Method for producing antigen-specific t cells, and kit for production

Abstract

Provided is a method for producing antigen-specific T cells that efficiently proliferates antigen-specific T cells from peripheral blood mononuclear cells.　The method for producing antigen-specific T cells according to the present invention is characterized by comprising: a first stimulation step for culturing peripheral blood mononuclear cells in a medium containing an antigen added thereto; and a second stimulation step for further culturing the cells after the first stimulation step in a medium to which an antigen has been newly added; and satisfying condition A. (Condition A) The medium of the first stimulation step contain a first group of cytokines, the medium of the second stimulation step contains a second group of cytokines, the first group of cytokines is a combination of IL-7, IL-15, and IL-21 and does not contain IL-21, IL-18 or TL1A, and the second group of cytokines is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A.

Description

Method for producing antigen-specific T cells, and kit for production.

[0001] This invention relates to a method for producing antigen-specific T cells and a kit for producing them.

[0002] After hematopoietic stem cell transplantation, there is a possibility of developing treatment-resistant opportunistic infections, making the establishment of treatment methods for such infections crucial. These infections can involve various viruses, such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV). Therefore, immunotherapy using multiple virus-specific T cells (hereinafter also referred to as MVST cells) that target various viruses is being implemented (Patent Document 1, Non-Patent Documents 1 and 2).

[0003] MVST cells can be prepared, for example, by preparing an antigenic peptide for a target virus and culturing peripheral blood mononuclear cells (PBMCs) in the presence of the antigenic peptide. Conventionally, MVST cell preparation typically involves using PBMCs derived from hematopoietic stem cell transplant donors or related individuals, and administering the resulting MVST cells to the patient. However, this method has several drawbacks, including the time required from collecting peripheral blood containing PBMCs to preparing MVST cells, the need to prepare MVST cells for each patient, and the impossibility of preparing MVST cells if the donor is not infected with the target virus. Therefore, in recent years, a method has been proposed and put into practical use in which unrelated healthy individuals act as donors and MVST cells are prepared from donor PBMCs.

[0004] International Publication No. 2013 / 088148

[0005] Tzannou, I et. al. Journal of Clinical Oncology. 2017Leen, AM et. al. Blood. 2013

[0006] As mentioned above, if the PBMCs are from a healthy donor, even MVST cells prepared from the PBMCs of a single healthy donor can be used to treat multiple patients. Therefore, it is desirable to be able to prepare a large quantity of highly purified MVST cells that can be used to treat multiple patients from a single culture of donor PBMCs. This is not limited to MVST cells, but is also true for immunotherapy using antigen-specific T cells, for example.

[0007] Therefore, the present invention aims to provide a method for producing antigen-specific T cells that efficiently proliferate antigen-specific T cells from PBMCs with high purity.

[0008] The present invention provides a method for producing antigen-specific T cells, comprising a first stimulation step of culturing peripheral blood mononuclear cells in a medium to which an antigen has been added, and a second stimulation step of further culturing the cells after the first stimulation step in a medium to which a new antigen has been added, and characterized in that it satisfies condition A. (Condition A) The medium in the first stimulation step contains a first cytokine group, the medium in the second stimulation step contains a second cytokine group, the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not contain IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A.

[0009] The antigen-specific T cell production kit of the present invention comprises a first cytokine group and a second cytokine group, wherein the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not include IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A, and is characterized for use in the antigen-specific T cell production method of the present invention.

[0010] According to the present invention, antigen-specific T cells can be efficiently proliferated from peripheral blood mononuclear cells with high purity.

[0011] Figure 1 is a graph showing the change in the number of cells (cell proliferation) over time during PBMC culture in Example 1. Figure 2A is a graph showing the number of T cells and CD8 ６ T cells in 1×10 ＋ cells of the MVST sample in Example 1. The left graph shows the number of T cells, and the right graph shows the number of CD8 ＋ T cells. Figure 2B is a graph showing the frequency of virus-specific T cells and CD8 ６ T cells in 1×10 ＋ cells of the MVST sample in Example 1. The left graph shows the number of virus-specific T cells, and the right graph shows the number of virus-specific CD8 ＋ T cells. Figure 3A is a graph showing the number of cells specific to each peptide contained in the CMV-pp65 antigen in 1×10 ６ cells of the MVST sample in Example 1. The multiple patterns in the bars indicate the responses to different peptides. Figure 3B is a graph showing the number of peptides shown in Figure 3A in Example 1. Figure 4A is a graph showing the viable cell rate in Example 2. Figure 4B is a graph showing the proportion of CD8 ＋ T cells and IFNγ-positive virus-specific T cells in viable cells in which apoptosis has not been initiated, as well as the frequency of MT-1-positive CD8 ＋ T cells representing the mitochondrial membrane potential in CD8 ＋ T cells in Example 2. Figure 4C is a graph showing cell exhaustion in CD8 ＋ T cells and IFNγ-positive virus-specific T cells in Example 2. Figure 4D is a graph showing cell differentiation in CD8 ＋ T cells in Example 2. Figure 4E is a graph showing the IFNγ, TNF-α, IL-2 production capacity, and CD107a expression indicating cytotoxicity of virus-specific T cells in CD8 ＋ T cells in Example 2. Figure 4F is a graph showing CD8 ＋This graph shows the ability of virus-specific T cells to simultaneously secrete cytokines. Figure 5A is a graph showing the viability rate in Example 3. Figure 5B is a graph showing the frequency of virus-specific T cells in the MVST sample in Example 3. Figure 6A is a graph showing the change in cell number (cell proliferation) after secondary stimulation in Example 4. Figure 6B is a graph showing the CD8 ＋ This graph shows cell exhaustion in T cells. Figure 6C shows CD8 in Example 4. ＋ This is a graph showing the degree of cell differentiation in T cells. Figure 6D shows CD8 in Example 4. ＋ Figure 6E shows graphs illustrating the IFNγ, TNF-α, and IL-2 production capacity and cytotoxicity of CD107a in virus-specific T cells, as well as the frequency of MVSTs that secrete or express at least one of these cytokines and CD107a. ＋ This graph shows the ability of virus-specific T cells to simultaneously secrete cytokines. Figure 7A is a graph showing the change in cell number (cell proliferation) after secondary stimulation in Example 5. Figure 7B is a graph showing the viability rate, T cell frequency, and IFNγ-positive virus-specific T cell frequency for the MVST sample in Example 5. Figure 8A is a graph showing the change in cell number (cell proliferation) over time during PBMC culture in Example 6. Figure 8B is a graph showing the cell frequency of cells with various cell surface markers contained in the MVST sample in Example 6. Figure 8C is a graph showing the respective percentages of IFNγ-producing cells, TNFα-producing cells, and CD107a-producing cells contained in the MVST sample in Example 6. Figure 9 is a graph showing cytotoxic activity in Example 6.

[0012] The present invention includes the following embodiments: [1] A method for producing antigen-specific T cells, comprising a first stimulation step of culturing peripheral blood mononuclear cells in a medium to which an antigen has been added, and a second stimulation step of further culturing the cells after the first stimulation step in a medium to which a new antigen has been added, and satisfying condition A. (Condition A) The medium in the first stimulation step contains a first cytokine group, the medium in the second stimulation step contains a second cytokine group, the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not contain IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A. [2] The method for producing antigen-specific T cells according to [1], satisfying condition B. (Condition B) At least one of the medium in the first stimulation step and the medium in the second stimulation step contains arginine. [3] A method for manufacturing according to [1] or [2] that satisfies condition C. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed. [4] A method for manufacturing according to [3], wherein the antigen-presenting cells are γδT cells. [5] A method for manufacturing according to any one of [1] to [4] that satisfies condition D. (Condition D) In ​​the first stimulation step, Flt3L and a pattern recognition receptor ligand are added to the culture medium and culture is performed. [6] A method for manufacturing according to any one of [1] to [5] that satisfies condition B, condition C, and condition D. (Condition B) At least one of the culture medium in the first stimulation step and the culture medium in the second stimulation step contains arginine. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed. (Condition D) In ​​the first stimulation step, Flt3L and a pattern recognition receptor ligand are added to the culture medium and culture is performed. [7] A method for manufacturing according to any one of [1] to [6], wherein the antigen is a viral antigen. [8] The method for producing a cancer antigen according to any one of items [1] to [6]. [9] The method for producing a cancer antigen according to any one of items [1] to [8].

[10] The method for producing a cancer antigen according to any one of items [1] to [9].

[11] The method for producing a drug according to

[10] , wherein the antigen-specific T cells are multiple virus-specific T cells.

[12] The method for producing a drug according to any one of [1] to

[11] , wherein the antigen added to the culture medium in the first stimulation step and the culture medium in the second stimulation step are the same antigen.

[0013]

[13] A kit for producing antigen-specific T cells, comprising a first cytokine group and a second cytokine group, wherein the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not include IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A, for use in a method for producing antigen-specific T cells according to any one of [1] to

[12] .

[0014]

[14] 600 x 10 ６ A group of antigen-specific T cells containing cells or more, with more than 90% being CD8-positive / CD45RO-positive.

[15] 600 x 10 ６ The antigen-specific T cell group described in

[14] , which contains cells or more, in which more than 90% of the total are CD8-positive / CD45RO-positive, mitochondrial biosynthesis is maintained, and antigen-specific T cells are cytotoxic.

[16] The antigen-specific T cell group obtained by the manufacturing method described in any one of [1] to

[12] .

[0015] Unless otherwise specified, terms used herein may be used in the sense commonly used in the art.

[0016] In this specification, the term "cell" includes at least one cell unless otherwise specified, and can be used, for example, to refer to a single cell or a group of cells.

[0017] In this specification, the terms “cell population” and “cell group” include at least one type of cell, unless otherwise specified, and are not limited to one type of cell, but can be used to refer to two or more types of cells.

[0018] In this specification, the target of the specificity to be conferred to antigen-specific T cells is also referred to as the "target" or "target antigen," and the antigen added to the culture medium in the production of antigen-specific T cells is also referred to as the "added antigen."

[0019] [First Invention] <Method for Producing Antigen-Specific T Cells> The production method of the present invention can be used for the production of antigen-specific T cells, and the type of specificity of the antigen-specific T cells produced is not particularly limited. That is, the antigen-specific T cells can be any type, number, and combination of target antigens that are not particularly limited. Furthermore, in the production method of the present invention, the type, number, and combination of antigens to be added can be set according to the desired type of specificity. The method for producing antigen-specific T cells of the present invention is also called the culture method for antigen-specific T cells of the present invention.

[0020] The manufacturing method of the present invention will be described below with specific examples. The present invention is not limited to these examples. Furthermore, each embodiment can be described by reference unless otherwise specified.

[0021] (Embodiment 1) The method for producing antigen-specific T cells of this embodiment is characterized by comprising a first stimulation step of culturing peripheral blood mononuclear cells in a medium to which an antigen has been added, and a second stimulation step of further culturing the cells after the first stimulation step in a medium to which an antigen has been newly added, and satisfying condition A. Antigen-specific T cells are also referred to as VSTs below, for example. (Condition A) The medium in the first stimulation step contains a first cytokine group, the medium in the second stimulation step contains a second cytokine group, the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not contain IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A.

[0022] According to the manufacturing method of this embodiment, by performing PBMC culture in two stages, a first stimulation step and a second stimulation step, using the above-described combination of cytokines, T cells that exhibit specificity to the antigen (antigen-specific T cells) can be efficiently proliferated. That is, for example, the number of cells included in the cell population obtained by culture can be increased, and the proportion (frequency) of the antigen-specific T cells in the cell population can be increased (purified). Furthermore, under condition A, for example, specificity can be improved.

[0023] (1) PBMCs Peripheral blood mononuclear cells are also referred to as PBMCs below. PBMCs are mononuclear cells found in peripheral blood. PBMCs are mainly composed of blood cells such as lymphocytes, monocytes, and dendritic cells. Examples of lymphocytes include T cells, B cells, and NK (natural killer) cells.

[0024] The PBMC used in the first stimulation step may be, for example, peripheral blood containing PBMC collected from a living organism, or a mononuclear cell population (mononuclear cell fraction) containing mononuclear cells separated from peripheral blood, preferably the latter mononuclear cell population. The mononuclear cell population is preferably, for example, a fraction obtained by removing plasma components, red blood cells, platelets, and granulocytes from peripheral blood. The method for separating mononuclear cells from peripheral blood is not particularly limited, and examples include density gradient centrifugation, specific gravity centrifugation, flow cytometry, magnetic cell separation, etc., with density gradient centrifugation being the most common.

[0025] For the culture in the first stimulation step, for example, PBMCs collected from living organisms may be used as is, PBMCs stored under refrigeration may be used, or PBMCs stored under freezing may be used.

[0026] The origin of PBMCs is not particularly limited and may be human or non-human animals, for example. The origin of PBMCs can be selected depending on the target to which the antigen-specific T cells obtained by the manufacturing method of this embodiment will be used. Specifically, when used for human organisms (living bodies), human organs, human tissues, or human cells, it is preferable to use human-derived PBMCs.

[0027] (2) Medium The medium used in the first stimulation step (hereinafter also referred to as the first medium) contains the first cytokine group. The first cytokine group includes a combination of IL-7, IL-15, and IL-21, but does not include IL-12, IL-18, and TL1A.

[0028] The IL-7 concentration in the first culture medium is, for example, 1 to 50 ng / mL, and specifically, 5 to 15 ng / mL. The IL-15 concentration in the first culture medium is, for example, 1 to 50 ng / mL, and specifically, 5 to 15 ng / mL. The IL-21 concentration in the first culture medium is, for example, 1 to 50 ng / mL, and specifically, 10 to 20 ng / mL. The concentration in the culture medium is, for example, the final concentration at the start of culture (the same applies hereinafter). The content ratio (molar ratio) of IL-7, IL-15, and IL-21 in the first culture medium is not particularly limited.

[0029] As described above, the first culture medium does not contain IL-12, IL-18, and TL1A. In the present invention, the absence of IL-12, IL-18, and TL1A in the first culture medium means, for example, in a broad sense, that they are not present to the extent that they would hinder the objective of the present invention, and in a narrow sense, that they are substantially absent. Substantially absent means that, preferably, the molar concentrations of IL-12, IL-18, and TL1A in the first culture medium are, for example, below the detection limit or 0 ng / mL, respectively.

[0030] The type of the first culture medium is not particularly limited, except for the conditions of the first cytokine group, and can be any culture medium that can be used for culturing PBMCs (hereinafter also referred to as the basic medium). Examples of the basic medium include RPMI1640, MEM, etc., except for the cytokine conditions. The first culture medium can be prepared, for example, by adding IL-7, IL-15, and IL-21 to the basic medium, and omitting IL-12, IL-18, and TL1A.

[0031] The basic culture medium may contain serum or not (be serum-free), with the latter being preferred. The basic culture medium may contain a serum substitute instead of animal-derived serum (e.g., fetal bovine serum, human serum, etc.). The basic culture medium may also contain, for example, insulin, transferrin, selenium, pyruvate, HEPES, ascorbic acid, etc., as components other than cytokines.

