Method for culturing t cells

Abstract

The purpose of the present invention is to maintain and proliferate T cells, which are in a composition containing the T cells, into a cell group suitable for gene transfer. The present invention pertains to a method for culturing T cells characterized by comprising a step for bringing a composition containing T cells into contact with a composition containing a T cell surface antigen identification substance. The method is also characterized in that, in the composition containing T cells, the amount of the T cell surface antigen identification substance added is 0.0001-2.0 μg and / or 0.0001-20 pmol in terms of antigen recognizing moiety, per 106 T cells.

Description

Method for culturing T cells

[0001] The present invention relates to a method for culturing T cells, and in particular to a method for culturing T cells that serve as a raw material for producing T cells (T cell preparations) for use in T cell therapy.

[0002] T cell therapy is known as an immunotherapy for cancer patients. In T cell therapy, T cells are extracted from the body, activated / expanded, and then returned. For example, a method using tumor-infiltrating lymphocytes (TIL) (Non-Patent Document 1) and a method of introducing a cancer antigen-specific T cell receptor (TCR) gene into T cells in the patient's peripheral blood (Non-Patent Document 2) are known. In recent years, a therapy using chimeric antigen receptor (CAR)-T cells (hereinafter simply referred to as CAR-T cells) has been developed, in which the T cell receptor (TCR) of cytotoxic T cells (CTL) is genetically modified to allow the CTL to directly and selectively recognize tumor cells and exert an antitumor effect (Non-Patent Document 3). In other words, CAR-T cells are artificially produced T cells obtained by introducing a CAR through genetic manipulation. Because cancer treatment with CAR-T cells kills cancer cells through a mechanism different from that of conventional anticancer drugs or radiation therapy, it is expected to be effective against intractable or treatment-resistant cancers. CAR-T cells have already been formulated for the treatment of some blood tumors, such as leukemia and malignant lymphoma.

[0003] Autologous T cells and allogeneic T cells are used to produce T cell preparations (activated T cells, CAR-T cells, etc.), but currently, autologous T cells collected from the patient's peripheral blood by apheresis or the like are used to produce CAR-T cells. When producing CAR-T cells, T cells are isolated and stimulated with cytokines or antibodies, and then the CAR gene is introduced using a viral vector or transposon.

[0004] The selection of the starting cell population is important for the stable production of T cell preparations. In the case of autologous transplants, even securing a large number of cells can be difficult depending on the patient. Since the amount of T cells collected from a patient is limited, it is important to isolate a cell population in a more suitable state to increase the efficiency of gene transfer.

[0005] Patent Documents 1 and 2 describe methods for isolating, culturing, and genetically manipulating immune cell populations, such as T cell populations, for adoptive therapy. Non-Patent Document 4 describes automated large-scale production of CAR-T cells incorporating a T cell population separation process.

[0006] Japanese Patent Application Laid-Open No. 2020-156495 Japanese Patent Application Laid-Open No. 2022-534921

[0007] Rosenberg SA. , Nat Rev Clin Oncol. 2011 8(10):577-585. Robbins PF. et al. , J Clin Oncol. 2011 29: 917-924. Eshhar Z. et al. , Proc Natl Acad Sci USA, 1993, 90: 720-724. Lock D. et al. , J Immunother Cancer. 2022 10(9):e005189.

[0008] When performing gene transfer, it is necessary to secure the required number of T cells, but because autologous T cells collected from the patient are used, it may not be possible to secure the desired cell population in the required amount. Therefore, an object of the present invention is to maintain and proliferate T cells collected from the patient into a cell population suitable for gene transfer.

[0009] In view of the above problems, the present inventors have conducted extensive research and have found that, when culturing T cells, a T cell population can be separated by contacting a composition containing T cells with a T cell surface antigen identifying substance, and that by setting the ratio of cells to T cell surface antigen identifying substance during contact within an appropriate range, the desired T cell population can be successfully separated, and the resulting T cell population has a high gene transfer efficiency and also a high survival rate, which has led to the completion of the present invention. That is, the present invention is as follows.

[0010] [1] A method for culturing T cells, comprising a step of contacting a composition containing T cells with a substance that identifies a T cell surface antigen, wherein the amount of the substance that identifies a T cell surface antigen added is such that it is 10 times the amount of T cells in the composition containing T cells. ６ [1A] A method for culturing T cells, comprising a step of contacting a composition containing T cells with a T cell surface antigen-recognizing substance, wherein the amount of the T cell surface antigen-recognizing substance added is 0.1 to 7.0 μg per 10 cells in the composition containing T cells. ６ [2] The method according to [1] or [1A], wherein the proportion of T cells contained in the composition containing T cells is 5% or more of the total number of cells contained in the composition. [3] A method for culturing T cells, comprising a step of contacting a composition containing T cells with a T cell surface antigen-recognizing substance, wherein the amount of the T cell surface antigen-recognizing substance added is 0.0001 to 2.0 μg and / or 0.0001 to 20 pmol per T cell, based on the antigen-recognizing portion. ６ [3A] A method for culturing T cells, comprising a step of contacting a composition containing T cells with a T cell surface antigen-recognizing substance, wherein the amount of the T cell surface antigen-recognizing substance added is 0.05 to 250 μg per 10 T cells in the composition containing T cells. ６[4] The method according to any one of [1] to [3], [1A] and [3A], wherein the T cell surface antigen recognizing substance comprises an antibody and / or an antigen-binding fragment thereof. [5] The method according to [4], wherein the antibody is an antibody against at least one selected from the group consisting of CD4, CD8, CD45, CD45RA, CD45RO, CD62L, CD127, CD28, CD3, CD27, CD44, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123 and CD235a. [6] The method according to any one of [1] to [5], [1A] and [3A], wherein the T cell surface antigen recognizing substance is present on a support. [7] The method according to [6], wherein the support is a bead or a nanomatrix. [8] The method according to any one of [1] to [7], [1A], and [3A], wherein the method for culturing T cells is a method for separating T cells. [9] The method according to [8], wherein the separation is by magnetic separation.

[10] The method according to any one of [1] to [9], [1A], and [3A], wherein the method for culturing T cells is a method for maintaining and expanding T cells.

[11] The method according to any one of [1] to

[10] , [1A], and [3A], wherein the T cells comprise naive and / or memory T cells.

[12] The method according to any one of [1] to

[11] , [1A], and [3A], wherein the T cells comprise naive and / or memory T cells at 60% or more.

[13] A method comprising expressing a chimeric antigen receptor in T cells obtained by the culture method according to any one of [1] to

[12] , [1A], and [3A].

