7) designed to be arranged in the order shown in Table 11 from the N-terminus was artificially synthesized.

[0229] [Table 11]

[0230] The artificial oligo DNA synthesized above was inserted into the multicloning site of the pMY retroviral vector. Viral vectors were produced using FLYRD18 cells for producing retroviral vectors. The retroviral vector carrying the IL-15Rα / IL-15 gene thus produced and the retroviral vector carrying the anti-CD19-CAR gene produced in 2-4 of Example 6 were infected into iγδTC and Vγ9Vδ2-iTC produced in Example 3 to produce CD19 / IL15iγδCARTC and CD19 / IL15iVγ9Vδ2CARTC, respectively.

[0231] Example 9: 1. T cell products 1-1. Expansion of T cells (CD19 / IL15iVγ9Vδ2CARTC) The CD19 / IL15iVγ9Vδ2CARTC obtained in Example 8 was suspended at 2,000,000 cells / mL in α-MEM medium containing 15% FBS and the cytokines listed in Table 6. The cells were then seeded onto plates coated with anti-CD3 antibody (UCHT1) and retronectin and cultured for 3 days at 5% CO₂ and 37°C. On day 3 of culture, the cells were harvested from the plates and counted using a NucleoCounter® NC-200 (ChemoMetec). The cells were then suspended in an appropriate volume in α-MEM medium containing 15% FBS and the cytokines listed in Table 5. The cells were then added to non-coated G-Rex® 6-well plates (WILSONWOLF) and cultured at 5% CO₂ and 37°C. Subsequently, 4 to 6 times on days 5, 6, 7, 8, 9, 10, 11, 14, and 17 of culture, a portion of the cells was harvested from the plates and counted using a hemocytometer. Anti-CD3 antibody and Retronectin were immobilized on culture plates as follows: Anti-CD3 antibody (UCHT1, final concentration 3000 ng / mL) and Retronectin (final concentration 150 μg / mL) dissolved in PBS at the required concentrations were added to the plate and then left to stand overnight at 4°C.

[0232] 1-2.Growth rate The proliferation rate of CD19 / IL15iVγ9Vδ2CARTC cells after repeating the expansion culture in 1-1 of [Example 9] three times is shown in Figure 13. CD19 / IL15iVγ9Vδ2CARTC cells were at least 10 9 It was possible to expand the culture to more than double its size.

[0233] In this way, CAR-T cells can be expanded from the T cell master cell bank and / or the T cell working cell bank to mass-produce and provide T cell products (e.g., CAR-T cell products). The expanded T cells of various types are filled into appropriate storage containers (such as sterile vials or transfusion bags), labeled, and packaged to produce the T cell product.

[0234] 1-3.Freezing of T cell products The containers containing the various types of expanded T cells are frozen in the same manner as in 1. of [Example 5] to produce a frozen T cell product.

[0235] 1-4. Building a T cell product collection The frozen T cell products obtained above are labeled so that their types can be distinguished, and stored to build a T cell product collection.

[0236] [Example 10] 1. Antigen Identification Biological samples such as tumor tissue fragments or body fluids (e.g., blood, serum, plasma, etc.) that may contain tumor cells are collected from the subject, mRNA is extracted from the sample, cDNA is synthesized from the mRNA as needed, and the cDNA is used to identify the tumor using nucleic acid amplification methods and / or nucleic acid detection methods such as digital PCR (e.g., ddPCR), RT-PCR, biochips (e.g., microarrays), and RNAseq.

[0237] 2. T Cell Product Selection Based on the documents (paper or electronic data media) showing the identification results according to 1. of [Example 10], a T cell product containing T cells expressing a CAR or exogenous TCR that recognizes and binds to the identified antigen is selected from a list of T cell products prepared in advance.

[0238] [Example 11] 1. CD19 / IL15iγδCARTC extends survival 5x10 NOD / Shi-scid, IL-2RγKO (NOG) mice (Central Institute for Experimental Animals, female, 7-8 weeks old) 5 Nalm6 xenograft mice were generated by transplanting 5 × 10 Nalm6 cells (ATCC) into the tail vein. Four days after transplantation, CD19 / IL15iγδCARTC (5 × 10 6A suspension of 100 cells (1000 cells) in 0.1 mL of HBSS-buffer or an equivalent volume of HBSS-buffer was administered via the tail vein, and the survival time was then determined. All mice in the control group that received CD19-positive Nalm6 cancer cells via the tail vein died within 3 weeks, whereas all mice in the CD19 / IL15iγδCARTC-administered group survived for at least 6 weeks (Figure 14).

[0239] 2. Antitumor effect of CD19 / IL15iVγ9Vδ2CARTC 5x10 NOD / Shi-scid, IL-2RγKO (NOG) mice (Central Institute for Experimental Animals, female, 7-8 weeks old) 5 Luciferase-expressing Nalm6 cells (ATCC) were transplanted into the tail vein to generate luciferase-expressing Nalm6 xenograft mice. Four days after transplantation, the CD19 / IL15iVγ9Vδ2CARTC (10 7 A suspension of 100 cells (1000 cells) in 0.2 mL of HBSS-buffer or an equal volume of HBSS-buffer was administered via the tail vein, and the survival time was then determined. All mice in the control group that received CD19-positive Nalm6 cancer cells via the tail vein died within 3 weeks, whereas all mice in the CD19 / IL15iγδCARTC-administered group survived for at least 6 weeks (Figure 15). [Industrial Applicability]

[0240] The present invention provides a system for providing high-quality off-the-shelf allogeneic T cell products (particularly multiple types of CAR-T cell products) that can reduce human, time, and financial costs and have minimal lot-to-lot variation, and the T cell products provided in this manner are useful for the prevention or treatment of diseases such as cancer and tumors.

[0241] This application is based on patent application No. 2019-212718 filed in Japan (filing date: November 25, 2019), the contents of which are incorporated herein by reference in their entirety.

Claims

[Claim 1] A T cell product delivery system including a T cell master cell bank and / or a T cell working cell bank.

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