[0032] The first culture medium may contain only the first group of cytokines as cytokines, or it may further contain other cytokines (excluding IL-12, IL-18, and TL1A). The first culture medium may further contain, for example, any of IL-2, IL-4, IL-33, IL-6, and IL-10, or a combination thereof, as other cytokines.

[0033] The culture medium used in the second stimulation step (hereinafter also referred to as the second culture medium) contains the second group of cytokines. The second culture medium can be described in the same way as the first culture medium, except that it contains the second group of cytokines.

[0034] The second cytokine group includes IL-7, IL-15, IL-21, IL-12, IL-18 (also known as IL-1F4), and TL1A. In the second stimulation step, unlike the first stimulation step, the second cytokine group includes IL-7, IL-15, and IL-21, as well as IL-12, IL-18, and TL1A, which were not included in the first medium. In other words, in the second stimulation step, a modified cytokine group is used from the cytokine group used in the first stimulation step.

[0035] The IL-7 concentration in the second medium is, for example, 1 to 50 ng / mL, and specifically, 5 to 15 ng / mL. The IL-15 concentration in the second medium is, for example, 1 to 50 ng / mL, and specifically, 5 to 15 ng / mL. The IL-21 concentration in the second medium is, for example, 1 to 50 ng / mL, and specifically, 10 to 20 ng / mL. The IL-12 concentration in the second medium is, for example, 1 to 100 ng / mL, and specifically, 20 to 50 ng / mL. The IL-18 concentration in the second medium is, for example, 10 to 100 ng / mL, and specifically, 20 ng / mL. The TL1A concentration in the second medium is, for example, 1 to 100 ng / mL, and specifically, 50 ng / mL. The concentrations in the medium are, for example, the final concentrations at the start of culture (the same applies hereinafter). In the second culture medium, the proportions (molar ratios) of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A are not particularly limited.

[0036] The type of the second medium is not particularly limited, except for the conditions of the second cytokine group, and can be any medium that can be used for culturing PBMCs (the basic medium), similar to the first medium. The second medium can be prepared, for example, by adding IL-7, IL-15, IL-21, IL-12, IL-18 and TL1A to the basic medium.

[0037] The second culture medium may contain only the second group of cytokines as cytokines, or it may further contain other cytokines. The second culture medium may further contain, for example, any of IL-2, IL-4, IL-33, IL-6, and IL-10, or a combination thereof, as other cytokines.

[0038] (3) Antigen The antigen added to the first medium in the first stimulation step and the antigen added to the second medium in the second stimulation step are not particularly limited and can be selected according to the specificity intended to be conferred to T cells.

[0039] The antigen may be, for example, one type or two or more types, and there are no particular restrictions on the combination thereof. The antigen added to the first medium and the antigen added to the second medium are, for example, the same.

[0040] In the first stimulation step, for example, the antigen may be added directly to the first culture medium and culture may be performed. The amount of antigen added to the culture medium is not particularly limited. The concentration of each antigen in the first culture medium is, for example, 10 ng / mL to 10 μg / mL, and specifically, from the viewpoint of effectiveness, for example, 1 μg / mL to 10 μg / mL is preferred, and from the viewpoint of cost-effectiveness, for example, 10 ng / mL to 10 μg / mL and 1 μg / mL to 10 μg / mL are also preferred. The antigen concentration in the culture medium is, for example, the final concentration at the start of culture in the presence of the antigen (the same applies hereinafter).

[0041] In the second stimulation step, for example, the antigen may be added directly to the second medium and culture may be performed, or cells pulsed with the antigen may be added and culture may be performed. The amount of antigen added to the medium is not particularly limited. The concentration of each antigen in the second medium is, for example, 10 ng / mL to 10 μg / mL, and specifically, from the viewpoint of effectiveness, for example, 1 μg / mL to 10 μg / mL is preferred, and from the viewpoint of cost-effectiveness, for example, 10 ng / mL to 10 μg / mL and 1 μg / mL to 10 μg / mL are also preferred. Note that the addition of the antigen to the second medium is not limited to this example, and for example, condition C of Embodiment 2 described later may also be applied.

[0042] The types of antigens (target antigens) that are the target of the specificity of the specific T cells are not particularly limited, and include, for example, viruses, fungi (e.g., bacteria, mycelium, etc.), cancer cells, proteins, peptides, etc. The types of viruses, fungi, and cancer cells are not particularly limited, and examples from other embodiments described later can be used. The proteins include, for example, viral proteins, fungal proteins, cancer proteins, etc., and the peptides include, for example, viral peptides, fungal peptides, cancer peptides, etc.

[0043] The antigen added to the culture medium (additive antigen) may be, for example, the target antigen itself, or a substance having a characteristic part of the structure of the target antigen. The form of the additive antigen is preferably, for example, a protein or a peptide, and more preferably a peptide. When the target antigen is a virus, fungus, or cancer cell, the additive antigen is preferably, for example, a protein or peptide characteristic of them, and when the target antigen is a protein, the additive antigen is preferably, for example, a peptide characteristic of it.

[0044] A specific example of a method for producing multiple antigen-specific T cells using two or more antigens is shown in Embodiment 3.

[0045] (4) Each step of the manufacturing method The manufacturing method of the present invention includes the first stimulation step and the second stimulation step as described above. Each step will be explained below with specific examples.

[0046] First, as the first stimulation step, the antigen is added to the first culture medium to create antigen (+) first culture medium, PBMC is added thereto, and culture is performed. The first stimulation step can also be called the first manufacturing step of the antigen-specific T cells.

[0047] The number of PBMC cells added to the antigen (+) first medium is not particularly limited, for example, 0.5 × 10 per 1 mL of the medium. ６ ~5.0 x 10 ６ These are cells. The culture conditions are not particularly limited, and general PBMC culture conditions can be selected, with the culture temperature being, for example, 37°C ± 5°C or 37°C.

[0048] The culture time in the antigen (+) first medium is not particularly limited, and the end of the culture can be determined, for example, by the number of cultured cells. For example, if the number of cells is 20 × 10 ６ It is preferable to continue culturing until the cells reach 20 × 10 ６ ~300 x 10 ６The culture may be continued until cells are formed. Specific examples of culture durations include, for example, less than 1 day or 1 to 12 days, less than 1 day or 1 to 14 days, less than 1 day or 1 to 16 days, less than 1 day or 1 to 18 days, less than 1 day or 1 to 20 days, less than 1 day or 1 to 22 days, or 10 to 16 days, or 10 to 22 days. The culture period for the first stimulation step may be, for example, 23 days or more.

[0049] The first stimulation step, as described above, is a step of culturing PBMCs in the antigen (+) first medium to which the antigen has been added. After the first stimulation step using the antigen (+) first medium, the process may proceed directly to the next second stimulation step, or after the first stimulation step, the process may proceed to the next second stimulation step via a culture step (also called the first culture step). The medium used in the culture step is not particularly limited and, for example, is an antigen (-) medium without added antigen, and specifically, a medium without added antigen that contains cytokines is an example. The cytokines may be, for example, the same first cytokine group as in the antigen (+) first medium, or a combination of fewer types of cytokines, for example, a medium containing IL-7 and IL-15 (IL7 / 15) can be exemplified. The duration of the culture step after the first stimulation step can be, for example, based on the example of the first stimulation step.

[0050] Next, as the second stimulation step, the cells from the first stimulation step are further cultured in the second medium to which the antigen has been newly added. This further increases the number of antigen-specific T cells. The second stimulation step can also be considered a second production step for the antigen-specific T cells.

[0051] Specifically, the same antigen as in the first stimulation step is newly added to the second medium to create the antigen (+) second medium. The antigen may be added as is, for example, or it may be added as cells pulsed with the antigen. Then, the antigen (+) first medium from the first stimulation step is replaced with the antigen (+) second medium, and further culture is performed. This example is not limited, and for example, components that are lacking in the second medium may be added to the first medium.

[0052] The amount of the antigen (+) second medium is not particularly limited and is, for example, the same amount as the antigen (+) first medium. That is, at the start of the second stimulation step, the number of cells after the first stimulation step added to the antigen (+) second medium is, for example, 0.5 × 10 per 1 mL of the medium. ６ ~5.0 x 10 ６ These are cells. Unless otherwise specified, the culture conditions can be set in the same manner as the first stimulation step described above.

[0053] The culture time in the antigen (+) second medium is not particularly limited, for example, when the number of antigen-specific T cells is 600 × 10 ６ It is preferable to continue culturing until cells or more are reached. The number of culturing days can be, for example, less than 1 day, 1 day or more, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, or 22 days or more. Specific examples include 10 to 16 days, 10 to 14 days, 12 to 14 days, 14 to 16 days, 16 to 18 days, 18 to 20 days, and 20 to 22 days. The culturing period for the second stimulation step may be, for example, 23 days or more.

[0054] The total number of culture days for the first stimulation step and the second stimulation step is, for example, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 22 days or more, 24 days or more, 26 days or more, 28 days or more, 30 days or more, 32 days or more, 34 days or more, and 36 days or more. Specific examples include 20-22 days, 22-24 days, 24-26 days, 26-28 days, 28-30 days, 30-32 days, 32-34 days, and 34-36 days. Since the manufacturing method of this embodiment allows for long-term cell culture, a larger quantity of cells can be produced. An example of a culture period is 14 days for the first stimulation step and 14 days for the second stimulation step, totaling 28 days. According to the manufacturing method of this embodiment, cell proliferation of, for example, 500 times or more, 1000 times or more, 1500 times or more, 2000 times or more, 2500 times or more, 3000 times or more, 3500 times or more, or 4000 times or more can be achieved by culturing for 28 days. Specifically, for example, cell proliferation of 1000 to 4000 times can be achieved. Furthermore, according to the manufacturing method of this embodiment, the proportion of T cells in the proliferated cell population can be, for example, 99% or more. Because efficient cell proliferation is possible according to the manufacturing method of this embodiment, the amount of blood collected from the donor can be reduced to, for example, 20 mL or less, 15 mL or less, 10 mL or less, or 5 mL or less.

[0055] The second stimulation step, as described above, is a step of further culturing the cells after the first stimulation step in the antigen (+) second medium to which the antigen has been added. This embodiment may also include a further culture step (also called a second culture step) after the second stimulation step using the antigen (+) second medium. The medium for the culture step is not particularly limited, and for example, it may be an antigen (-) medium without the antigen, and specifically, a medium without the antigen that contains cytokines. The cytokines may be, for example, the same second cytokine group as in the antigen (+) second medium, or a combination of fewer types of cytokines, for example, a medium containing IL-7 and IL-15 (IL7 / 15) can be exemplified. The duration of the culture step after the second stimulation step can be, for example, based on the example of the second stimulation step.

[0056] This embodiment includes, for example, the initiation of PBMC culture, the first stimulation step, the first culture step after the first stimulation, the second stimulation step, and the second culture step after the second stimulation. In this case, the total period from the start to the end of the culture is, for example, 28 days ± 5 days. Specific examples include: the culture until the start of the first stimulation step is 1 to 3 days, the first stimulation step is 1 to 3 days, the first culture step is 14 days ± 3 days, the second stimulation step is 1 to 3 days, and the second culture step is 14 days ± 3 days.

[0057] Thus, the manufacturing method of this embodiment involves culturing PBMCs in a first medium containing the first cytokine group in the presence of the antigen, and then culturing the cultured cells in a second medium containing a second cytokine group different from the first cytokine group in the presence of the antigen. As described above, this makes it possible to efficiently obtain the antigen-specific T cells.

[0058] In this embodiment, for example, the cell population obtained by the completion of the second stimulation step may be applied to the usage method described later as a cell population including the antigen-specific T cells.

[0059] (5) Method of Use The antigen-specific T cells obtained by this embodiment are not particularly limited in their use and can be used, for example, in medical care, livestock farming, research, etc.

[0060] In this embodiment, for example, the cell population obtained at the end of the second stimulation step may be used as a cell population including the antigen-specific T cells, or the antigen-specific T cells may be isolated from the cell population and used. In the former case, the cell population including the antigen-specific T cells may include other cells (for example, PBMC, which is the raw material) in addition to the antigen-specific T cells. According to the manufacturing method of the present invention, the antigen-specific T cells can be efficiently proliferated, so the cell population obtained at the end of the second stimulation step can be used without isolating the antigen-specific T cells, for example. The cell population including the antigen-specific T cells is also called an antigen-specific T cell population, and as a specific example, in the case of virus-specific T cells (VST cells), it is also called a VST cell population, and in the case of multiple virus-specific T cells (MVST cells), it is also called an MVST cell population. Hereinafter, the use of antigen-specific T cells includes, for example, the meaning of using an antigen-specific T cell population. The same applies to other embodiments and examples.

[0061] Furthermore, the antigen-specific T cells may be used, for example, in vitro against cells, ex vivo against organs or tissues, or in vivo against living organisms.

[0062] The antigen-specific T cells are preferably used in vivo, i.e., administered to an individual, as a treatment method for various diseases. The individual is a living organism, such as a human or a non-human animal. The individual is also referred to as a patient (human), a diseased animal (non-human animal), a test subject, a recipient, etc.

[0063] When the antigen-specific T cells are used in vivo, for example, the PBMC used in the production method of the present invention is preferably from an individual of the same species as the individual to which the antigen-specific T cells are administered (recipient). That is, if the recipient is human, for example, human PBMC is preferred.

[0064] The PBMC used in the manufacturing method of the present invention is, for example, a PBMC from another individual of the same species (donor). The donor is, for example, an individual not infected with the target antigen.

[0065] According to the manufacturing method of the present invention, for example, a large quantity of antigen-specific T cells that can be administered to many individuals (recipients) can be prepared from PBMCs of one individual (donor). PBMCs from healthy individuals are preferred. Specifically, when the goal is to administer antigen-specific T cells to many individuals (recipients), the origin of the PBMCs (donor) is not limited to blood relations, and PBMCs from healthy individuals with one or more matching HLA types are particularly preferred.

[0066] (Embodiment 2) The manufacturing method of the present invention preferably satisfies, for example, in addition to condition A, any one of the following optional conditions B, C, and D, more preferably satisfies condition A and any two of the optional conditions (conditions B and C, condition C and D, or condition B and D), and preferably satisfies condition A and all of the optional conditions (conditions B, C, and D).

[0067] (1) Condition B The manufacturing method of this embodiment is preferably further satisfied with condition B. That is, the manufacturing method of this embodiment may further contain arginine in the culture medium used. (Condition B) At least one of the culture medium of the first stimulation step and the culture medium of the second stimulation step contains arginine.

[0068] Thus, under condition B, the culture medium further contains arginine, which can, for example, further promote the proliferation of antigen-specific T cells and increase the viability of antigen-specific T cells. Furthermore, the inclusion of arginine in the culture medium can improve, for example, anti-apoptotic effects, anti-exhaustion effects, differentiation inhibitory effects (also known as stemness-enhancing effects), cytotoxic effects, and the ability to simultaneously secrete and express cytokines.

[0069] Arginine may be included, for example, in the first medium of the first stimulation step, in the second medium of the second stimulation step, or in both mediums (first medium and second medium). In this embodiment, for example, it is preferable to include arginine in both the first medium and the second medium.