[14] An additive for T cell culture containing a T cell surface antigen-recognizing substance, wherein 10 cells are cultured in a composition containing T cells. ６ [14A] A formulation characterized by using a T cell surface antigen identifying substance at a dose of 0.1 to 7.0 μg per cell on a complex basis, wherein the T cell surface antigen identifying substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody. [14B] An additive for T cell culture containing a T cell surface antigen identifying substance, wherein the additive is a composition containing T cells, and the composition contains 10 cells. ６

[15] A formulation characterized by using a T cell surface antigen-recognizing substance at a dose of 0.0001 to 2.0 μg and / or 0.0001 to 20 pmol per cell based on the antigen-recognizing portion, wherein the T cell surface antigen-recognizing substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody.

[16] An additive for T cell culture, comprising a T cell surface antigen-recognizing substance, for culturing T cells in a composition containing T cells at a concentration of 10 to 1500 μg of T cells. ６ [15A] A formulation characterized by using a T cell surface antigen identifying substance at a dose of 0.05 to 250 μg per cell on a complex basis, wherein the T cell surface antigen identifying substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody. [15B] An additive for T cell culture containing a T cell surface antigen identifying substance, wherein the additive is used to culture 10 T cells in a composition containing T cells. ６

[16] A formulation characterized by using a T cell surface antigen-recognizing substance at a dose of 0.0001 to 2.0 μg and / or 0.0001 to 20 pmol per cell based on the antigen-recognition portion, wherein the T cell surface antigen-recognizing substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody.

[16] The formulation according to

[14] , [14A],

[15] , or [15A], which is for producing T cells.

[17] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the following steps: (1) a step of culturing T cells by the method described in any of [1] to

[12] , [1A], and [3A] (pre-culture step), (2) a step of introducing a CAR gene into the pre-cultured T cells (CAR introduction step), and, optionally, (3) a step of expanding the cells into which the CAR gene has been introduced (expansion step).

[0011] According to the method of the present invention, it is possible to obtain T cells with high gene transfer efficiency and high survival rate. As a result, it is possible to obtain a sufficient amount of T cells suitable for T cell therapy or CAR-T cell therapy. Furthermore, when separating T cells using beads, aggregation of beads and / or cells can be suppressed.

[0012] The present invention will be described below. Terms used in this specification have the meanings normally used in the art unless otherwise specified. The present invention provides a method for culturing T cells (hereinafter also referred to as the "culturing method of the present invention"). The method comprises the step of contacting a composition containing T cells with a T cell surface antigen-recognizing substance, and the amount of the T cell surface antigen-recognizing substance added to the composition containing T cells is 0.1 to 7.0 μg (10 cells) on a complex basis. ６ per cell), 0.05-250 μg (T cells 10 ６ 0.0001-2.0 μg (per 10 cells) of antigen-recognizing moiety ６ per cell), 0.0001-20 pmol (per 10 cells ６ per cell), 0.0001-2.0 μg (per 10 T cells ６ per cell), 0.0001-20 pmol (T cells 10 ６ (per piece).

[0013] Composition Comprising T Cells The T cells in the "composition comprising T cells" of the present invention (hereinafter also simply referred to as "T cell-containing composition") may be either autologous or allogeneic cells. In the present invention, "autologous cells" means cells obtained from a subject who will receive a T cell preparation (e.g., activated T cells, CAR-T cells) produced using T cells cultured and expanded by the method of the present invention as a starting material, or cells derived from the obtained cells, and "allogeneic cells" means cells that are not the "autologous cells" described above. Preferably, the T cells are autologous cells. T cells in the T cell-containing composition include CD4-positive CD8-negative (CD4+CD8-) T cells, CD4-negative CD8-positive (CD4-CD8+) T cells, CD4-positive CD8-positive (CD4+CD8+) T cells, CD4-negative CD8-negative (CD4-CD8-) T cells, αβ-T cells, γδ-T cells, Treg cells, NK-like T cells, NKT cells, and the like. The T cells may be a subset such as naive T cells, effector T cells, or memory T cells. The T cells may be cells isolated from a human, or may be cells obtained by differentiation from cells such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells. Considering that a T cell preparation is produced using the T cell-containing composition as a starting material, the T cells are preferably young, non-exhausted T cells, such as naive and / or memory T cells.

[0014] Naive T cells are immature T cells that have not yet encountered an antigen. When naive T cells encounter foreign substances such as pathogens or cancer cells, they proliferate and become activated, but many eventually die. However, some remain as memory T cells in preparation for the next reinfection or recurrence. Memory T cells are classified into several types with different stages of differentiation: stem cell memory T cells (Tscm), central memory T cells (Tcm), and effector memory T cells (Tem). Among these, Tems are the most differentiated cells with the strongest attacking power against cancer cells, while Tscm are memory T cells that are similar to naive T cells, have the longest lifespan, and are thought to retain immune memory for a long period of time. Tscm can give rise to many highly differentiated Tcms and Tems.

[0015] In one embodiment, in the T cell-containing composition, the percentage of naive T cells is greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or 95% of the total T cells in the T cell-containing composition, preferably greater than 60%, 65%, 70%, 80%, 90%, or 95%. In certain embodiments, the naive T cells comprise CCR7+CD45RA+, CD27+CCR7+, or CD62L+CCR7+ T cells. In some embodiments, the naive T cells comprise CCR7+CD45RA+ T cells. In certain embodiments, the naive T cells comprise CD27+CCR7+ T cells. In certain embodiments, the naive T cells comprise CD4+, CD8+, or CD4+CD8+ T cells.

[0016] In one embodiment, in the T cell-containing composition, the percentage of memory T cells is greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or 95% of the total T cells in the T cell-containing composition, preferably greater than 60%, 65%, 70%, 80%, 90%, or 95%. In a particular embodiment, the memory T cells comprise CCR7+CD45RA+, CCR7+CD45RA-, CCR7-CD45RA-, or CCR7-CD45RA+ T cells. In some embodiments, the memory T cells comprise CCR7+CD45RA+, or CCR7+CD45RA- T cells. In some embodiments, the memory T cells comprise CCR7+CD45RA+ T cells. In certain embodiments, memory T cells include CD4+, CD8+, and CD4+CD8+ T cells.

[0017] In one embodiment, the percentage of naive and memory T cells is greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, or 95% of the total T cells in the T cell-containing composition. The ratio of naive T cells to memory T cells is not particularly limited, but may be 1:0.1-10, preferably 1:0.5-5.