[0070] The concentration of arginine in the first culture medium is, for example, 1.2 mM to 10 mM. The unit M can be expressed in mol / L (the same applies hereinafter). The concentration in the culture medium is, for example, the final concentration at the start of cultivation (the same applies hereinafter). The proportion of arginine in the first culture medium is not particularly limited.

[0071] The concentration of arginine in the second medium is, for example, 1.2 mM to 20 mM or 1.2 mM to 10 mM. The concentration in the medium is, for example, the final concentration at the start of culture (the same applies hereinafter). The proportion of arginine in the second medium is not particularly limited.

[0072] (2) Condition C The manufacturing method of this embodiment is preferably further satisfied with condition C. That is, in the manufacturing method of this embodiment, antigen-presenting cells may be further added to the second culture medium in the second stimulation step. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed.

[0073] Thus, under condition C, by further adding antigen-presenting cells to the second medium in the second stimulation step, for example, the proliferation of antigen-specific T cells can be further promoted, and the viability of antigen-specific T cells can be further increased. In addition, by adding the antigen-presenting cells, for example, differentiation inhibitory effect, anti-exhaustion effect, specificity, and simultaneous cytokine secretion and expression ability can be improved. The antigen-presenting cells are, for example, antigen-presenting cells that present the target antigen, and specifically, antigen-presenting cells that have been peptide-pulsed with the target antigen are preferred. Under condition C, antigen-presenting cells that present the target antigen may be added as the antigen to be added to the second medium, or both the added antigen and the antigen-presenting cells may be added.

[0074] The antigen-presenting cells are not particularly limited and, for example, are γδ T cells. Preferably, the antigen-presenting cells are in a state in which the antigen (additional antigen) to be added in the second stimulation step has been added (pulsed) in advance. By pulsing the antigen, the antigen-presenting cells can be made to present the antigen.

[0075] The number of antigen-presenting cells added to the second medium is not particularly limited. The number of antigen-presenting cells is not particularly limited and can be set according to, for example, the total number of cells (including the antigen-specific T cells) increased in the first medium during the first stimulation step. For example, an equivalent number of antigen-presenting cells may be added to the total number of cells increased in the first medium. The number of cells increased in the first medium and the number of antigen-presenting cells added are not particularly limited and can be, for example, 0.1 × 10⁶ per 500 μL of the second medium, respectively. ６ ~8 x 10 ６ A specific example of the range of cells is 1 x 10⁻⁶. ６ These are cells, and the number of cells in both groups may be roughly the same or different.

[0076] (3) Condition D The manufacturing method of this embodiment is preferably further satisfied with condition D, for example. That is, in the manufacturing method of this embodiment, Flt3L and pattern recognition receptor ligand (hereinafter also referred to as PRR ligand) may be added to the first culture medium in the first stimulation step. (Condition D) In ​​the first stimulation step, Flt3L and PRR ligand are added to the culture medium and culture is performed.

[0077] Thus, by further adding Flt3L and PRR ligand to the first culture medium in the first stimulation step, the activation and proliferation of T cells by dendritic cells, which are antigen-presenting cells present in PBMCs, can be further promoted. This allows for, for example, the proliferation of cytotoxic lymphocytes (CTLs) such as CD8 ＋ The proportion of T cells can be increased further. Furthermore, when using multiple antigens to produce multi-antigen-specific T cells that are specific to multiple antigens, for example, it is possible to induce T cells specific to a variety of antigens, thus further improving antigen diversity.

[0078] Flt3L, or Flt3 ligand, is a growth factor. The origin of Flt3L is not particularly limited and can be, for example, human or non-human animals. Examples of non-human animals include rats, mice, cats, dogs, guinea pigs, rabbits, sheep, horses, pigs, cattle, camels, and monkeys. Preferably, the origin of Flt3L is the same as that of PBMCs, for example. Specifically, in the case of human-derived PBMCs, it is preferable to use human-derived Flt3L. Flt3L may be, for example, a commercially available product or may be prepared in-house based on sequence information (database GenBank accession No. U04806.1).

[0079] PRR ligands are ligands for pattern recognition receptors (also called PRRs). PRRs are generally a collective term for receptors that recognize pathogen-derived molecular patterns in cells, such as Toll-like receptors (TLRs), cGAS (cyclic GMP-AMP), and STING (stimulator of interferon genes). Examples of PRR ligands include TLR ligands, cGAS ligands, and STING ligands. A specific example is ssRNA40, which binds to TLR7 and TLR8. ssRNA40 is a single-stranded RNA. An example is HIV-I derived ssRNA40, whose sequence information is registered in the Genbank database as JA792524.1.

[0080] In the first culture medium, the Flt3L concentration is, for example, 5 to 500 ng / mL, and specifically, 10 to 100 ng / mL. In the first culture medium, the PRR concentration is, for example, 0.1 to 5 μg / mL, and specifically, 0.2 to 1 μg / mL. The concentration in the culture medium is, for example, the final concentration at the start of culture (the same applies hereinafter). In the first culture medium, the content ratio (molar ratio) of Flt3L to the PRR ligand is not particularly limited.

[0081] (Embodiment 3) As described above, a treatment method has been proposed that utilizes multiple virus-specific T cells that have specificity for various viruses causing treatment-resistant opportunistic infections after hematopoietic stem cell transplantation. Furthermore, not limited to viruses, for example, in the treatment of cancer, the use of multiple cancer-specific T cells that have specificity for multiple cancer antigens has also been proposed. The manufacturing method of the present invention can be applied, for example, to the production of multiple antigen-specific T cells that exhibit specificity for such multiple antigens (target antigens).

[0082] According to the manufacturing method of the present invention, for example, multiple antigen-specific T cells that exhibit specificity and diversity (specificity targeting multiple antigens) to target antigens can be efficiently proliferated, enabling the preparation of a larger number of cells. Multiple antigen-specific T cells are also called, for example, multi-antigen-specific T cells or multi-antigen-specific T cells.

[0083] (1) Use of two or more antigens This embodiment is an example of a method for producing multiple antigen-specific T cells, and uses two or more antigens. In the production method of this embodiment, when two or more antigens (additional antigens) are used, these together are also called an antigen set (additional antigen set).

[0084] The method of adding the antigen set to the first medium and the second medium is not particularly limited. Examples of the addition method include the following: for example, adding the same antigen to the first medium and the second medium. In this case, the antigen set containing all types of antigens is added to the first medium, and the same antigen set is also added to the second medium.

[0085] In this embodiment, the types of antigens (additional antigens) included in the antigen set are not particularly limited, with a lower limit of, for example, two types and an upper limit of, not particularly limited. When producing the multiple antigen-specific cells, the types of target antigens to be conferred to the cells are not particularly limited, for example, two or more types, with an upper limit of, not particularly limited, and examples include 2 to 20 types. As a specific example, if the target antigen is a protein such as a viral protein, the number of types is 2 to 20 types in terms of protein units. When the target antigen is a protein, the additional antigen added to the culture medium is preferably a peptide, as described above, and the number of additional antigens (peptides) set per type of target antigen is not particularly limited, and may be one or more, two or more, 100 or more, or 1000 or more, with no upper limit.

[0086] In the manufacturing method of this embodiment, the total amount of each antigen (added antigen) in the antigen set may be the same or different. The concentrations of each antigen in the first medium and the second medium are not particularly limited, and the examples of Embodiment 1 can be used, for example.

[0087] (2) Overlapping peptides This embodiment is another example of a method for producing multiple antigen-specific T cells, in which, for example, peptides with overlapping amino acid sequences are used as the added antigens. Such peptides are also called overlapping peptides.

[0088] In this embodiment, for example, two or more overlapping peptides may be used as the added antigen, or the overlapping peptide and a non-overlapping peptide may be used.

[0089] (3) Multiple antigen-specific T cells The types of multiple antigen-specific T cells are not particularly limited, and specific examples include multiple virus-specific T cells (MVST cells), multiple fungus-specific T cells, and multiple cancer-specific T cells.

[0090] (3-1) Multiple virus-specific T cells When producing multiple virus-specific T cells, the target viruses for which the specificity is to be conferred to the cells are not particularly limited and can be set arbitrarily. Examples of target viruses include viruses for which specific antigens have been identified, and the following are some examples.

[0091] CARV (Respiratory Virus) IFV (Influenza Virus) PIV (Parainfluenza Virus) RSV (Synocytosis Virus) hMPV (Human Metapneumovirus) Polyomavirus JCV (JC Virus) BKV (BK Virus) EBV (Epstein-Barr Virus) CMV (Cytomegalovirus) AdV (Adenovirus) HHV (Herpes Virus) HHV6 HHV8 Herpes Simplex Virus BoV (Boka Virus) CoV (Coronavirus) SARS CoV-1 SARS CoV-2 HRV (Human Rhinovirus) LCMV (Lymphocytic Choriomeningitis Virus) Hepatitis Virus Hepatitis C Virus Hepatitis B Virus HIV (Human Immunodeficiency Virus) HPV (Human Papillomavirus) HTLV (Human T-lymphophobia) HTLV1 Mumps virus Measles virus PBV (Parvovirus) PBV B Rotavirus Merkel cell virus West Nile virus Zika virus Ebola virus Dengue virus

[0092] The number of target antigens (target viruses) that are the subject of specificity and are conferred to the aforementioned multiple virus-specific T cells (including the cell population) is not particularly limited, with a lower limit of, for example, 2 and an upper limit that is not particularly limited. The range is, for example, 2 to 10 types.

[0093] The additive antigens (viral antigens) used in the production of the aforementioned multiple virus-specific T cells include, for example, viral proteins that constitute a virus and their peptides (viral peptides). Examples of the viral proteins include capsid proteins (also called coat proteins), and their constituents VP1, VP2, and VP3. The viral peptides may also be, for example, fragments of the viral proteins. The viral antigens may be prepared from a virus, for example, or artificially prepared based on sequence information, etc.

[0094] If the target antigen is one of multiple viruses, the added antigen may be, for example, a peptide characteristic of each virus, a peptide that overlaps among the viruses, or both. Furthermore, for example, two or more types of added antigens may be used for a single virus.

[0095] (3-2) When producing multiple fungus-specific T cells, the target antigen (target fungus) to which the specificity is to be conferred to the cells is not particularly limited and can be set arbitrarily. Examples of fungi include fungi for which specific antigens have been identified, such as the following bacteria and fungi.

[0096] Bacteria: Mycobacterium tuberculosis; Fungi: Candida albicans, Aspergillus fumigatus, Borrelia afzelii (Lyme disease)

[0097] The number of fungi to which specificity is to be conferred to the aforementioned multi-fungus specific T cells (including the cell population thereof) is not particularly limited, with a lower limit of, for example, 2 and an upper limit that is not particularly limited. The range is, for example, 2 to 10 species.

[0098] Examples of additive antigens (additive fungal antigens) used in the production of the aforementioned multi-fungus-specific T cells include fungal proteins that constitute fungi and their peptides (fungal peptides). The fungal antigens may be prepared from fungi, for example, or artificially prepared based on sequence information, etc.

[0099] If the target antigen is from multiple fungi, the added antigen may be, for example, a peptide characteristic of each fungus, a peptide that overlaps among the fungi, or both. Furthermore, for example, two or more antigens may be used for a single fungus.

[0100] (3-3) Multiple cancer-specific T cells When producing multiple cancer-specific T cells, the cancers to which the specificity is to be conferred to the cells are not particularly limited and can be set arbitrarily. Examples of cancers include cancers for which specific antigens have been identified, cancers targeted by CAR-T therapy, CTL therapy, peptide therapy, etc., and the following cancers can be given as examples.

[0101] Neuroblastoma (antigen: e.g., GD2) Breast and ovarian cancer (antigen: e.g., Mucin-1) Diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma-transformed low-grade non-Hodgkin lymphoma, high-grade B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia (CD19-positive ALL) Multiple myeloma Osteosarcoma Brain tumor Ovarian cancer Germ cell carcinoma WT1-expressing cancer NY-ESO-1-expressing cancer

[0102] <Kit for the Production of Antigen-Specific T Cells> The production kit of the present invention comprises a first cytokine group and a second cytokine group, wherein the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not include IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A, and is a kit used for the production method of antigen-specific T cells of the present invention.

[0103] The manufacturing kit of the present invention includes, for example, the first medium used in the first stimulation step and the second medium used in the second stimulation step. The manufacturing kit of the present invention may also include at least one or more of arginine, the antigen-presenting cells (e.g., γδ T cells), Flt3L, and ssRNA40. The first and / or second mediums may further include any or a combination thereof of IL-2, IL-4, IL-33, IL-6, and IL-10.

[0104] The various elements in the manufacturing kit of the present invention may, for example, be incorporated into the kit so that the manufacturing method of the present invention can be carried out by referring to the description of the manufacturing method of the present invention. Furthermore, the manufacturing kit of the present invention may include, for example, instructions for the manufacturing method of the present invention.

[0105] [Second Invention] In addition to the first invention (a method for producing a first specific T cell) that satisfies the above-mentioned condition A, this application further includes the following second invention. That is, the second antigen-specific T cell production method of the present invention comprises a first stimulation step of culturing peripheral blood mononuclear cells in a medium to which an antigen has been added, and a second stimulation step of further culturing the cells after the first stimulation step in a medium to which an antigen has been newly added, and is characterized in that it satisfies at least one of the above conditions B, C, and D.

[0106] (Condition B) At least one of the culture medium in the first stimulation step and the culture medium in the second stimulation step contains arginine. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed. (Condition D) In ​​the first stimulation step, Flt3L and the pattern recognition receptor ligand are added to the culture medium and culture is performed.

[0107] With regard to the second method for producing antigen-specific T cells of the present invention, unless otherwise specified, the description of the first method for producing antigen-specific T cells can be applied, for example.

[0108] The second antigen-specific T cell production method of the present invention may be, for example, a form that satisfies any one of conditions B, C, and D, a form that satisfies any two of conditions B, C, and D (conditions B and C, C and D, or B and D), a form that satisfies all of conditions B, C, and D, and as described above, may also satisfy condition A.

[0109] [Third Invention] In addition to the first and second inventions, this application further includes the following third invention. That is, the third method for producing antigen-specific T cells of the present invention includes a stimulation step of culturing peripheral blood mononuclear cells in a culture medium to which an antigen has been added, and is characterized in that it satisfies at least one of conditions B3, C3, and D3. Unlike the first and second inventions, in which the stimulation step consists of two steps (the first stimulation step and the second stimulation step), the third method for producing antigen-specific T cells of the present invention consists of one stimulation step.

[0110] (Condition B3) The culture medium in the stimulation step contains arginine. (Condition C3) In the stimulation step, antigen-presenting cells are added to the culture medium and culture is performed. (Condition D3) In the stimulation step, Flt3L and pattern recognition receptor ligand are added to the culture medium and culture is performed.

[0111] The third manufacturing method of the present invention can be adapted from the descriptions of the first and second inventions, except that the stimulation step is one step, unless otherwise specified.

[0112] Unless otherwise specified, condition B3 can be provided by referring to the description of condition B in the first invention; unless otherwise specified, condition C3 can be provided by referring to the description of condition C in the first invention; and unless otherwise specified, condition D3 can be provided by referring to the description of condition D in the first invention.

[0113] A third method of production of the present invention preferably includes, for example, a step of satisfying condition B3, that is, culturing peripheral blood mononuclear cells in a culture medium containing arginine.

[0114] A third method of production of the present invention preferably includes, for example, a step of satisfying condition C3, that is, culturing in a medium to which antigen-presenting cells have been added. The antigen-presenting cells are not particularly limited and may be, for example, γδ T cells.

[0115] A third method of production according to the present invention preferably includes, for example, a step of satisfying condition D3, that is, adding Flt3L and PRR ligand to the culture medium and performing cultivation.

[0116] The third manufacturing method of the present invention preferably satisfies, for example, one of the conditions B3, C3, and D3, preferably two of the conditions (conditions B3 and C3, conditions C3 and D3, or conditions B3 and D3), and preferably all of the conditions (conditions B3, C3, and D3).

[0117] The culture medium in the stimulation step is preferably a culture medium containing a group of cytokines. The group of cytokines may be, for example, the first group of cytokines in the first invention, the second group of cytokines, or both the first and second groups of cytokines. The group of cytokines may further include, for example, any one of IL-2, IL-4, IL-33, IL-6, and IL-10, or a combination thereof. The description of the group of cytokines can be referenced from the description of condition A in the first invention.

[0118] The conditions for adding the antigen to the culture medium are not particularly limited, and the description in the first invention can be incorporated. The culture conditions in the stimulation step are not particularly limited, and the description in the first invention can be incorporated. For example, the culture time may be the time of the first stimulation step in the first invention, the time of the second stimulation step in the first invention, or the total time of the first and second stimulation steps in the first invention.

[0119] According to the first to third inventions, for example, a cell population containing antigen-specific T cells possessing fatigue-inhibiting effects, anti-apoptotic effects, differentiation-inhibiting effects, and cytotoxic properties can be obtained. Furthermore, according to the first to third inventions, for example, a cell population containing the antigen-specific T cells can be obtained at a high frequency and in large quantities. The fatigue-inhibiting effect is the effect of suppressing cell fatigue, which generally means a decrease in functions such as cytokine production ability that should be performed in response to antigen stimulation. The anti-apoptotic effect is the effect of cells becoming resistant to stress and damage, and is also called, for example, an effect of improving viability. The differentiation-inhibiting effect is also called, for example, a stemness-enhancing effect.

[0120] The aforementioned cell population can be described, for example, as a group of cells in which mitochondrial biosynthesis is maintained. Mitochondria are central organelles that govern cell survival, exhaustion, and differentiation, and the mitochondrial-derived anti-apoptotic factor MCL-1 is particularly important for T cell survival. In condition AB, which adds condition B to condition A, for example, the expression of MCL-1 increases, and the proportion of cells that are not apoptotic (for example, cCasp-3 negative and annexin V negative) can be further increased. In addition, in condition AB, for example, in addition to suppression of cell exhaustion and differentiation, the frequency of MT-1 positive cells, indicated by mitochondrial membrane potential, can also be further increased. From these points, it can be said that "mitochondrial biosynthesis is maintained" means, for example, a state in which anti-apoptotic function is maintained, exhaustion and differentiation are suppressed, and the number of cells with mitochondrial membrane potential increases.

[0121] [Fourth Invention] In addition to the first to third inventions described above, this application further includes the following fourth invention. That is, the fourth antigen-specific T cell population of the present invention is 600 × 10 ６ It is characterized by containing cells of cell count or higher, with more than 90% of the total being CD8-positive / CD45RO-positive.

[0122] The antigen-specific T cell population of the present invention is, for example, the CD8-positive / CD45RO-positive cells which are antigen-specific T cells that have relatively preserved mitochondrial biosynthesis and are cytotoxic.

[0123] The antigen-specific T cell population of the present invention can be obtained, for example, by any of the manufacturing methods described in the first to third inventions. That is, for example, by any of the manufacturing methods described in the first to third inventions, an antigen-specific T cell population can be obtained that effectively contains the CD8-positive / CD45RO-positive antigen-specific T cells that have relatively preserved mitochondrial biosynthesis and cytotoxicity. As described above, the antigen-specific T cell population of the present invention can be obtained by referring to the descriptions in the first to third inventions.

[0124] The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these.

[0125] [Example 1] PBMCs were cultured using a culture method that satisfies condition A, and a culture method that satisfies conditions A and D, to produce a population of multiple virus-specific T cells (MVST cells), and their proliferative capacity and function were confirmed.

[0126] <Materials> Culture medium and reagents RPMI-1640 Fetal Bovine Serum (FBS) Penicillin-Streptomycin solution HEPES solution (1M), SIGMA, H0887-100ML L-glutamine solution (200mM), SIGMA, G7513-100ML L-ascorbic acid 2-phosphate, SIGMA, A8960 Insulin-Transferrin-Selenium Supplement (ITS) (100X), GIBCO, 41400-045 Condition A: T cell growth factor (cytokine) Human IL-7, premium grade, Miltenyi Biotech, 130-095-363 Human IL-12, premium grade, Miltenyi Biotech, 130-096-705 Human IL-15, premium grade, Miltenyi Biotech, 130-095-765 Human IL-18 / IL-1F4 Protein, MBL, B001-5 Human IL-21, Pepro Tec, AF-200-21 Human TL-1A, Pepro Tec, 310-23 Condition D: Maturation and activation of dendritic cells ssRNA40 (HIV-1 LTR-derived ssRNA / LyoVec (trademark)), InvivoGen, tlrl-lrna Human FLT3 Ligand (FLT3L), R&D, 308-FK-025 Comparative example (conventional method): T cell growth factor (cytokine) Human IL-4, premium grade, Miltenyi Biotech, 130-093-922 Human IL-7, premium grade, Miltenyi Biotech, 130-095-363 Viral antigen overlap peptide (viral peptide) PepMix CMV pp65, JPT,PM-PP65-2 PepMix CMV IE1, JPT, PM-IE1 PepMix EBV EBNA1, JPT,PM-EBV-EBNA1 PepMix EBV LMP2, JPT, PM-EBV-LMP2 PepMix EBV BZLF1, JPT, PM-EBV-BZLF1 PepMix AdV Hexon, JPT, PM-HAdV3 PepMix AdV Penton, JPT, PM-HADV5-L2 PepMix HHV6 (U90), JPT, PM-HHV6-U90 PepMix HHV6 (U54), JPT, PM-HHV6-U54 PepTivator BKV VP1, Myltenyi, 130-131-251 PepTivator BKV Large T, Myltenyi, 130-131-249,

[0127] <Basic Medium> 500 mL of RPMI1640 was mixed with 5 mL of penicillin / streptomycin solution, 5 mL of HEPES solution, and 5 mL of L-glutamine solution. Immediately before use, L-ascorbic acid was added to a final concentration of 50 mg / mL. Each added reagent was used at the following final concentrations (final concentrations in the medium). Condition A: T cell growth factors (cytokines) IL-7 10 ng / mL IL-15 5 ng / mL IL-21 20 ng / mL IL-12 50 ng / mL IL-18 / IL-1F4 50 ng / mL TL-1A 50 ng / mL Condition D: Dendritic cell maturation and activation Flt3L 50 ng / mL ssRNA40 0.5 μg / mL Comparative example (conventional method): T cell growth factors (cytokines) IL-4 800 U / mL (160 ng / mL) IL-7 900 U / mL (18 ng / mL) Viral peptides were used at 100 ng / mL each under all conditions.

[0128] <Cells> Peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of healthy donors were frozen and stored in liquid nitrogen. <Culture conditions> Culture conditions were 37°C, CO2 ２ We set it at 5% (the same applies below).

[0129] (I) Preparation of MVST samples MVST samples for Example 1A satisfying condition A, Example 1AD satisfying conditions A and D, and Comparative Example 1 were prepared by the following method.

[0130] (Example 1A) (1) Pre-culture (day 0) For pre-culture, PBMCs were used, which were obtained by lysing the frozen cells and culturing them overnight in the basic medium. A 48-well plate was used, and the PBMCs were 1 × 10 ６ Cells were cultured in 500 μL (1 well) of the basic medium for 24 hours.

[0131] (2) First stimulation step and culture step (day 1) 50 μL of the basic medium was prepared containing IL-7 (100 ng / m), IL-15 (50 ng / mL), IL-21 (200 ng / mL), and viral peptide (1 μg / mL) at 10 times the final concentration. This was added to the wells of (1) so that the final concentration of each component was 1, and the cells were cultured for 24 hours. (day 2) 500 μL of the basic medium containing IL-7 and IL-15 was added to the wells to make the culture volume in one well 1 mL, and the cells were cultured for 24 hours. (day 3) The entire amount of cultured cell solution (1 well) was transferred to one well of a 12-well plate, 3 mL of the basic medium containing IL-7 and IL-15 was added to make the culture volume 4 mL, and the cells were cultured. (Day 6) The entire volume of cultured cell saturation (1 well) was transferred to a vertically oriented T25 flask (with the inlet facing upwards; the same applies hereafter), and 4 mL of basic medium containing IL-7 and IL-15 was added to make a total culture volume of 8 mL, and the cells were cultured. (Day 9) The entire volume of cell saturation cultured in the flask was transferred to a horizontally oriented T75 flask (with the inlet facing sideways; the same applies hereafter), and 22 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 30 mL, and the cells were cultured. (Day 13) The cell concentration in the cultured cell saturation was measured, and the total number of cells was calculated. (Day 14) The entire volume of cell saturation was collected in a 50 mL centrifuge tube, the supernatant was removed by centrifugation, the precipitated cells were suspended in cell freezing solution, and the cells were cryopreserved in liquid nitrogen.

[0132] (3) Second stimulation step and culture step (day14: 2nd day0) The cells frozen on day14 were lysed and suspended in 250 μL of the basic medium containing IL-7, IL-15, IL-21, IL-12, IL-18, TL-1A, and viral peptides, and cultured overnight in one well of a 48-well plate. (day15: 2nd day1) 250 μL of the basic medium containing IL-7 and IL-15 was added to the well to make a culture volume of 500 μL, and the cells were cultured. (day16: 2nd day2) The entire amount of cultured cell solution (1 well) was transferred to one well of a 12-well plate, 2 mL of the basic medium containing IL-7 and IL-15 was added to make a culture volume of 2.5 mL, and the cells were cultured. (Day 19: 2nd Day 5) The entire volume of cultured cell saturation (1 well) was transferred to a T25 flask inverted vertically, and 7.5 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 10 mL, and the cells were cultured. (Day 23: 2nd Day 9) The entire volume of cell saturation cultured in the flask was transferred to a T75 flask inverted horizontally, and 22 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 30 mL, and the cells were cultured. (Day 27: 2nd Day 13) The cell concentration in the cultured cell saturation was measured, and the total number of cells was calculated.

[0133] (4) Preparation of analysis sample (day 28) 1.5 × 10⁶ of the cell fluid after the second stimulation (day 27) ６ Cells were separated and the supernatant was removed by centrifugation. The precipitated cells were suspended in the basic medium, which does not contain cytokines, etc., and cultured overnight. The entire cell saturation from day 28 was collected in a 50 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were suspended in cell freezing solution and cryopreserved in liquid nitrogen. The cell population (total cells) obtained in this way was used as the cell population containing MVST cells (hereinafter referred to as the MVST sample) for the evaluation described later.

[0134] (Example 1AD) Example 1AD, which satisfies conditions A and D, was carried out in the same manner as Example 1A, except for the conditions shown below. (1) Pre-culture (day 0) 500 μL of the basic medium containing Flt 3L was used as the culture medium. (2) First stimulation step and culture step (day 1 to day 14) 50 μL of the basic medium containing IL-7 (100 ng / m), IL-15 (50 ng / mL), IL-21 (200 ng / mL), ssRNA 40 (5 mg / mL), and viral peptide (1 mg / mL) at 10 times the final concentration was used as the culture medium.

[0135] (Comparative Example 1) Comparative Example 1 was carried out in the same manner as Example 1A, except for the following conditions: (1) Pre-culture (day 0) 500 μL of the same basic medium as in Example 1A was used as the culture medium.

[0136] (2) First stimulation step and culture step (day 1) 50 mL of the basic medium containing IL-4 (8000 U / mL), IL-7 (9000 U / mL), and viral peptide (1 mg / mL) at 10 times the final concentration was used. (day 2) 500 μL of the basic medium containing IL-4 and IL-7 was used. (day 3) 3 mL of the basic medium containing IL-4 and IL-7 was used. (day 6) 4 mL of the basic medium containing IL-4 and IL-7 was used. (day 9) 22 mL of the basic medium containing IL-4 and IL-7 was used.

[0137] (3) Second stimulation step and culture step (day14: 2nd day0) 250 μL of the basic medium containing IL-4, IL-7, and viral peptide was used. (day15: 2nd day1) 250 μL of the basic medium containing IL-4 and IL-7 was used. (day16: 2nd day2) 2 mL of the basic medium containing IL-4 and IL-7 was used. (day19: 2nd day5) 2.5 mL of the basic medium containing IL-4 and IL-7 was added to make a culture volume of 5 mL, and the cells were cultured. (day23: 2nd day9) 3 mL of the culture medium was removed from the top of the flask, 3 mL of the basic medium containing IL-4 and IL-7 was added, and the cells were cultured. (Day 27: 2nd Day 13) The cell concentration in the cultured cell solution was measured, and the total number of cells was calculated.

[0138] (II) Evaluation As described above, MVST samples (n=7) prepared from PBMCs of seven healthy donors were used to evaluate the following items. In this example, cells were cryopreserved after the first stimulation (day 14), and the frozen cells were thawed and cultured at the start of the second stimulation. Therefore, the descriptions such as "day 1 to day 28" above exclude the cryopreservation period and represent the actual culture period during which the cells were cultured.

[0139] (1) In the cell proliferation cultures of Example 1AD, Example 1A, and Comparative Example 1, the number of cells was counted at the start of pre-culture (day 0), after the first stimulation (day 14), and after the second stimulation (day 27).

[0140] These results are shown in Figure 1. Figure 1 is a graph showing the change in cell number over time (cell proliferation). In Figure 1, the vertical axis represents the cell number (×10 ６ The cells are labeled as follows: pre represents the result at the start of the pre-culture, 1st represents the result after the first stimulation, and 2nd represents the result after the second stimulation.

[0141] As shown in Figure 1, compared to Comparative Example 1, Example 1A, which satisfies condition A, showed an increase in the number of cells after the first stimulation (1st) for each of the PBMCs (n=7) derived from healthy individuals, and exhibited more significant proliferation after the second stimulation (day 27). Furthermore, Example 1AD, which also satisfies condition D, showed overall proliferation equivalent to or better than Example 1A for each of the PBMCs (n=7) derived from healthy individuals.

[0142] (2) Cell frequency: For the MVST samples (prepared on day 28) after the second stimulation of Example 1AD, Example A, and Comparative Example 1, T cells (CD3 ＋ cells), CD8 ＋ T cells, multiple virus-specific T cells, multiple virus-specific CD8 ＋ The frequency of T cells was evaluated. CD8 ＋ T cells can be evaluated as cells that have the activity to damage cells expressing specific antigens. Specifically, this was done using the following method.