[0018] The proportion of T cells in a T cell-containing composition is not particularly limited as long as the T cell surface antigen-identifying substance of the present invention, described below, can exert the desired effect, but is preferably 5% or more. In one embodiment, the proportion of T cells in a T cell-containing composition may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%. As described below, when a T cell-containing composition is obtained from or derived from a subject (i.e., a patient) with a specific disease or condition, in need of cell therapy, or to be administered cell therapy, the proportion of T cells in the T cell-containing composition may be less than 5%, or may be as low as a few percent. In such cases, the T cells may be concentrated, as appropriate, by various methods known per se.

[0019] The T cell-containing composition may be a T cell source itself, or a processed product obtained by isolating, selecting, or enriching T cells from a T cell source. When the T cells are autologous, a biological sample obtained or derived from a T cell source, such as a subject with a particular disease or condition, in need of cell therapy, or to whom cell therapy is being administered, is used as the T cell-containing composition. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, as well as processed samples derived therefrom. Preferably, the biological sample is blood or a blood-derived sample, or a product of apheresis or leukapheresis (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product). The T cell-containing composition may be cryopreserved.

[0020] A preferred T cell-containing composition uses peripheral blood mononuclear cells (PBMCs) or a processed product thereof. The PBMCs are preferably autologous PBMCs. PBMCs can be prepared by conventional methods; see, for example, Saha S, Nakazawa Y, Huye LE, Doherty JE, Galvan DL, Rooney CM, Wilson MH. J Vis Exp. 2012 Nov 5; (69): e4235. Unless otherwise specified, various cells (e.g., T cells) used herein are human cells.

[0021] T Cell Surface Antigen Identifying Substance The culture method of the present invention includes a step of contacting a T cell-containing composition with a T cell surface antigen identifying substance. This step may involve isolating T cells from the T cell-containing composition by contacting the composition with a T cell surface antigen identifying substance. According to the present invention, by culturing T cells, particularly by selecting, isolating, or enriching T cells, it is possible to obtain T cells with high gene transfer efficiency and high viability. This step can be carried out, for example, by separating and obtaining T cells from a T cell source based on the characteristics of T cells and / or non-T cells, or by separating and removing non-T cells from a T cell source. As an example, T cells can be separated from a T cell source, such as a PBMC sample or leukapheresis sample, by negative selection for markers expressed on B cells, monocytes, or other leukocytes, e.g., non-T cells such as CD14. As another example, T cells can be separated from a T cell source, such as a PBMC sample or leukapheresis sample, by positive selection for markers expressed on T cells, such as CD4.

[0022] The proportion of naive T cells, Tscm, Tcm, and / or Tems in a T cell-containing composition can also be adjusted by positive or negative selection based on surface antigens associated with each subpopulation of T cells.

[0023] In the present invention, a "T cell surface antigen identifying substance" refers to a substance capable of recognizing a cell surface antigen expressed on T cells or non-T cells, and may be a T cell surface antigen capturing substance or a non-T cell surface antigen capturing substance. When a T cell surface antigen capturing substance is used as the T cell surface antigen identifying substance, T cells can be separated by positive selection. When a non-T cell surface antigen capturing substance is used as the T cell surface antigen identifying substance, T cells can be separated by negative selection. Furthermore, cell surface antigen identifying substances can also be selected based on surface antigens associated with each subpopulation of T cells. For example, when a substance that captures a surface antigen specifically expressed on naive T cells or memory T cells is used as the T cell surface antigen identifying substance, young T cells such as naive T cells and memory T cells can be separated by positive selection. When a substance that captures a surface antigen that is not specific to naive T cells or memory T cells but is generally expressed on T cells is used, young T cells such as naive T cells and memory T cells can be separated by negative selection.

[0024] Specific methods for selecting, isolating, or enriching T cells from a T cell source include the following. Here, we describe the use of a T cell surface antigen capture substance as a T cell surface antigen-identifying substance. T cells expressing a specific cell membrane surface antigen (T cell surface antigen) are separated from cells that do not express that antigen using the T cell surface antigen capture substance. This separation process can be performed using methods commonly used to separate cells based on the expression status of cell membrane surface antigens. Preferred methods include density gradient separation, immunological cell separation, magnetic cell separation, nylon wool separation, and adhesion. Density gradient separation (also known as density gradient centrifugation) is a technique that enables cells to be separated according to their size, shape, and density. A density gradient is formed in a container, such as a centrifuge tube, by layering solutions of various densities that become dense at the bottom of the container. Magnetic cell separation, also known as magnetic cell sorting (MACS), involves preparing a cell suspension from tissue containing a mixture of various cells, magnetically labeling specific cells within the suspension, and then separating and collecting the magnetically labeled cells from non-magnetically labeled cells using magnetic force. Immunological cell separation utilizes antigen-antibody reactions to separate cells. Nylon wool separation utilizes the property of B cells to adhere well to nylon wool. The adhesion method utilizes differences in the adhesive ability of cells to substrates (e.g., culture flasks) to separate cells. Specifically, when a T cell source is placed in a culture flask, adhesive monocytes and other lymphocytes adhere to the bottom of the flask, while non-adhesive T cells and other lymphocytes float in the culture medium. Therefore, the adhesion method is used to separate the two cell populations. When cells adhering to a culture vessel are removed, a T cell source is placed in the culture vessel for a certain period of time (e.g., 1 second to 7 days), and after the certain period of time has passed, only the floating cells are collected and used as the T cell source, without using the adherent cells. Examples of culture vessels include, but are not limited to, culture flasks, culture chambers, culture bags, culture plates, culture dishes, bioreactors, etc. Furthermore, the material of the culture vessel is not particularly limited, and examples thereof include polystyrene, TPP, PETG, glass, etc.The culture vessel may or may not be coated, surface treated, or surface-processed with a coating agent (eg, collagen, fibronectin, polylysine, plasma treatment, charge treatment, etc.).

[0025] These separation methods and techniques may be used alone or in combination with two or more of them. Examples include immunodensity gradient separation, which combines density gradient separation and immunological cell separation; immunomagnetic cell separation, which combines magnetic cell separation and immunological cell separation; and a technique that combines adhesion and immunological cell separation to separate cells by adhering them to a culture vessel coated with an antibody or the like. Cell separation using immunological cell separation techniques is performed by labeling the cells to be separated with an antibody or ligand specific to the surface of the cells, and then using the label as an indicator for density gradient separation. One type of immunomagnetic cell separation technique is magnetic bead separation, which uses magnetic beads.

[0026] In the present invention, a T cell surface antigen identifying substance (hereinafter also simply referred to as "identification substance") is used by contacting it with T cells, particularly naive and / or memory T cells, contained in a T cell-containing composition. Here, "contacting" includes incubating and / or culturing cells with the identification substance. By contacting the cells with the identification substance, desired T cells can be efficiently isolated from the T cell-containing composition. In the case of positive selection, the identification substance contains a T cell surface antigen capture substance (hereinafter also simply referred to as "capture substance"), and desired T cells can be obtained by obtaining T cells captured by the capture substance. In the case of negative selection, the identification substance contains a non-T cell surface antigen capture substance or a substance that captures a surface antigen expressed on undesired T cells, and desired T cells can be obtained by removing cells captured by the capture substance. As will be described in detail in the Examples, T cells isolated using the identification substance in the present invention have a high survival rate and a high efficiency of gene transfer, such as of a CAR gene.

[0027] In the present invention, the capture substance is not particularly limited as long as it can capture a marker factor expressed in T cells desired to be separated, but a substance capable of capturing a marker factor expressed in young T cells such as naive T cells and memory T cells is preferred (positive selection). Such substances are not particularly limited as long as they have affinity for the desired marker factor, and examples thereof include RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, etc.

[0028] In some embodiments, the capture agent is attached to a support and contains one or more agents (e.g., antibodies) that specifically bind to one or more of the following molecules on cells: [For positive selection] CD3, CD4, CD8, CD45, CD45RA, CD27, CD127, CD62L, CD28, CD44, CCR7, CD95, ICOS, HVEM, OX40, 4-1BB, CD40L, GITR, CD38, CXCR5, BTLA, PD-L1, CX3CR1, CD248, CD327, CD79a, CD35, CD73, CD318, CD305, CD242, CD158d, CD42d, CD56, CD257, CD49f, CD201, CD7, CD328, CD300c, CD 11d, CD228, CD336, CD289, CD172b, CD167a, CD353, CD355, CD217, CD172g, CD183, CD11c, CD159a, CD69, CD366, CD258, CD254, CXCR1, CXCR2, CXCR4, CCR2, CCR5, CCR6, CD6, CCL5, CCL19, CD45RB, CD45RC, CD80, 4-1BB, CD40, CD52, CD70, CD49, CD51, CD103, CD11b, CD31 and CD18. Preferred examples include CD3, CD4, CD8, CD45, CD45RA, CD27, CD127, CD62L, CD28, CD44, CCR7, CD95, CXCR5, CCL5, CCL19, CD45RB, CD45RC, CD80, and 4-1BB. More preferred examples include CD3, CD4, CD8, CD45, CD45RA, CD27, CD127, CD62L, CD28, and CD44.[Negative selection] CD45RO, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, CD235a, CD58, KLRG1, TIM3, LAG3, PD-1, CD11a, CD26, CD122, CXCR3, IL-2Rβ, HLA-DR, CD86, 4-1BBL, CXCL10, ID2, CCL4L2, CTLA4, CD160, CCL3, CD249, CD150, CD283, CD131, C CD66a, CD253, CD120b, CD61, CD87, CD280, CD195, CD54, CD265, CD203a, CD271, CD68, CD307c, CD357, CD196, CD25, CD154, CD203c, CD134, CD106, CD244, CD71, CD220, CD158e, CD123, CD16b, CD364, CD230, CD41, CD361, CD161, CCR4, and CD292. Preferred examples include CD45RO, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, CD235a, LAG3, CXCR3, and IL-2Rβ. More preferred are CD45RO, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a. As a substance for capturing CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a, a mixture (antibody cocktail) of anti-CD14 antibody, anti-CD15 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD34 antibody, anti-CD36 antibody, anti-CD56 antibody, anti-CD123 antibody, and anti-CD235a antibody is commercially available.

[0029] In a preferred embodiment of the present invention, the capture agent is a substance (e.g., an antibody or an antigen-binding fragment thereof) that captures CD3, CD4, CD8, CD45, CD45RA, CD27, CD127, CD62L, CD28, and CD44, as well as CD45RO, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a. In some embodiments, these capture agents are used in a state attached to a support.

[0030] Antibodies include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments (e.g., Fab, F(ab')). ２ In some aspects, the identifying agent can comprise an antibody fragment (including an antigen-binding fragment), such as Fab, Fab'-SH, Fv, scFv, or F(ab'). ２ It is recognized that constant regions of any isotype can be used for the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species (e.g., murine species). In the present invention, when the T cell surface antigen-recognizing substance comprises an antibody, the antibody used may be an antigen-binding fragment thereof, so long as it is capable of recognizing the antigen of interest.

[0031] In some embodiments, the conditions for contacting the T cell-containing composition with the T cell surface antigen identifying substance are appropriately set depending on the type of identifying substance used and the proportion of cells, particularly T cells, in the T cell-containing composition. One embodiment of the T cell surface identifying substance is a complex composed of a support (e.g., beads), a capture substance, a linker (e.g., PEG linker) connecting them, and a substance for labeling them. The amount of the T cell surface antigen identifying substance to be contacted with the T cell-containing composition may be determined either as the amount per complex (complex basis) or as the amount per capture substance, which is the portion that recognizes T cells, i.e., the antigen recognition portion (antigen recognition portion basis). In this specification, the terms "antigen recognition portion" and "capture substance" are used interchangeably. Unless otherwise specified, the term "T cell surface identifying substance" in this specification encompasses both a complex containing a capture substance and the capture substance alone. Typically, on a complex basis, 10 cells in the T cell-containing composition are identified. ６0.1-7.0μg, 0.2-6.8μg, 0.3-6.7μg, 0.4-6.6μg, 0.5-6.5μg, 0.6-6.4μg, 0.7-6.3μg, 0.8-6.2μg, 0.9- 6.1μg, 1.0-6.0μg, 1.1-5.9μg, 1.2-5.8μg, 1.3-5.7μg, 1.3-5.6μg, 1.3-5.5μg, 1.3-5.4μg, 1.3-5.3μg, 1.3 Up to 5.2 μg, 1.3 to 5.1 μg, 1.3 to 5.0 μg, 1.3 to 4.9 μg, 1.3 to 4.8 μg, 1.3 to 4.7 μg, 1.3 to 4.6 μg, 1.3 to 4.5 μg, 1.3 to 4.4 μg, 1.3 to 4.3 μg, 1.3 to 4.2 μg, 1.3 to 4.1 μg, 1.3 to 4.0 μg, 1.3 to 3.9 μg, 1.31 to 3.8 μg, or 1.32 to 3.7 μg of a T cell surface antigen-recognizing substance is used. In another embodiment, 1.33 to 6.5 μg, preferably 1.34 to 6.0 μg, more preferably 1.34 to 5.0 μg, even more preferably 1.34 to 4.0 μg, even more preferably 1.35 to 3.0 μg, and particularly preferably 1.35 to 2.0 μg of T cell surface antigen-recognizing substance can be used on a complex basis.