[0143] Flow cytometry analysis was performed using the aforementioned MVST samples. The expression frequencies of CD3, CD4, and CD8 on the cell surface of cells contained in the MVST samples after the second stimulation were measured using antibodies corresponding to the target organisms. In addition, the frequency of cells that produce IFNγ or TNFα in response to viral peptide stimulation, i.e., multiple virus-specific T cells, was measured by intracellular staining of the MVST samples using antibodies corresponding to the target organisms.

[0144] These results are shown in Figure 2. Figure 2 shows the MVST sample (1 × 10⁻¹⁰ ６ This graph shows the cell frequency of specific cells (per cell). Figure 2A shows the frequency of T cells and CD8 in the MVST sample. ＋ This graph shows the frequency of T cells. The graph on the left shows the number of T cells, and the graph on the right shows CD8 ＋ Figure 2B shows the number of T cells. Figure 2B shows the frequency of multiple virus-specific T cells and multiple virus-specific CD8 in the MVST sample. ＋ These graphs show the frequency of T cells. The graph on the left shows the number of multiple virus-specific T cells, and the graph on the right shows the number of multiple virus-specific CD8 cells.＋ This shows the number of T cells. The two graphs in Figure 2B show the number of cells that produce at least one of either IFNγ or TNFα after viral peptide stimulation.

[0145] As shown in the left graph of Figure 2A (T cell frequency), all MVST samples were composed almost entirely of T cells. And, as shown in the right graph of Figure 2A (CD8 ＋ As shown in the T cell frequency, compared to Comparative Example 1, Example 1A satisfying condition A, and Example 1AD satisfying conditions A and D, both had CD8 for each of the MVST samples (n=7). ＋ The frequency of T cells increased. In particular, Example 1AD showed an increase in CD8 compared to Example 1A. ＋ The frequency of T cells was higher. Also, as shown in both graphs in Figure 2B, compared to Comparative Example 1, both Example 1A and Example 1AD produced multiple virus-specific T cells and multiple virus-specific CD8 cells that produced IFNγ or TNFα for each of the MVST samples (n=7). ＋ The frequency of T cells increased.

[0146] (3) Diversity in CMV Specificity The MVST samples (prepared on day 28) from Example 1AD and Example 1A after the second stimulation were examined to determine whether T cells specific to each peptide constituting CMV-pp65 existed. In other words, diversity in CMV specificity was examined. Specifically, the following method was used. The CMV pp65 viral peptide used in the stimulation culture consisted of 138 types of overlapping peptides. The MVST samples were stimulated with the 138 types of overlapping peptides constituting the CMV-pp65 antigen and then cultured overnight. After the culture, the number of cells producing IFNγ in response to each overlapping peptide stimulation was measured using an ELISpot assembly.

[0147] These results are shown in Figure 3. Figure 3A is a graph showing the specificity of MVST samples (n=7) prepared from PBMCs derived from healthy donors (n=7) to individual overlapping peptides in the CMV-pp65 antigen, with the vertical axis representing CMV-pp65-specific T cells (IFNγ). ＋ This is the number of cells in the MVST sample (n=7). Figure 3A is a graph for each MVST sample (1 × 10⁻¹⁰). ６ The number of cells specific to each overlapping peptide in the cells is shown for each overlapping peptide. Figure 3B is a graph summarizing Figure 3A. Specifically, CMV-pp65 specific T cells (IFNγ ＋ This graph shows the diversity of CMV-pp65-specific T cells. In Figure 3B, the vertical axis represents the number of peptides targeted by CMV-pp65-specific T cells contained in the MVST sample, i.e., the diversity of CMV-pp65-specific T cells. In Figure 3B, identical plots for Example 1A and Example 1AD indicate that the MVST samples were obtained from the same healthy individual's PBMC (the same applies to other figures below).

[0148] As shown in Figures 3A and 3B, the MVST sample obtained in Example 1A, which satisfies condition A, contains a variety of multiple virus-specific T cells (IFNγ). ＋ ) was included. Furthermore, in Example 1AD, by satisfying condition D in addition to condition A, the diversity of MVST samples derived from 6 out of 7 donors was further improved compared to Example 1A.

[0149] As described above, compared to the comparative example, in Examples 1A and 1AD, CD8 ＋ T cells, or MVST cells (multiple virus-specific T cells, or multiple virus-specific CD8 cells) ＋ It was found that a large number of MVST cell populations containing a high frequency of T cells can be produced. Furthermore, in Example 1AD, which satisfies condition D in addition to condition A, a greater diversity of multiple virus-specific CD8 cells was observed compared to Example 1A. ＋ It was found that it is possible to create a population of MVST cells, including T cells.

[0150] [Example 2] PBMCs were cultured using a culture method that satisfies conditions A and B, and a population of multiple virus-specific T cells (MVST cells) was produced. Their proliferative capacity and function were then confirmed.

[0151] <Materials> Culture medium and reagents RPMI-1640 Fetal Bovine Serum (FBS) Penicillin-Streptomycin solution HEPES solution (1M), SIGMA, H0887-100ML L-glutamine solution (200mM), SIGMA, G7513-100ML Condition A: T cell growth factor (cytokine) Same as in Example 1 Condition B: L-arginine medium L-arginine, Sigma-Aldrich, A5006-100G D-PBS, Wako, 045-29795 Viral antigen overlap peptide (viral peptide) PepTivator CMV pp65, Miltenyi Biotec, 130-093-435 PepTivator CMV IE1, Miltenyi Biotec, 130-093-494 PepTivator EBV EBNA1, Miltenyi Biotec, 130-093-614 PepTivator EBV LMP2A, Miltenyi Biotec, 130-093-616 PepTivator EBV BZLF1, Miltenyi Biotec, 130-093-612 PepTivator AdV Hexon, Miltenyi Biotec, 130-093-496 PepTivator AdV Penton, Miltenyi Biotec, 130-093-777 PepMix HHV6 (U90), JPT, PM-HHV6-U90 PepMix HHV6 (U54), JPT, PM-HHV6-U54 PepTivator BKV VP1, Miltenyi Biotec, 130-131-250 PepTivator BKV Large T, Miltenyi Biotec, 130-131-541

[0152] <Basic Medium> ・Basic medium (+) for the L-arginine-supplemented group: 50 mL of FBS, 5 mL of Sodium Pyruvate, 5 mL of HEPES, 5 mL of L-glutamine solution, and 5 mL of penicillin / streptomyceline solution were added to 500 mL of RPMI1640, and then the entire volume of 250 mg of L-arginine diluted in 10 mL of PBS was added. ・Basic medium (-) for the L-arginine-unsupplemented group: 50 mL of FBS, 5 mL of Sodium Pyruvate, 5 mL of HEPES, 5 mL of L-glutamine solution, and 5 mL of penicillin / streptomyceline solution were added to 500 mL of RPMI1640, and then 10 mL of PBS was added.

[0153] The viral peptide was used at a concentration of 100 ng / mL under all conditions, as in Example 1.

[0154] <Cells> Peripheral blood from healthy donors was subjected to density centrifugation at room temperature, and the fraction containing peripheral blood mononuclear cells (PBMCs) was collected as a pellet.

[0155] (I) Preparation of MVST samples MVST samples of Example 2AB satisfying conditions A and B, and Example 2A satisfying condition A were prepared by the following method. For Example 2AB, the basic medium (+) of the L-arginine-added group was used as the basic medium, and for Example 2A, the basic medium (-) of the L-arginine-not-added group was used.

[0156] (1) Pre-culture (day 0) Use a 24-well plate and PBMC 2 × 10 ６ Cells were cultured overnight in 1 mL (1 well) of each of the basic media mentioned above.

[0157] (2) First stimulation step and culture step (day 1) 100 μL of each basic medium containing IL-7 (100 ng / mL), IL-15 (50 ng / mL), IL-21 (200 ng / mL), and viral peptide (1 μg / mL) at 10 times the final concentration was prepared. This was added to the wells of (1) so that the final concentration of each component was 1, and the cells were cultured for 24 hours. (day 2) 1 mL of each basic medium containing IL-7 and IL-15 was added to the wells to make the culture volume in each well 1 mL, and the cells were cultured for 24 hours. (day 3) The entire amount of cultured cell solution (1 well) was transferred to one well of a 12-well plate, 2 mL of each basic medium containing IL-7 and IL-15 was added to make the culture volume 4 mL, and the cells were cultured. (Day 6) The entire volume of cultured cell saturation (1 well) was transferred to a T25 flask inverted vertically, and 4 mL of each basic medium containing IL-7 and IL-15 was added to make a total culture volume of 8 mL, and the cells were cultured. (Day 9) The entire volume of cell saturation cultured in the flask was transferred to a T25 flask inverted horizontally, and 8 mL of each basic medium containing IL-7 and IL-15 was added to make a total culture volume of 16 mL, and the cells were cultured. (Day 13) If the growth was good, the cells were transferred to a T75 flask inverted horizontally, and 14 mL of each basic medium containing IL-7 and IL-15 was added to make a total culture volume of 30 mL, and the cells were cultured. On the other hand, if cell clusters could not be seen with the naked eye, or if there was no change in the color of the medium, it was judged that the growth was poor. If cell growth was poor, instead of transferring to a new flask, 8 mL of the 16 mL of culture medium was removed from the flask being used, and 8 mL of each basic medium containing IL-7 and IL-15 was added (half-volume medium exchange), and the culture was continued. (Day 15) The entire cell saturation was collected in a 50 mL centrifuge tube, the supernatant was removed by centrifugation, and then the cells were suspended in each of the basic media and the cell concentration was measured.

[0158] (3) Second stimulation step and culture step (day 15: 2nd day 0) Following the first stimulation (day 15), the second stimulation step was started (2nd day 0). Specifically, 2 × 10⁻¹⁶ of the culture medium from day 15 was used. ６Cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were suspended in 500 μL of the basic medium containing IL-7, IL-15, IL-21, IL-12, IL-18, TL-1A, and viral peptides, and cultured overnight in one well of a 24-well plate. (day16: 2nd day1) 500 μL of the basic medium containing IL-7 and IL-15 was added to the well to make a culture volume of 500 μL, and the cells were cultured. (day18: 2nd day3) The entire cultured cell saturation (1 well) was transferred to one well of a 12-well plate, 3 mL of the basic medium containing IL-7 and IL-15 was added to make a culture volume of 4 mL, and the cells were cultured. (Day 21: 2nd Day 6) The entire volume of cultured cell saturation (1 well) was transferred to a T25 flask inverted vertically, and 4 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 8 mL, and the cells were cultured. (Day 24: 2nd Day 9) The entire volume of cell saturation cultured in the flask was transferred to a T25 flask inverted horizontally, and 8 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 16 mL, and the cells were cultured. (Day 27: 2nd Day 12) The entire volume of cell saturation cultured in the flask was transferred to a T25 flask inverted horizontally, and 14 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 30 mL, and the cells were cultured. (Day 29: 2nd Day 14) The cell concentration in the cultured cell saturation was measured, and the total number of cells was calculated.

[0159] (4) Preparation of analysis samples 1.5 × 10⁻¹⁶ samples from the cell solution at each of the predetermined time periods (day 15 and day 29) ６ Cells were separated and the supernatant was removed by centrifugation. The precipitated cells were suspended in the basic medium, which does not contain cytokines, etc., and cultured overnight. The entire volume of the cultured cell solution was collected in 15 mL centrifuge tubes, and the supernatant was removed by centrifugation. The precipitated cells were used as MVST samples for the evaluation described below.

[0160] (II) Evaluation As described above, MVST samples prepared from PBMCs of seven healthy donors were used to evaluate the following items by flow cytometry analysis. In the figures of this example, Example 2A is also referred to as L-arg(-) and Example 2AB as L-arg(+). (1) Viability Rate Dead cells were stained using a staining reagent (product name Fixable Viability Stain 575V, BD Biosciences) on the MVST samples (day 15) before the start of the second stimulation. Unstained cells were considered viable cells, and viable and dead cells were counted. The viability rate (%) was then calculated as the survival rate using the following formula: Viability rate (%) = [Number of viable cells / (Number of viable cells + Number of dead cells)] × 100

[0161] These results are shown in Figure 4A. Figure 4A is a graph showing the viable cell rate (%) before the start of the second stimulation (day 15). As shown in Figure 4A, compared to Example 2A (L-arg(-)) which satisfies condition A, Example 2AB (L-arg(+)) further satisfies condition B, and thus already showed a higher viable cell rate before the start of the second stimulation (day 15).

[0162] (2) Regarding the MVST sample (day 15) before the start of the second apoptosis stimulus, CD8 ＋ T cells, and MVST (multiple virus-specific CD8 ＋ IFNγ (T cell) ＋ CD8 ＋ T cells were extracted, and the expression of annexin V and cCasp-3 (cleavage caspase 3), which are apoptosis markers, and MCL-1, a mitochondrial anti-apoptotic marker indicating resistance to apoptosis, was evaluated in each cell using the corresponding antibodies.

[0163] These results are shown in Figure 4B. Figure 4B shows the cell population (CD8) before the start of the second stimulus (day 15). ＋ T cells, IFNγ ＋ CD8 ＋ In T cells, cells in which apoptosis signaling has not been initiated (AnnexinV － cCasp-3 －The graph shows the percentage (%) of cells expressing MCL-1, and the graph shows the frequency of expression of the anti-apoptotic marker (MCL-1). Specifically, the frequency of MCL-1 expression (%) refers to the percentage of cells expressing MCL-1 in the aforementioned cell group (MCL-1 ＋ The percentage of cells was expressed as (%).

[0164] As shown in Figure 4B, in either cell group (CD8 ＋ T cells, IFNγ ＋ CD8 ＋ In T cells, compared to Example 2A (L-arg(-)) which met condition A, Example 2AB (L-arg(+)) further met condition B, resulting in enhanced expression of the anti-apoptotic marker (MCL-1). Corresponding to these results, compared to Example 2A (L-arg(-)), Example 2AB (L-arg(+)) showed enhanced expression of cells (AnnexinV) in which the apoptotic signal had not been initiated. － cCasp-3 － A further increase in the proportion of (1) viable cells was confirmed. Furthermore, based on the (1) viable cell rate and the (2) apoptosis evaluation results, it can be said that by satisfying condition A and also condition B, the MVST sample better prevents the initiation of apoptosis and exhibits a superior anti-apoptotic effect. CD8 ＋ The frequency of MT-1 staining in T cells was higher in all samples of Example 2AB (L-arg(+)) compared to Example 2A (L-arg(-)) (n=5, p=0.0625). This is because Example 2AB, by further adding condition B, showed CD8 staining. ＋ This indicates an increase in mitochondrial membrane potential (MMP) in T cells. Furthermore, since the anti-apoptotic marker MCL-1 is a mitochondrial marker, the maintenance of membrane potential and increased MCL-1 expression suggest that the mitochondrial biosynthesis system is maintained in the aforementioned cell population.