[0032] In addition, the cells 10 in the T cell-containing composition can be identified based on the antigen recognition moiety. ６ 0.0001-2.0μg, 0.0002-1.8μg, 0.0004-1.6μg, 0.0006-1.4μg, 0.0008-1. 2μg, 0.0010-1.0μg, 0.0012-0.8μg, 0.0014-0.6μg, 0.0016-0.5μg, 0.0018-0. In another embodiment, 0.002 to 0.4 μg, preferably 0.002 to 0.35 μg, more preferably 0.0020 to 0.3 μg, even more preferably 0.0020 to 0.1 μg, still more preferably 0.0020 to 0.05 μg, and particularly preferably 0.002 to 0.02 μg of T cell surface antigen identifying substance can be used, based on the antigen recognition portion.

[0033] In yet another embodiment, the antigen recognition moiety-based T cell-containing composition is ６ 0.0001 to 20 pmol, 0.0005 to 15 pmol, 0.001 to 10 pmol, 0.002 to 9 pmol, 0.003 to 8 pmol, 0.004 to 7 pmol, 0.005 to 6 pmol, 0.006 to 5 pmol, 0.007 to 4 pmol, 0.008 to 3 pmol, 0.009 to 2.5 pmol, 0.010 to 2.0 pmol, 0.010 to 1.5 pmol, 0.010 to 1.0 pmol, 0.010 to 0.5 pmol, 0.010 to 0.4 pmol, 0.010 to 0.3 pmol, 0.010 to 0.2 pmol, or 0.010 to 0.1 pmol of T cell surface antigen-recognizing substance is used per particle. In another embodiment, 0.009 to 3.0 pmol, preferably 0.009 to 2.5 pmol, more preferably 0.010 to 2.0 pmol, even more preferably 0.010 to 0.5 pmol, still more preferably 0.010 to 0.2 pmol, and particularly preferably 0.01 to 0.1 pmol of T cell surface antigen-recognizing substance can be used, based on the antigen-recognizing portion.

[0034] In some embodiments, the conditions for contacting the T cell-containing composition with the T cell surface antigen-identifying substance are appropriately set depending on the type of identifying substance used. ６ Per cell, 0.05 to 250 μg, 0.05 to 100 μg, 0.05 to 50 μg, 0.05 to 30 μg, 0.05 to 20 μg, 0.05 to 15 μg, 0.1 to 10 μg, 0.1 to 9.0 μg, 0.1 to 8.0 μg, 0.1 to 7.0 μg, 0.1 to 6.0 μg, 0.1 to 5.0 μg, 0.1 to 4.0 μg, 0.1 to 3.0 μg, 0.1 to 2.0 μg, 0.2 to 2.5 μg, or 0.2 to 3.0 μg of T cell surface antigen-recognizing substance can be used on a conjugate basis. In another embodiment, 0.2 to 9.0 μg, preferably 0.2 to 7.0 μg, more preferably 0.2 to 5.0 μg, and even more preferably 0.2 to 3.0 μg of the T cell surface antigen-recognizing substance can be used on a complex basis.

[0035] In addition, T cells in a T cell-containing composition can be stimulated based on the antigen recognition moiety. ６0.0001 to 2.0 μg, 0.0005 to 1.8 μg, 0.0005 to 1.6 μg, 0.001 to 1.4 μg, 0.001 to 1.2 μg, 0.002 to 1.0 μg, 0.003 to 0.5 μg, 0.003 to 0.4 μg, 0.003 to 0.3 μg, 0.003 to 0.2 μg, 0.003 to 0.1 μg, 0.003 to 0.05 μg, 0.003 to 0.03 μg, or 0.004 to 0.03 μg of T cell surface antigen-recognizing substance is used per cell. In another embodiment, 0.003 to 0.5 μg, preferably 0.003 to 0.1 μg, more preferably 0.003 to 0.08 μg, even more preferably 0.003 to 0.05 μg, still more preferably 0.003 to 0.04 μg, and particularly preferably 0.003 to 0.03 μg of T cell surface antigen-recognizing substance can be used, based on the antigen-recognizing portion.

[0036] In yet another embodiment, T cells in a T cell-containing composition are stimulated with an antigen recognition moiety-based ６ 0.0001 to 20 pmol, 0.0005 to 15 pmol, 0.001 to 10 pmol, 0.005 to 5 pmol, 0.010 to 4 pmol, 0.011 to 3.8 pmol, 0.012 to 3.6 pmol, 0.013 to 3.4 pmol, 0.014 to 3.2 pmol, 0.015 to 3 pmol, 0.015 to 2 pmol, 0.015 to 1 pmol, 0.015 to 0.5 pmol, 0.015 to 0.25 pmol, or 0.015 to 0.15 pmol of T cell surface antigen-recognizing substance is used per particle. In another embodiment, 0.015 to 3 pmol, preferably 0.015 to 2 pmol, more preferably 0.015 to 1 pmol, even more preferably 0.015 to 0.5 pmol, still more preferably 0.015 to 0.25 pmol, and particularly preferably 0.015 to 0.15 pmol of T cell surface antigen-recognizing substance can be used based on the antigen recognition portion.

[0037] When two or more substances are used in combination as T cell surface antigen-recognizing substances, the total amount of each substance used is preferably within the above range, but may be increased or decreased as appropriate depending on the type of substance used. For example, when two or more types of T cell surface antigen-recognizing substances are used in combination, the dose of each substance is 100 mg / mL on a complex basis. ６The amounts per particle are 0.01 to 5.0 μg, 0.03 to 2.5 μg, 0.05 to 1.0 μg, 0.06 to 0.9 μg, 0.07 to 0.8 μg, 0.08 to 0.7 μg, 0.09 to 0.69 μg, 0.1 to 0.68 μg, 0.11 to 0.67 μg, 0.12 to 0.66 μg, 0.13 to 0.65 μg, 0.14 to 0.64 μg, 0.15 to 0.63 μg, 0.16 to 0.62 μg, 0.17 to 0.61 μg, 0.18 to 0.6 μg, 0.19 to 0.5 μg, and 0.3 to 0.4 μg. In another embodiment, 0.14 to 0.7 μg, preferably 0.4 to 0.7 μg, more preferably 0.5 to 0.7 μg, even more preferably 0.6 to 0.7 μg, and even more preferably 0.65 to 0.69 μg of each T cell surface antigen-recognizing substance can be used on a complex basis.

[0038] In addition, each T cell surface antigen-recognizing substance was identified based on the antigen-recognizing portion of the cell 10 ６ 0.001-0.5μg, 0.001-0.3μg, 0.001-0.25μg, 0.001-0.1μg, 0.001-0.09μg, 0.001-0.08μ per piece g, 0.001-0.07μg, 0.001-0.06μg, 0.001-0.05μg, 0.001-0.04μg, 0.001-0.03μg, 0.001-0.02 μg, 0.001 to 0.019 μg, 0.001 to 0.018 μg, 0.001 to 0.017 μg, 0.001 to 0.016 μg, 0.001 to 0.015 μg, 0.001 to 0.014 μg, 0.001 to 0.013 μg, 0.001 to 0.012 μg, 0.001 to 0.011 μg, or 0.001 to 0.01 μg can be used.

[0039] In another embodiment, 0.001 to 0.07 μg, preferably 0.001 to 0.05 μg, more preferably 0.001 to 0.04 μg, even more preferably 0.001 to 0.03 μg, still more preferably 0.001 to 0.2 μg, and particularly preferably 0.001 to 0.015 μg of each T cell surface antigen recognition substance can be used, based on the antigen recognition portion. In another embodiment, 0.001 to 0.50 pmol, preferably 0.003 to 0.40 pmol, more preferably 0.003 to 0.30 pmol, even more preferably 0.003 to 0.20 pmol, even more preferably 0.003 to 0.15 pmol, and particularly preferably 0.003 to 0.10 pmol of each T cell surface antigen recognition substance can be used, based on the antigen recognition portion.

[0040] T cell 10 ６ When two or more T cell surface antigen-recognizing substances are used in combination, the dose of each substance is calculated per 10 T cells on a complex basis. ６ In another embodiment, 0.05 to 6.5 μg, preferably 0.05 to 6.0 μg, more preferably 0.08 to 3.0 μg of each T cell surface antigen-recognizing substance can be used per complex.

[0041] In addition, each T cell surface antigen-recognizing substance was identified based on the antigen-recognizing portion of the T cell 10 ６0.001-1.0μg, 0.001-0.8μg, 0.001-0.6μg, 0.001-0.25μg, 0.001-0.1μg, 0.001-0. 09μg, 0.001-0.08μg, 0.001-0.07μg, 0.001-0.06μg, 0.001-0.05μg, 0.001-0.04μg, 0.00 Doses of 1 to 0.038 μg, 0.001 to 0.036 μg, 0.001 to 0.034 μg, 0.001 to 0.032 μg, 0.001 to 0.03 μg, 0.001 to 0.028 μg, 0.001 to 0.026 μg, 0.001 to 0.024 μg, 0.001 to 0.022 μg, and 0.001 to 0.02 μg can be used. In another embodiment, 0.001 to 0.1 μg, preferably 0.001 to 0.05 μg, more preferably 0.001 to 0.045 μg, even more preferably 0.001 to 0.04 μg, still more preferably 0.001 to 0.035 μg, and particularly preferably 0.001 to 0.03 μg of each T cell surface antigen-recognizing substance can be used, based on the antigen-recognizing portion.

[0042] In yet another embodiment, T cells in a T cell-containing composition are stimulated with an antigen recognition moiety-based ６ 0.002 to 5.0 pmol, 0.002 to 4.0 pmol, 0.002 to 3.0 pmol, 0.003 to 2.0 pmol, 0.003 to 1.5 pmol, 0.004 to 1.0 pmol, 0.004 to 0.8 pmol, 0.004 to 0.6 pmol, 0.004 to 0.4 pmol, 0.004 to 0.3 pmol, 0.004 to 0.2 pmol, 0.004 to 0.2 pmol, 0.005 to 0.2 pmol, or 0.005 to 0.1 pmol of T cell surface antigen-recognizing substance is used per particle. In another embodiment, 0.003 to 1.0 pmol, preferably 0.003 to 0.8 pmol, more preferably 0.003 to 0.6 pmol, even more preferably 0.003 to 0.4 pmol, still more preferably 0.004 to 0.3 pmol, and particularly preferably 0.004 to 0.2 pmol of each T cell surface antigen-recognizing substance can be used, based on the antigen-recognizing portion.

[0043] In one embodiment, the distinguishing agent comprises an anti-CD4 antibody and / or an anti-CD8 antibody. In this embodiment, the dose of the anti-CD4 antibody and / or the anti-CD8 antibody as the T cell surface antigen distinguishing agent is as follows: (on a conjugate basis) The anti-CD4 antibody or the anti-CD8 antibody is administered to 10 cells, respectively. ６ Per particle, 0.01 to 5.0 μg, 0.03 to 2.5 μg, 0.05 to 1.0 μg, 0.06 to 0.9 μg, 0.07 to 0.8 μg, 0.08 to 0.7 μg, 0.09 to 0.69 μg, 0.1 to 0.68 μg, 0.11 to 0.67 μg, 0.12 to 0.66 μg, 0.13 to 0.65 μg, 0.14 to 0.64 μg, 0.15 to 0.63 μg, 0.16 to 0.62 μg, 0.17 to 0.61 μg, 0.18 to 0.60 μg, 0.19 to 0.5 μg, and 0.3 to 0.4 μg are used. In another embodiment, a dose of 0.14 to 0.7 μg, preferably 0.4 to 0.7 μg, more preferably 0.5 to 0.7 μg, even more preferably 0.6 to 0.7 μg, and even more preferably 0.65 to 0.69 μg can be used. ６ Converted to per cell, it is 10 T cells ６ The amount of each antibody used is 0.05 to 125 μg, 0.05 to 50 μg, 0.05 to 25 μg, 0.05 to 15 μg, 0.05 to 10 μg, 0.05 to 9.0 μg, 0.05 to 8.0 μg, 0.05 to 7.0 μg, 0.05 to 6.0 μg, 0.06 to 5.0 μg, 0.07 to 4.0 μg, 0.08 to 3.0 μg, or 0.09 to 2.0 μg per cell. In another embodiment, the amount of each antibody used is 0.05 to 6.5 μg, preferably 0.05 to 6.0 μg, and more preferably 0.08 to 3.0 μg. When both an anti-CD4 antibody and an anti-CD8 antibody are used, the total amount of each antibody is preferably 0.05 to 6.5 μg, more preferably 0.05 to 6.0 μg, and most preferably 0.08 to 3.0 μg per 10 cells in the T cell-containing composition. ６0.1 to 7.0 μg, 0.2 to 6.8 μg, 0.3 to 6.7 μg, preferably 0.4 to 6.6 μg, 0.5 to 6.5 μg, 0.6 to 6.4 μg, 0.7 to 6.3 μg, 0.8 to 6.2 μg, 0.9 to 6.1 μg, 1.0 to 6.0 μg, 1.1 to 5.9 μg, 1.2 to 5.8 μg, 1.3 to 5.7 μg, 1.3 to 5.6 μg, 1.3 to 5.5 μg, 1.3 to 5.4 μg, 1.3 In another embodiment, the amount of T cells used is 1.0 to 6.0 μg, preferably 1.3 to 6.5 μg, and more preferably 1.35 to 2.0 μg. ６ Converted to per cell, it is 10 T cells ６ 0.05 to 250 μg, 0.05 to 100 μg, 0.05 to 50 μg, 0.05 to 30 μg, 0.05 to 20 μg, 0.05 to 15 μg, 0.1 to 10 μg, 0.1 to 9.0 μg, 0.1 to 8.0 μg, 0.1 to 7.0 μg, 0.1 to 6.0 μg, 0.1 to 5.0 μg, 0.1 to 4.0 μg, 0.1 to 3.0 μg, or 0.1 to 2.0 μg can be used per cell. In another embodiment, 0.2 to 9.0 μg, preferably 0.2 to 5.0 μg, and more preferably 0.2 to 3.0 μg can be used.