[0165] (3) Regarding the MVST sample (day 15) before the start of the second cell exhaustion stimulus, cCasp3 － CD8 ＋ T cells, MVST (multiple virus-specific CD8 ＋ Casp3 (T cell) －IFNγ ＋ CD8 ＋ CD8 T cells were extracted, and for each cell, the expression of cell exhaustion markers (PD-1, TIM-3) was evaluated.

[0166] These results are shown in FIG. 4C. FIG. 4C is a graph showing the expression frequencies of the cell exhaustion markers (PD-1, TIM-3) in the cell group (Casp3 － CD8 ＋ T cells, Casp3 － IFNγ ＋ CD8 ＋ T cells) before the start of the second stimulation (day 15). The expression frequency (%) of the marker is specifically the ratio (%) of the cells (PD-1 ＋ cells, TIM-3 ＋ cells) expressing the marker in the cell group.

[0167] As shown in FIG. 4C, for any cell group (Casp3 － CD8 ＋ T cells, Casp3 － IFNγ ＋ CD8 ＋ T cells), Example 2AB (L-arg(+)) further satisfies condition B compared to Example 2A (L-arg(-)) that satisfies condition A, and the expression of the cell exhaustion marker is more suppressed.

[0168] (4) Cell differentiation For the MVST sample (day 15) before the start of the second stimulation, CD8 ＋ CCR7 － CD45RA ＋／－ T cells were extracted, and the expression of differentiation-related markers (CD27, CD28, CD62L) was evaluated.

[0169] These results are shown in FIG. 4D. FIG. 4D is a graph showing the expression frequencies of the differentiation-related markers (CD27, CD28, CD62L) in the cell group (CD8 ＋ CR7 － CD45RA ＋／－ T cells) before the start of the second stimulation. The expression frequency (%) of the marker is specifically the ratio (%) of the cells (CD27 ＋ cells, CD28＋ cells, CD62L ＋ The percentage of cells was expressed as (%).

[0170] As shown in Figure 4D, compared to Example 2A (L-arg(-)) which satisfies condition A, Example 2AB (L-arg(+)) maintained significantly higher expression of the differentiation-related markers (CD27, CD62L). Since the expression of these differentiation-related markers decreases as differentiation progresses, these results confirm an effect of suppressing hyperdifferentiation. Therefore, even when condition B is added, for example from the viewpoint of anti-apoptotic effect, it was found that the resulting cell population did not induce hyperdifferentiation, and a differentiation-suppressing effect was observed.

[0171] (5) For the MVST samples before the start of the second stimulus (day 15) and after the second stimulus (day 29) of the effector function, CD8 ＋ T cells were extracted, and the production and expression of cytokines involved in effector function in immunity (IFNγ production, TNF-α production, CD107a expression) were evaluated.

[0172] These results are shown in Figure 4E. Figure 4E shows CD8 before the start of the second stimulus (day 15) and after the second stimulus (day 29). ＋ This graph shows the production rate and expression frequency of cytokines in T cells. The production rate (%) of IFNγ or TNF-α specifically refers to the total CD8 ＋ The expression frequency of CD107a is expressed as the percentage of IFNγ or TNF-α production relative to T cells, and specifically, the CD8 ＋ T cells expressing the aforementioned CD107a (CD107a ＋ The percentage of cells was expressed as (%).

[0173] As shown in Figure 4E, compared to Example 2A (L-arg(-)) which satisfies condition A, Example 2AB (L-arg(+)) further satisfies condition B, resulting in a greater increase in the cytokine. Specifically, before the second stimulation (day 15), CD107a expression tended to be low, but after the second stimulation (day 29), an increase in CD107a expression, which indicates cytotoxicity, was confirmed in all cases, and an increase in IFNγ production and TNF-α production was also confirmed in 6 out of 7 cases. This result means that, according to Example 2AB, before the second stimulation (Day 15), cytotoxicity temporarily decreased due to the anti-exhaustion effect of (3) and the hyperdifferentiation inhibitory effect of (4), but after the second stimulation (day 29), effector function was acquired.

[0174] (6) Simultaneous secretion and expression of cytokines Based on the data obtained in (5) above, the simultaneous secretion and expression of the cytokines was evaluated using SPICE and PESTLE software.

[0175] These results are shown in Figure 4F. Figure 4F shows CD8 before the start of the second stimulus (day 15) and after the second stimulus (day 29). ＋ This graph shows the ability of T cells to simultaneously secrete and express cytokines. In Figure 4F, "g" represents IFNγ, "T" represents TNF-α, and "7" represents CD107a. "+" means that the secretion or production of the corresponding cytokine was confirmed, and "-" means that the secretion or production of the corresponding cytokine was not confirmed. In Figure 4F, the vertical axis is (%) simultaneous secretion cells / CD8 ＋ It is a T cell, and all CD8 ＋ This is shown as the proportion of cells that simultaneously secrete and express each type of T cell (the same applies below).

[0176] As shown in Figure 4F, in both Example 2A (L-arg(-)) and Example 2AB (L-arg(+)), the frequency of simultaneous cytokine secretion and expression increased from day 15 to day 29. In particular, compared to Example 2A (L-arg(-)) which met condition A, Example 2AB (L-arg(+)) further met condition B, resulting in an even greater increase in the frequency of simultaneous secretion and expression. Specifically, before the start of the second stimulation (day 15), the frequency of simultaneous occurrence of two or more of IFNγ secretion, TNF-α secretion, and CD107a expression was low, but as with (5) above, after the second stimulation (day 29), the frequency of simultaneous occurrence of two or more of IFNγ secretion, TNF-α secretion, and CD107a expression increased further. In addition, after the second stimulation (day 29), the expression of CD107a alone, which represents cytotoxic activity, also increased. In particular, Example 2AB showed a greater increase in the simultaneous secretion and expression frequency of the three types of cytokines.

[0177] [Example 3] PBMCs were cultured using a culture method that satisfies conditions A and B, and further using a culture method that satisfies condition D, to produce a group of multiple virus-specific T cells (MVST cells), and their proliferative capacity and function were confirmed.

[0178] <Materials> Culture medium and culture medium additive reagents RPMI-1640 (containing L-glutamine and phenol red), Fujifilm Wako Pure Chemical Industries, 183-02023 Fetal bovine serum (FBS) NB, Nichirei Bioscience, 174012 Penicillin-Streptomycin (penstrep), liquid, GIBCO, 15140-122 Sodium Pyruvate, GIBCO, 11360070 Insulin-Transferrin-Selenium Supplement (ITS) (100X), GIBCO, 41400-045 Condition A: T cell growth factor (cytokine) Same as in Example 1 Condition B: L-arginine medium L-arginine, Sigma-Aldrich, A5006-100G D-PBS, Wako, 045-29795 Viral antigen overlap peptide (viral peptide) PepTivator CMV pp65, Miltenyi Biotec, 130-093-435 PepTivator CMV IE1, Miltenyi Biotec, 130-093-494 PepTivator EBV EBNA1, Miltenyi Biotec, 130-093-614 PepTivator EBV LMP2A, Miltenyi Biotec, 130-093-616 PepTivator EBV BZLF1, Miltenyi Biotec, 130-093-612 PepTivator AdV Hexon, Miltenyi Biotec, 130-093-496 PepTivator AdV Penton, Miltenyi Biotec, 130-093-777 PepMix HHV6 (U90), JPT, PM-HHV6-U90 PepMix HHV6 (U54), JPT, PM-HHV6-U54 PepTivator BKV VP1, Miltenyi Biotec, 130-131-250 PepTivator BKV Large T, Miltenyi Biotec, 130-131-541 Condition D: Maturation and activation of dendritic cells Same as in Example 1 above

[0179] <Culture Medium> ・Basic medium (+) for the L-arginine-supplemented group: 50 mL of FBS, 5 mL of ITS, 5 mL of Sodium Pyruvate, and 5 mL of penicillin / streptomyceline solution were added to 500 mL of RPMI1640, and then the entire volume of 250 mg of L-arginine diluted in 10 mL of PBS was added. ・Basic medium (-) for the L-arginine-free group: 500 mL of RPMI1640, 50 mL of FBS, 5 mL of ITS, 5 mL of Sodium Pyruvate, and 5 mL of penicillin / streptomyceline solution were added, and then 10 mL of PBS was added.

[0180] The viral peptide was used at a concentration of 100 ng / mL under all conditions, as in Example 1.

[0181] <Cells> Peripheral blood from healthy donors was subjected to density centrifugation at room temperature, and the fraction containing peripheral blood mononuclear cells (PBMCs) was collected as a pellet.

[0182] (I) MVST samples were prepared for Example 3ABD, satisfying conditions A, B, and D, and for Example 3AD, satisfying conditions A and D. For Example 3ABD, the basic medium (+) of the L-arginine-added group was used as the basic medium, and for Example 3AD, the basic medium (-) of the L-arginine-free group was used.

[0183] (1) Pre-culture (day 0) The same procedure as in Example 1AD in Example 1 above was followed.

[0184] (2) First stimulation step and culture step Culture was carried out in the same manner as in Example 1AD from day 1 to day 14. On day 14, the entire amount of cultured cell solution was collected in a 50 mL centrifuge tube, the cell concentration was measured, and the total number of cells was calculated.

[0185] (3) Second stimulation step and culture step (day 14: 2nd day 0) After the first stimulation (day 14), the second stimulation step was started (2nd day 0). Of the culture medium from day 14, 1 × 10 ６The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were suspended in 250 μL of the basic medium containing IL-7, IL-15, IL-21, IL-12, IL-18, TL-1A, and viral peptides, and cultured overnight in one well of a 48-well plate. (day15: 2nd day1) 250 μL of the basic medium containing IL-7 and IL-15 was added to the well to make a culture volume of 500 μL, and the cells were cultured. (day18: 2nd day4) The entire cultured cell solution (1 well) was transferred to one well of a 12-well plate, 3.5 μL of the basic medium containing IL-7 and IL-15 was added to make a culture volume of 4 mL, and the cells were cultured. (Day 21: 2nd Day 7) The entire volume of cultured cell saturation (1 well) was transferred to a T25 flask inverted vertically, and 4 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 8 mL, and the cells were cultured. (Day 24: 2nd Day 10) The entire volume of cell saturation cultured in the flask was transferred to a T75 flask inverted horizontally, and 22 mL of the basic medium containing IL-7 and IL-15 was added to make a total culture volume of 30 mL, and the cells were cultured. (Day 28: 2nd Day 14) The cell concentration in the cultured cell saturation was measured, and the total number of cells was calculated.

[0186] (4) Preparation of analysis sample (day 29) 1.5 × 10⁶ of the cell fluid after the second stimulation (day 28) ６ Cells were separated and the supernatant was removed by centrifugation. The precipitated cells were suspended in the basic medium, which does not contain cytokines, etc., and cultured overnight. The entire volume of the cell saturation from day 29 was collected in a 15 mL centrifuge tube, the supernatant was removed by centrifugation, and the precipitated cells were used as an MVST sample for the evaluation described below.

[0187] (II) Evaluation Using MVST samples prepared from PBMCs of five healthy donors as described above, the following items were evaluated by flow cytometry analysis.

[0188] (1) Viability Rate The viability rate (%) of the MVST sample (prepared on day 29) after the second stimulation was evaluated. Specifically, dead cells in the MVST sample were stained using a staining reagent (product name LIVE / DEAD Fixable aqua dead cell stein kit). Unstained cells were considered viable cells, and the number of viable and dead cells was counted. The spermatocyte rate (%) was then calculated as the survival rate using the following formula: Viability rate (%) = [Number of viable cells / (Number of viable cells + Number of dead cells)] × 100

[0189] These results are shown in Figure 5A. Figure 5A is a graph showing the viability rate (%). As shown in Figure 5A, compared to Example 3AD, which satisfies conditions A and D, Example 3ABD showed a higher viability rate by also satisfying condition B.

[0190] (2) For the MVST sample (day 29) after the second cell frequency stimulation, virus-specific T cells (CD3) were analyzed in the same manner as in Example 1. ＋ The frequency of MVST cells was evaluated.

[0191] These results are shown in Figure 5B. Figure 5B shows the frequency of multiple virus-specific T cells showing IFNγ production in MVST samples. As shown in Figure 5B, compared to Example 3AD which satisfies conditions A and D, Example 3ABD, which also satisfies condition B, showed a further increase in the frequency of multiple virus-specific T cells showing IFNγ production in 4 out of 5 MVST samples.

[0192] [Example 4] PBMCs were cultured using a culture method that satisfies condition C, and a population of multiple virus-specific T cells (MVST cells) was produced. Their proliferative capacity and function were then confirmed.

[0193] In the aforementioned Examples 1 to 3, IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A were used as the second stimulus cytokines under Condition A. On the other hand, in Example 4, a new Condition A (hereinafter referred to as Condition A') was set in which IL-7, IL-15, and IL-21 were used as the second stimulus cytokines, with IL-12, IL-18, and TL1A not being used.

[0194] (I) Preparation of antigen-presenting cells (I-1) Preparation of peptide-pulsed γδT cells As antigen-presenting cells under condition C, peptide-pulsed γδT cells were prepared by the method shown below.

[0195] <Materials> Culture medium and culture medium additive reagents RPMI-1640 (containing L-glutamine and phenol red), Fujifilm Wako Pure Chemical Industries, 183-02023 Fetal bovine serum (FBS) NB, Nichirei Bioscience, 174012 Penicillin-Streptomycin (penstrep), liquid, GIBCO, 15140-122 HEPES solution (1M), SIGMA, H0887-100ML Sodium Pyruvate, GIBCO, 11360070 Zometa® intravenous infusion 4 mg / 100 mL, 3999423A2024, Novartis Pharma K.K. Human IL-2 IS, premium grade, Miltenyi Biotech, 130-097-748

[0196] <Culture Medium> ・Basic medium 500 mL of RPMI1640 was mixed with 50 mL of FBS, 5 mL of penicillin / streptomycin solution, 5 mL of sodium pyruvate, and 5 mL of HEPES solution. <Cytokines> IL-2 was added to the basic medium to a final concentration of 1000 U / mL.

[0197] <Culture Method> (Day 0) Peripheral blood from healthy donors was subjected to density centrifugation at room temperature, and the fraction containing peripheral blood mononuclear cells (PBMCs) was collected as a pellet. Collected PBMCs 1 × 10 ６(day 2) Cells were suspended in 1 mL of the basic medium containing IL-2, and 34 μL of Zometa was added. The cells were then cultured in one well of a 48-well plate for 48 hours. (day 4) Half of the medium in the wells of the well plate was replaced with 0.5 mL of the basic medium containing IL-2, and the cells were cultured for 48 hours. (day 6) The entire volume of cultured cells (1 well) was transferred to one well of a 24-well plate, and 1 mL of the basic medium containing IL-2 was added to make a total culture volume of 2 mL. The cells were then cultured. (day 8) The entire volume of cultured cells (1 well) was transferred to one well of a 12-well plate, and 2 mL of the basic medium containing IL-2 was added to make a total culture volume of 4 mL. The cells were then cultured. (day 8) The entire volume of cultured cells (1 well) was transferred to a vertically oriented T25 flask, and 4 mL of the basic medium containing IL-2 was added to make a total culture volume of 8 mL. The cells were then cultured. (Day 10) The flask was turned from vertical to horizontal, and 8 mL of the basic medium containing IL-2 was added to bring the culture volume to 16 mL, and the cells were cultured. (Day 12) The entire volume of cell solution cultured in the flask was transferred to a horizontally positioned T75 flask, and 16 mL of the basic medium containing IL-2 was added to bring the culture volume to 32 mL, and the cells were cultured.