[0044] (In the case of antigen recognition moiety / capture substance-based) Cell 10 ６0.0001-0.2μg, 0.0005-0.1μg, 0.0005-0.05μg, 0.001-0.04μg, 0.001-0.03μg, 0.001-0.02μg, 0.001-0.01μg, 0.001-0.008μg, 0.001-0.007μg, 0.001-0.006μg, 0.00 In another embodiment, the anti-CD4 antibody can be used at a dose of 0.001 to 0.03 μg, preferably 0.001 to 0.02 μg, more preferably 0.001 to 0.01 μg, even more preferably 0.001 to 0.008 μg, even more preferably 0.001 to 0.006 μg, and particularly preferably 0.001 to 0.004 μg. T cell 10 ６ Converted to per cell, it is 10 T cells ６ 0.001 to 0.2 μg, 0.001 to 0.15 μg, 0.001 to 0.1 μg, 0.001 to 0.08 μg, 0.001 to 0.06 μg, 0.001 to 0.05 μg, 0.001 to 0.04 μg, 0.001 to 0.03 μg, 0.001 to 0.02 μg, 0.001 to 0.01 μg, 0.001 to 0.009 μg, 0.001 to 0.008 μg, 0.001 to 0.007 μg, 0.001 to 0.006 μg, 0.001 to 0.005 μg, 0.001 to 0.004 μg, or 0.001 to 0.003 μg of anti-CD4 antibody is used per cell. In another embodiment, an anti-CD4 antibody can be used at a dose of 0.001 to 0.05 μg, preferably 0.001 to 0.03 μg, more preferably 0.001 to 0.01 μg, even more preferably 0.001 to 0.008 μg, even more preferably 0.001 to 0.006 μg, and particularly preferably 0.001 to 0.004 μg.

[0045] cell 10 ６0.0001 to 0.1 μg, 0.0001 to 0.08 μg, 0.00001 to 0.06 μg, 0.0001 to 0.04 μg, 0.0001 to 0.03 μg, 0.0001 to 0.02 μg, 0.0005 to 0.01 μg, 0.0005 to 0.008 μg, 0.001 to 0.007 μg, 0.001 to 0.006 μg, 0.001 to 0.005 μg, 0.001 to 0.0048 μg, 0.001 to 0.0046 μg, 0.001 to 0.0044 μg, 0.001 to 0.0042 μg, 0.001 to 0.004 μg, 0.001 to 0.0038 μg, 0.001 to 0.0036 μg, or 0.001 to 0.0034 μg of anti-CD8 antibody is used. In another embodiment, the anti-CD8 antibody can be used at a dose of 0.0001 to 0.02 μg, preferably 0.001 to 0.015 μg, more preferably 0.001 to 0.01 μg, even more preferably 0.001 to 0.008 μg, even more preferably 0.001 to 0.006 μg, and particularly preferably 0.001 to 0.004 μg. ６ Converted to per cell, it is 10 T cells ６ 0.0001-0.1μg, 0.0001-0.08μg, 0.00001-0.06μg, 0.0001-0.05μg, 0.0001-0.04μg, 0.0001-0.03μg, 0.0001-0.02μg, 0.0005-0.01μg, 0.0005-0.008μg, 0.001-0.007μg, 0.001-0.008μg 0.006 μg, 0.001 to 0.005 μg, 0.001 to 0.0048 μg, 0.001 to 0.0046 μg, 0.001 to 0.0044 μg, 0.001 to 0.0042 μg, 0.001 to 0.004 μg, 0.001 to 0.0038 μg, 0.001 to 0.0036 μg, or 0.001 to 0.0034 μg of anti-CD8 antibody can be used. In another embodiment, an anti-CD8 antibody can be used at a dose of 0.0001 to 0.05 μg, preferably 0.0005 to 0.01 μg, more preferably 0.0005 to 0.008 μg, even more preferably 0.001 to 0.005 μg, even more preferably 0.001 to 0.0045 μg, and particularly preferably 0.001 to 0.004 μg.

[0046] In yet another embodiment, the antigen recognition moiety-based T cell-containing composition is ６ In another embodiment, 0.001 to 1 pmol, 0.002 to 0.5 pmol, 0.003 to 0.1 pmol, 0.004 to 0.08 pmol, 0.005 to 0.06 pmol, 0.006 to 0.04 pmol, 0.007 to 0.03 pmol, 0.007 to 0.02 pmol, or 0.007 to 0.01 pmol of anti-CD4 antibody per cell is used. ６ An anti-CD4 antibody of 0.008 to 0.08 pmol, 0.009 to 0.09 pmol, 0.01 to 0.10 pmol, 0.01 to 0.15 pmol, or 0.01 to 0.2 pmol per cell is used. In another embodiment, an anti-CD4 antibody of 0.001 to 1 pmol, preferably 0.003 to 0.1 pmol, more preferably 0.005 to 0.06 pmol, even more preferably 0.006 to 0.04 pmol, even more preferably 0.007 to 0.02 pmol, and particularly preferably 0.007 to 0.01 pmol can be used based on the antigen recognition portion. In another embodiment, it is also preferable to use an anti-CD4 antibody of 0.01 to 0.2 pmol based on the antigen recognition portion. ６ Converted to per cell, it is 10 T cells ６ 0.01 to 1.5 pmol, 0.01 to 1.0 pmol, 0.01 to 0.5 pmol, 0.01 to 0.4 pmol, 0.01 to 0.3 pmol, 0.01 to 0.2 pmol, 0.01 to 0.15 pmol, 0.01 to 0.10 pmol, 0.01 to 0.08 pmol, 0.01 to 0.06 pmol, 0.01 to 0.05 pmol, 0.01 to 0.04 pmol, or 0.01 to 0.03 pmol of anti-CD4 antibody per cell is used. In another embodiment, 0.001 to 0.3 pmol, preferably 0.001 to 0.15 pmol, more preferably 0.001 to 0.10 pmol, even more preferably 0.01 to 0.08 pmol, still more preferably 0.01 to 0.06 pmol, and particularly preferably 0.01 to 0.04 pmol of anti-CD4 antibody can be used, based on the antigen-recognition portion.