[0198] (Day 14: Peptide pulse to γδT cells) The entire volume of cultured cell saturation was collected in a 50 mL centrifuge tube, and 1 × 10⁶ cells were selected from the obtained cells. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were added to 131 μL of the IL-2-free basic medium, 1 × 10⁶ cells. ６ The cell solution was diluted to cells / mL. Each antigen peptide was added to this cell solution at a final concentration of 350 ng / mL. The entire cell solution was then transferred to a 96-well flat plate and cultured for 3 hours. This process was designated as peptide pulse (X). After peptide pulse (X), the entire cell solution from one well of the plate was placed in a 15 mL centrifuge tube, and 5 mL of the basic medium was added. Centrifugation was performed at 1500 rpm for 5 minutes to remove excess peptides and supernatant. This centrifugation process was repeated a total of three times. The cell population obtained as the precipitate fraction was used as "peptide-pulsed γδ T cells" in the second stimulation step of condition C, which will be described later.

[0199] (I-2) Peptide-pulsed PHA-BLAST cells were prepared as antigen-presenting cells under preparation condition C' by the following method.

[0200] <Materials> Culture medium and reagents: Lectin derived from Phaseolus vulgaris (bean), 61764-5MG, Sigma-Aldrich Human IL-2 IS, premium grade, Miltenyi Biotech, 130-097-748; RPMI-1640 (containing L-glutamine and phenol red), Fujifilm Wako Pure Chemical Industries, 183-02023; Fetal bovine serum (FBS) NB, Nichirei Bioscience, 174012; Penicillin-Streptomycin (penstrep), liquid, GIBCO, 15140-122; Human IL-2 IS, premium grade, Miltenyi Biotech, 130-097-748

[0201] <Cells> PHA-BLAST cells were prepared from PBMCs using the following culture method. <Basic medium> 500 mL of RPMI1640 was mixed with 50 mL of FBS, 5 mL of penicillin / streptomycin solution, 5 mL of sodium pyruvate, and 5 mL of HEPES solution. <Cytokines> IL-2 was added to the basic medium to a final concentration of 100 U / mL.

[0202] <Culture Method> (Day 0) Peripheral blood from healthy donors was subjected to density centrifugation at room temperature, and the fraction containing peripheral blood mononuclear cells (PBMCs) was collected as a pellet. Collected PBMCs 1 × 10 ６(day 2) Cells were suspended in 1 mL of the basic medium containing IL-2, and 0.5 μL of PHA (phytohemoaglutinin) was added, followed by incubation in one well of a 48-well plate for 48 hours. (day 4) Half of the medium in the wells of the well plate was replaced with 0.5 mL of the basic medium containing IL-2, and the cells were incubated for 48 hours. (day 6) The entire volume of cultured cells (1 well) was transferred to one well of a 24-well plate, and 1 mL of the basic medium containing IL-2 was added to make a total culture volume of 2 mL, and the cells were incubated. (day 6) The entire volume of cultured cells (1 well) was transferred to one well of a 12-well plate, and 2 mL of the basic medium containing IL-2 was added to make a total culture volume of 4 mL, and the cells were incubated. (Day 8) The entire volume of cultured cell saturation (1 well) was transferred to a T25 flask turned vertically, and 4 mL of the basic medium containing IL-2 was added to make a total culture volume of 8 mL, and the cells were cultured. (Day 10) The flask was turned from vertical to horizontal, and another 8 mL of the basic medium containing IL-2 was added to make a total culture volume of 16 mL, and the cells were cultured. (Day 12) The entire volume of cell saturation cultured in the flask was transferred to a T75 flask turned horizontally, and 16 mL of the basic medium containing IL-2 was added to make a total culture volume of 32 mL, and the cells were cultured.

[0203] (Day 14: Peptide pulse to PHA-BLAST cells) The entire volume of cultured cell saturation was collected in a 50 mL centrifuge tube, and 1 × 10⁶ cells were selected from the obtained cells. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The peptide pulse (X) was performed on the precipitated cells in the same manner as in (I-1), except that the culture medium was used. Then, the cells were centrifuged in the same manner as in (I-1), and the cell population obtained as the precipitated fraction was used as "peptide-pulsed PHA-BLAST cells" in the second stimulation step of condition C' described later.

[0204] (II) Preparation of MVST samples MVST samples of Example 3A' satisfying condition A', Example 3A'C satisfying conditions A' and C, and Example 3A'C' satisfying conditions A' and C' were prepared by the following method.

[0205] <Materials> Culture medium and reagents under the same conditions as in Example 1 A': T cell growth factor (cytokine) Human IL-7, premium grade, Miltenyi Biotech, 130-095-363 Human IL-15, premium grade, Miltenyi Biotech, 130-095-765 Human IL-21, Pepro Tec, AF-200-21

[0206] <Basic Medium> 500 mL of RPMI1640 was mixed with 50 mL of FBS, 5 mL of penicillin / streptomycete solution, 4 mL of ITS, 5 mL of sodium pyruvate, and 5 mL of HEPES solution. <Reagent Concentrations> Unless otherwise specified, the reagents were used at the same final concentrations as in Examples 1 and 2. <Cells> A pellet of peripheral blood mononuclear cells (PBMCs) prepared in the same manner as in Example 2 was used.

[0207] (Example 4A') (1) Pre-culture (day 0) This was carried out in the same manner as in Example 2A of Example 2. (2) First stimulation step and culture step On day 1, the first stimulation was started in the same manner as in Example 1A, and on day 14, the entire amount of cultured cell solution was collected in a 50 mL centrifuge tube and the cell concentration was measured. (3) Second stimulation step and culture step (day 14: 2nd day 0) After the first stimulation (day 14), 1 × 10 of the cultured cells were collected from the cell solution. ６ Cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were suspended in 500 μL of the basic medium containing IL-7, IL-15, IL-21, and the same viral peptide as in the first stimulation step, as in Example 1A, and cultured overnight in the wells of a 48-well plate 1. (day 15-28: 2nd day 1-2nd day 14) Culture was carried out in the same manner as in Example 1A. (4) Preparation of analysis sample (day 28) An MVST sample was prepared from the cell solution on day 28 in the same manner as in Example 1A.

[0208] (Example 4A'C) (1) Pre-culture (day 0) This was carried out in the same manner as in Example 4A'. (2) First stimulation step and culture step This was carried out in the same manner as in Example 4A'. (3) Second stimulation step and culture step (day 14: 2nd day 0) In the same manner as in Example 4A', 1 × 10 of the cultured cells were added to the cell solution on day 14. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were divided into 1 × 10⁶ peptide-pulsed γδ T cells. ６ The cells were suspended in 500 μL of the basic medium containing IL-7, IL-15, and IL-21, and cultured overnight in one well of a 48-well plate. (day 15-28: 2nd day 1-2nd day 14) Culture was carried out in the same manner as in Example 1A. (4) Preparation of analytical samples (day 28) An MVST sample was prepared from the cell saturation on day 28 in the same manner as in Example 1A.

[0209] (Example 4A'C') (1) Pre-culture (day 0) This was carried out in the same manner as in Example 4A'. (2) First stimulation step and culture step This was carried out in the same manner as in Example 4A'. (3) Second stimulation step and culture step (day 14: 2nd day 0) In the same manner as in Example 4A', 1 × 10 of the cultured cells were added to the cell solution on day 14. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The collected cells were divided into 1 × 10⁶ peptide pulsed PHA-BLAST cells. ６ The cells were suspended in 500 μL of the basic medium containing IL-7, IL-15, and IL-21, and cultured overnight in one well of a 48-well plate. (day 15-28: 2nd day 1-2nd day 14) Culture was carried out in the same manner as in Example 1A. (4) Preparation of analytical samples (day 28) An MVST sample was prepared from the cell saturation on day 28 in the same manner as in Example 1A.

[0210] (III) Evaluation Using the MVST samples prepared from PBMCs of seven healthy donors as described above, the following items were evaluated by flow cytometry analysis. Hereinafter, each example will also be referred to as Example 4A' (+pep), Example 4A'C (+γδT), and Example 4A'C' (+PHA).

[0211] (1) In the cell proliferation cultures of Example 4A' (+pep), Example 4A'C (+γδT), and Example 4A'C' (+PHA), the number of cells after the second stimulation (day 28) was counted.

[0212] These results are shown in Figure 6A. Figure 6A is a graph showing the number of cells after the second stimulus (day 28). In Figure 6A, the vertical axis represents the number of cells (×10). ６ (cells). As shown in Figure 6A, compared to Example 4A' (+pep) which satisfies condition A', Examples 4A'C (+γδT) and 4A'C' (+PHA) showed higher cell proliferation when further satisfying condition C or condition C'.

[0213] (2) Regarding the MVST sample after the second cell exhaustion stimulation (prepared on day 28), CD8 ＋ T cells were extracted, and the expression of cell exhaustion markers (PD-1, TIM-3) was evaluated.

[0214] These results are shown in Figure 6B. Figure 6B shows CD8 after the second stimulus (day 28). ＋ This graph shows the expression frequency of the aforementioned cell exhaustion markers (PD-1, TIM-3) in T cells. Specifically, the expression frequency (%) of the markers is CD8 ＋ Cells expressing the aforementioned marker in T cells (PD-1 ＋ cells, TIM-3 ＋ The expression was expressed as a percentage (%) of cells. As shown in Figure 6B, compared to Example 4A' (+pep) which satisfies condition A', Example 4A'C (+γδT) and Example 4A'C' (+PHA) showed further suppression of the cell exhaustion marker when they also satisfied condition C or condition C'. Among these, Example 3A'C (+γδT) showed the best anti-exhaustion effect.

[0215] (3) Content ratio of naive T cells The MVST samples (day 28) of Example 4A' (+pep), Example 4A'C (+γδT), and Example 4A'C' (+PHA) were analyzed by flow cytometry to determine CD8 ＋ T cells were extracted, and the expression of CD45RA and CCR7 was evaluated.

[0216] These results are shown in Figure 6C. Figure 6C shows CD8 after the second stimulus (day 28). ＋ CCR7 in T cells ＋ CD45RA ＋ Cell content (naive T cells) and CCR7 － CD45RA ＋ This graph shows the proportion of cells (TEMRA cells). As shown in Figure 6C, compared to Examples 4A' (+pep) and 4A'C' (+PHA) which satisfy condition A', Example 4A'C (+γδT) tended to have the lowest proportion of naive T cells, which can be a risk factor for graft-versus-host disease (GVHD). Furthermore, Example 4A'C (+γδT) contained the most differentiated TEMRA cells (Effector Memory T cell with CD45RA, CCR7), suggesting a decrease in effector function. － CD45RA ＋ The proportion of ) was also the lowest.

[0217] (4) Regarding the MVST sample (day 28) after the second stimulation of the effector function, CD8 ＋ T cells were extracted, and the production or expression of cytokines involved in effector function (IFNγ, TNF-α, and CD107a) was evaluated. Specifically, cells expressing at least one of the above cytokines (IFNγ, TNF-α, and CD107a) were defined as Virus-Specific T cells (VSTs), and the total number of MVSTs was evaluated. The total number of MVSTs was defined by the following formula: Total number of MVSTs = [Total number of cells] × [Visibility rate by flow cytometry] × [CD3 ＋ T cell frequency] × [CD8 ＋ T cell frequency] × [CD8 ＋[MVST frequency in T cells]

[0218] These results are shown in Figure 6D. Figure 6D shows CD8 after the second stimulus (day 29). ＋ This graph shows the production or expression of cytokines in T cells.

[0219] As shown in Figure 6D, Example 3A'C (+γδT) showed higher production and expression of all cytokines compared to Example 3A' (+pep) which met condition A'. Furthermore, compared to Example 3A'C' (+PHA), Example 3A'C (+γδT) showed a further increase in the production rate of IFNγ and TNF-α, and a further increase in CD107a expression in all MVST samples. Also, when multiple virus-specific T cells (MVSTs) were defined as cells expressing at least one of IFNγ, TNF-α, and CD107a, the total number of MVSTs was highest in Example 3A'C (+γδT). These results indicate that co-culturing γδT cells with PBMCs is the most efficient way to induce VSTs with effector function.

[0220] (5) Simultaneous secretion and expression of cytokines Based on the data obtained in (4) above, the simultaneous secretion and expression of the cytokines was evaluated using SPICE and PESTLE software.

[0221] These results are shown in Figure 6E. Figure 6E shows CD8 after the second stimulus (day 29). ＋ This graph shows the ability of T cells to simultaneously secrete and express cytokines. In Figure 4F, "g" represents IFNγ, "T" represents TNF-α, and "7" represents CD107a. "+" means that the corresponding cytokine was secreted or produced, and "-" means that the corresponding cytokine was not secreted or produced. In Figure 6E, the vertical axis is (%) simultaneous secretion cells / CD8 ＋ This is the frequency (%) of simultaneous secretion and expression of T cells, and all CD8 ＋ This is shown as the proportion of T cells that simultaneously secrete and express each type of secretion.

[0222] As shown in Figure 6E, compared to Example 3A' (+pep) which satisfies condition A', Examples 3A'C (+γδT) and 3A'C' (+PHA), which were further co-cultured with the antigen-presenting cells, showed a further increase in the frequency of simultaneous expression and secretion of two or more cytokines. In particular, Example 3A'C (+γδT), which was co-cultured with γδT cells as the antigen-presenting cells, showed an even greater increase in the frequency of simultaneous occurrence of two or more IFNγ secretion, TNF-α secretion, and CD107a expression. Thus, it was found that co-culture with the antigen-presenting cells, especially with γδT cells, can very efficiently induce VSTs with high simultaneous cytokine secretion and expression capabilities.

[0223] [Example 5] PBMCs were cultured using a culture method that satisfies conditions A, B, and D, and also a culture method that satisfies condition C, to produce a population of multiple virus-specific T cells (MVST cells), and their proliferative capacity and function were confirmed.

[0224] (I) Preparation of antigen-presenting cells Peptide-pulsed γδT cells were prepared in the same manner as in Example 4, except that the peptide pulse to γδT cells on day 14 was performed under the following conditions.

[0225] (Day 14: Peptide pulse to γδT cells) The entire volume of cultured cell saturation was collected in a 50 mL centrifuge tube, and 2 × 10⁶ cells were selected from the obtained cells. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were placed in 666 μL of the IL-2-free basic medium, 3 × 10⁶ cells. ６ The cell solution was diluted to cells / mL. Each antigen peptide was added to this cell solution at a final concentration of 100 ng / mL. The entire cell solution was then transferred to a 48-well plate and cultured for 3 hours. This process was designated as peptide pulse (Y). After peptide pulse (Y), the entire cell solution from one well of the plate was placed in a 15 mL centrifuge tube, and 10 mL of the basic medium was added. Centrifugation was performed at 1500 rpm for 5 minutes to remove excess peptides and supernatant. This centrifugation process was repeated a total of three times. The cell population obtained as the precipitate fraction was used as "peptide-pulsed γδ T cells" in the second stimulation step of condition C, which will be described later.

[0226] (II) Preparation of MVST samples MVST samples of Example 5ABCD satisfying conditions A, B, C, and D, and Example 5ABD satisfying conditions A, B, and D were prepared by the following method.

[0227] <Materials> Culture medium and culture medium additive reagents Same as in Example 1 Condition A: T cell growth factor (cytokine) Same as in Example 1 Condition B: L-arginine medium Same as in Example 2 Condition C: γδ T cells γδ T cells prepared in (I) above, pulsed with the peptide (Y) Condition D: Maturation and activation of dendritic cells Same as in Example 1

[0228] <Basic Medium> The basic medium (+) of the L-arginine supplement group used in Example 3 was used. <Reagent Concentration> Unless otherwise specified, the various reagents were used at the same final concentrations as in Example 1 and Example 3. <Cells> A pellet of peripheral blood mononuclear cells (PBMCs) prepared in the same manner as in Example 3 was used.

[0229] (Example 5ABCD) (1) Pre-culture was carried out in the same manner as in Example 1AD of Example 1. (2) First stimulation step and culture step On day 1, the first stimulation was started in the same manner as in Example 3ABD, and on day 14, the entire amount of cultured cell solution was collected in a 50 mL centrifuge tube, the cell concentration was measured, and the total number of cells was calculated. (3) Second stimulation step and culture step (day 14: 2nd day 0) In the same manner as in Example 3ABD, after the first stimulation (day 14), 1 × 10 of the cultured cells were collected from the cell solution. ６ The cells were collected in a 15 mL centrifuge tube, and the supernatant was removed by centrifugation. The precipitated cells were 1 × 10 γδ T cells treated with the peptide pulse (Y). ６ Together with cells, the cells were suspended in 250 μL of the basic medium containing IL-7, IL-15, IL-21, IL-12, IL-18, and TL-1A, and cultured overnight in one well of a 48-well plate. (day 15-28: 2nd day 1-2nd day 14) Culture was carried out in the same manner as in Example 3ABD. (4) Preparation of analytical samples (day 29) The MVST sample for day 29 was prepared in the same manner as in Example 3ABD.

[0230] (Example 5ABD) (1) Pre-culture was carried out in the same manner as in Example 5ABCD. (2) First stimulation step and culture step were carried out in the same manner as in Example 5ABCD. (3) Second stimulation step and culture step were carried out in the same manner as in Example 3ABD. (4) Preparation of analysis sample The MVST sample for day 29 was prepared in the same manner as in Example 3ABD.

[0231] (III) Evaluation Using the MVST samples prepared from PBMCs of two healthy donors as described above, the following items were evaluated by flow cytometry analysis.

[0232] (1) In the cell proliferation cultures of Examples 5ABD and 5ABCD, the number of cells was counted at the start of pre-culture (day 0), after the first stimulation (day 14), and after the second stimulation (day 28).

[0233] (2) Evaluation of cell function The viable cell rate % and the frequency % of multiple virus-specific T cells showing IFNγ production in the MVST sample were evaluated in the same manner as in Example 3, and the cell frequency % of T cells was evaluated in the same manner as in Example 1.

[0234] These results are shown in Figure 7. Figure 7A shows the changes in cell number over time (cell proliferation) for each donor, with the vertical axis representing the cell number (×10). ６ Figure 7B shows the cell count, T cell frequency, and the frequency of multiple virus-specific T cells (%) that produce IFNγ in the MVST sample from each donor.

[0235] As shown in Figure 7A, both Donor 1 and Donor 2 showed sufficient cell proliferation in the MVST samples of Example 5ABD and Example 5ABCD. Furthermore, Example 5ABCD showed even higher cell proliferation compared to Example 5ABD when co-cultured with γδ T cells. Also, as shown in Figure 7B, both Donor 1 and Donor 2 showed sufficient viability, cell frequency, and frequency of multiple virus-specific T cells producing IFNγ in the MVST samples of Example 5ABD and Example 5ABCD. Furthermore, Example 5ABCD showed a higher viability and T cell frequency compared to Example 5ABD when co-cultured with γδ T cells, and the percentage of multiple virus-specific T cells producing IFNγ in the MVST sample was equivalent to or higher than that of Example 5ABD. From this, it was found that co-culturing with γδ T cells can further increase the proliferation of MVST cells in the MVST cell population (total cells) and improve their proportion.

[0236] [Example 6] PBMCs were cultured using a culture method that satisfies the above conditions A, B, C, and D, and a population of multiple virus-specific T cells (MVST cells) was produced. Their proliferative capacity and function were then confirmed.

[0237] (I) Preparation of MVST samples Following the same procedure as in Examples 3ABCD above, PBMCs were cultured to prepare MVST samples for day 29, and the following evaluations were performed.

[0238] (II) Evaluation The following items were evaluated for the MVST samples prepared from PBMCs of healthy donors as described above.

[0239] (1) In the culture of cell proliferation example 6ABCD, the number of cells was counted at the start of pre-culture (day 0), after the first stimulation (day 14), and after the second stimulation (day 28), and the proliferation rate (fold expansion) was calculated. PBMCs from 15 healthy donors were used to evaluate cell proliferation (n=15).

[0240] (2) Cell frequency 1 (cell surface markers) For the MVST samples from day 29, cell surface markers were evaluated by flow cytometry analysis using antibodies corresponding to the target of detection. For this evaluation, MVST samples obtained from PBMCs of 10 healthy donors were used (n=10).

[0241] (3) Cell frequency 2 (specificity) The cell frequency of the MVST sample from day 29 was evaluated using the same method as in Example 1. That is, the frequency of multiple virus-specific T cells (MVST cells) in the MVST sample was measured. For this evaluation, MVST samples obtained from PBMCs of 15 healthy donors were used (n=15).

[0242] The results of the evaluations described in (1) to (3) above are shown in Figures 8A, 8B, and 8C.

[0243] Figure 8A shows the changes in cell number over time (cell proliferation) for each donor, with the vertical axis representing the cell proliferation rate. As shown in Figure 8A, all donors showed an average cell proliferation of 2000 times after 28 days of culture. Compared to previous literature (Tzannou, Ifigeneia, et al. “Mini” bank of only 8 donors supplies CMV-directed T cells to diverse recipients. Blood advances 3.17 (2019): 2571-2580.), this result indicates the realization of stable large-scale culture.

[0244] Figure 8B shows the cell frequencies of cells possessing various cell surface markers contained in the MVST sample. In Figure 8B, the horizontal axis represents the type of cell surface marker, and the vertical axis represents the cell frequency (%). As shown in Figure 8B, the MVST sample contained less than 1% NK cells, more than 99% T cells, and less than 10% of the T cells were γδ T cells used in the second stimulation step. In addition, nearly 100% of the T cells other than γδ T cells were single T cells positive for CD45RO.

[0245] Figure 8C shows the proportions of IFNγ-producing cells, TNFα-producing cells, and CD107a-producing cells in the MVST sample. The horizontal axis represents the type of specificity indicator, and the vertical axis represents the proportion (%) of each producing cell, i.e., specificity (%). A higher specificity value indicates higher specificity. As shown in the figure, the proportion of IFNγ-producing cells was around 80%, and the proportions of TNFα-producing cells and CD107a-producing cells were similarly high. Thus, from the results in Figures 8A, 8B, and 8C, it was confirmed that the MVST sample contained MVST cells that possessed both specificity and functionality.

[0246] (4) Cytotoxic activity The cytotoxic activity of the MVST sample from day 29 was evaluated. For this evaluation, MVST samples obtained from PBMCs of six healthy donors were used (n=6).

[0247] <Materials> Multiwell plate, flat bottom, 6 wells, BD FALCON, 353046 Basic medium, same as (3) Cell frequency 2 above Viral peptide, same as in Example 3 above Antibody reagent for staining, Calcein AM, Dojindo P378-Cellstain (registered trademark) - PI solution, Fujifilm Wako Pharmaceutical, 34107881 Flow cytometer MACSquant (trademark)

[0248] (4-1) Preparation of target cell solution PHA-BLAST derived from healthy PBMCs used in the preparation of the MVST sample was used as the target cell solution, and the target cell solution was stained with Calcein AM. A new basic medium was added to the stained target cell solution, and 1 × 10⁻⁶ cells were prepared. ６ A target cell solution with cells / mL was prepared.

[0249] (4-2) Preparation of MVST cell solution Add fresh basic medium to the MVST sample from day 29, and 1 × 10 ６ cells / mL, 5×10 ６ cells / mL, 10×10 ６ cells / mL, 20×10 ６ An MVST cell solution containing MVST cells at a concentration of cells / mL was prepared.

[0250] (4-3) Preparation of cell mixtures 100 μL of the target cell solution and 100 μL of four different MVST cell solutions with varying cell concentrations were seeded in each well of a 96-well U plate. This resulted in cell ratios (T:M) of the target cells (T) to the cells in the MVST cell solution (M) of 1:1, 1:5, 1:10, and 1:20, respectively. Seeding was performed in two wells for each ratio. In one of the two wells, all of the viral peptides (final concentration 100 ng / mL) used in the preparation of the MVST sample were added to form the peptide-added group pep(+), and in the other well, no viral peptides were added to form the peptide-free group pep(-). Furthermore, for each of the peptide-added group (pep(+)) and the peptide-non-added group (pep(-)), a control (A) was prepared by seeding 100 μL of the target cell solution with 100 μL of the basic medium without the MVST cell population. The cell ratio (T:M) in control (A) was 1:0. pep(+) T:M 1:0, 1:1, 1:5, 1:10, 1:20 pep(-) T:M 1:0, 1:1, 1:5, 1:10, 1:20

[0251] (4-4) Analysis method The plate is CO ２ After standing for 4 hours under 5% humidity and 37°C, each well was stained with PI and classified into the following three cell populations by flow cytometry: • Viable target cells (= Calcein AM positive and PI negative cells) • Viable MVST cell population (= Calcein AM negative and PI negative cells) • Target cells or MVST cell population in which cell death was induced (= Calcein AM negative and PI positive cells)

[0252] Cytotoxic activity refers to the ability of the created MVST cells to induce cell death in target cells (cytotoxicity), and can be quantified using the following formula, based on the number of target cells surviving in each well. Formulas (1) and (2) below have the same meaning.

[0253]

[0254] A = Number of surviving target cells in control (A) = Number of Calcein AM-positive and PI-negative cells in control (A) Number of target cells in which cell death was induced by MVST cells = A - (Number of surviving target cells in the mixture) = A - (Number of Calcein AM-positive and PI-negative cells in the mixture)

[0255] (4-5) Analysis Results These results are shown in Figure 9. Figure 9 is a graph showing cytotoxic activity. In Figure 9, the horizontal axis shows the mixing ratio (T:M) of target cells and cells in the MVST cell solution, and the vertical axis shows the cytotoxic activity (%). As shown in Figure 9, in the pep(-) system, the cytotoxic activity remained around 0%, regardless of the cell ratio of the target cells (T) and cells in the MVST cell solution (M). In contrast, in the pep(+) system, an increase in cytotoxic activity % was observed with increasing cell ratio of the MVST cell solution. From these results, it was found that the cells contained in the MVST sample obtained in this example did not exhibit cytotoxicity to uninfected cells, but only to infected cells. Therefore, it is thought that the MVST cell group obtained in this example can suppress the development of graft-versus-host disease (GVHD) while maintaining damage to infected cells.

[0256] Although the present invention has been described above with reference to embodiments, the present invention is not limited to the above embodiments. Various modifications to the structure and details of the present invention are possible within the scope of the present invention as can be understood by those skilled in the art.

[0257] This application claims priority based on Japanese Patent Application No. 2024-167547, filed on 26 September 2024, and incorporates all of its disclosures herein.

[0258] According to the present invention, antigen-specific T cells can be efficiently proliferated from peripheral blood mononuclear cells. Therefore, for example, in immunotherapy, it is possible to produce a large quantity of antigen-specific T cells for administration to multiple patients from peripheral blood mononuclear cells of healthy individuals, which is useful in the medical field.

Claims

1. A method for producing antigen-specific T cells, comprising a first stimulation step of culturing peripheral blood mononuclear cells in a medium to which an antigen has been added, and a second stimulation step of further culturing the cells after the first stimulation step in a medium to which a new antigen has been added, wherein the method satisfies condition A. (Condition A) The medium in the first stimulation step contains a first cytokine group, the medium in the second stimulation step contains a second cytokine group, the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not contain IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A.

2. A manufacturing method according to claim 1 that satisfies condition B. (Condition B) At least one of the culture medium in the first stimulation step and the culture medium in the second stimulation step contains arginine.

3. A method for manufacturing according to claim 1 or 2, satisfying condition C. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed.

4. The manufacturing method according to claim 3, wherein the antigen-presenting cell is a γδ T cell.

5. A manufacturing method according to any one of claims 1 to 4, satisfying condition D. (Condition D) In ​​the first stimulation step, Flt3L and pattern recognition receptor ligand are added to the culture medium and culture is performed.

6. A method for manufacturing according to any one of claims 1 to 5, satisfying conditions B, C, and D. (Condition B) At least one of the culture medium in the first stimulation step and the culture medium in the second stimulation step contains arginine. (Condition C) In the second stimulation step, antigen-presenting cells are added to the culture medium and culture is performed. (Condition D) In ​​the first stimulation step, Flt3L and a pattern recognition receptor ligand are added to the culture medium and culture is performed.

7. The manufacturing method according to any one of claims 1 to 6, wherein the antigen is a viral antigen.

8. The manufacturing method according to any one of claims 1 to 6, wherein the antigen is a cancer antigen.

9. The manufacturing method according to any one of claims 1 to 8, wherein the antigen added to the culture medium is a combination of two or more antigens.

10. The manufacturing method according to any one of claims 1 to 9, wherein the antigen-specific T cells are multiple antigen-specific T cells.

11. The manufacturing method according to claim 10, wherein the antigen-specific T cells are multiple virus-specific T cells.

12. The manufacturing method according to any one of claims 1 to 11, wherein the antigen added to the culture medium in the first stimulation step and the culture medium in the second stimulation step are the same antigen.

13. A kit for producing antigen-specific T cells, used in a method for producing antigen-specific T cells according to any one of claims 1 to 12, comprising a first cytokine group and a second cytokine group, wherein the first cytokine group is a combination of IL-7, IL-15, and IL-21, and does not include IL-12, IL-18, and TL1A, and the second cytokine group is a combination of IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A.

14. 600 x 10 ６ A group of antigen-specific T cells containing cells or more, with over 90% being CD8-positive / CD45RO-positive.

15. 600 x 10 ６ The antigen-specific T cell group according to claim 14, comprising cells of a certain size or larger, wherein more than 90% of the total are CD8-positive / CD45RO-positive, mitochondrial biosynthesis is maintained, and the antigen-specific T cells are cytotoxic.

16. A group of antigen-specific T cells obtained by the manufacturing method described in any one of claims 1 to 12.

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