[0047] In yet another embodiment, the antigen recognition moiety-based T cell-containing composition is ６ 0.0001 to 0.5 pmol, 0.0005 to 0.4 pmol, 0.001 to 0.3 pmol, 0.001 to 0.2 pmol, 0.0015 to 0.15 pmol, 0.002 to 0.1 pmol, 0.002 to 0.05 pmol, 0.002 to 0.01 pmol, 0.002 to 0.005 pmol, 0.002 to 0.0045 pmol, 0.002 to 0.004 pmol, or 0.001 to 0.003 pmol of anti-CD8 antibody per cell is used. In another embodiment, 0.002 to 0.1 pmol, preferably 0.002 to 0.09 pmol, more preferably 0.002 to 0.08 pmol, even more preferably 0.002 to 0.07 pmol, even more preferably 0.002 to 0.06 pmol, and particularly preferably 0.002 to 0.005 pmol of anti-CD8 antibody can be used based on the antigen recognition portion. ６ Converted to per cell, it is 10 T cells ６ 0.001 to 1 pmol, 0.002 to 0.5 pmol, 0.003 to 0.4 pmol, 0.004 to 0.3 pmol, 0.004 to 0.2 pmol, 0.004 to 0.1 pmol, 0.004 to 0.05 pmol, 0.004 to 0.045 pmol, 0.004 to 0.04 pmol, 0.004 to 0.035 pmol, 0.004 to 0.03 pmol, 0.004 to 0.025 pmol, 0.004 to 0.02 pmol, 0.004 to 0.015 pmol, or 0.004 to 0.01 pmol of anti-CD8 antibody per cell is used. In another embodiment, 0.004 to 0.2 pmol, more preferably 0.004 to 0.1 pmol, even more preferably 0.004 to 0.05 pmol, still more preferably 0.004 to 0.03 pmol, and particularly preferably 0.004 to 0.01 pmol of anti-CD8 antibody can be used based on the antigen recognition portion.

[0048] In one embodiment, the identifying substance comprises an anti-CD45RA antibody. In this embodiment, the dose of the anti-CD45RA antibody as a T cell surface antigen identifying substance is as follows: (Antigen recognition moiety / capture substance-based) The anti-CD45RA antibody is used at a dose of 100 mg / mL for 100 cells.６ 0.001~2μg, 0.002~1.5μg, 0.004~1.0μg, 0.006~0.8μg, 0.008~0.6μg, 0.00μg per piece. 01-0.5μg, 0.01-0.4μg, 0.01-0.3μg, 0.01-0.2μg, 0.01-0.1μg, 0.01-0.09μg, In another embodiment, a dose of 0.01 to 0.08 μg, 0.01 to 0.07 μg, 0.01 to 0.06 μg, 0.01 to 0.05 μg, 0.01 to 0.045 μg, 0.01 to 0.04 μg, 0.01 to 0.035 μg, 0.01 to 0.03 μg, 0.01 to 0.025 μg, or 0.01 to 0.02 μg is used. In another embodiment, a dose of 0.01 to 0.3 μg, preferably 0.01 to 0.1 μg, more preferably 0.01 to 0.05 μg, even more preferably 0.01 to 0.04 μg, even more preferably 0.01 to 0.03 μg, and particularly preferably 0.01 to 0.02 μg can be used. ６ Converted to per cell, it is 10 T cells ６ 0.001~2μg, 0.002~1.5μg, 0.004~1.0μg, 0.006~0.8μg, 0.008~0.6μg, 0.00μg per piece. 01-0.5μg, 0.01-0.4μg, 0.01-0.3μg, 0.01-0.2μg, 0.01-0.1μg, 0.01-0.09μg, In another embodiment, a dose of 0.01 to 0.5 μg, preferably 0.01 to 0.1 μg, more preferably 0.01 to 0.06 μg, even more preferably 0.01 to 0.05 μg, still more preferably 0.01 to 0.04 μg, and particularly preferably 0.01 to 0.03 μg can be used.

[0049] In yet another embodiment, the antigen recognition moiety-based T cell-containing composition is ６0.01 to 20 pmol, 0.02 to 10 pmol, 0.03 to 5 pmol, 0.04 to 3 pmol, 0.05 to 2 pmol, 0.05 to 1.0 pmol, 0.05 to 0.8 pmol, 0.05 to 0.4 pmol, 0.05 to 0.3 pmol, 0.05 to 0.2 pmol, 0.05 to 0.15 pmol, 0.05 to 0.1 pmol, 0.05 to 0.09 pmol, or 0.05 to 0.08 pmol of anti-CD45RA antibody is used per cell. In another embodiment, 0.05 to 1.0 pmol, preferably 0.05 to 0.5 pmol, more preferably 0.05 to 0.2 pmol, even more preferably 0.05 to 0.1 pmol, even more preferably 0.05 to 0.09 pmol, and particularly preferably 0.05 to 0.08 pmol of anti-CD45RA antibody can be used based on the antigen recognition portion. ６ Converted to per cell, it is 10 T cells ６ 0.01 to 20 pmol, 0.02 to 18 pmol, 0.03 to 16 pmol, 0.04 to 14 pmol, 0.05 to 12 pmol, 0.06 to 10 pmol, 0.07 to 8 pmol, 0.08 to 6 pmol, 0.09 to 5 pmol, 0.1 to 3 pmol, 0.1 to 1 pmol, 0.1 to 0.8 pmol, 0.1 to 0.6 pmol, 0.1 to 0.4 pmol, or 0.1 to 0.2 pmol of anti-CD45RA antibody per cell is used. In another embodiment, 0.1 to 3 pmol, 0.1 to 1 pmol, 0.1 to 0.8 pmol, 0.1 to 0.6 pmol, 0.1 to 0.4 pmol, and particularly preferably 0.1 to 0.2 pmol of anti-CD45RA antibody can be used based on the antigen recognition portion.

[0050] Examples of the support to which the identification substance (herein synonymous with the capture substance) is bound include films, sponges, fibers, rods, beads, and colloids (e.g., gels), with beads (particle size 10 nm to 500 μm, preferably 10 nm to 100 μm, more preferably 10 nm to 50 μm) or gels being particularly preferred. A nanomatrix structure is also a preferred embodiment. Examples of materials include magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, polystyrene, retronectin, biopolymers (collagen, gelatin, fibrin, chitosan, alginic acid, silk fibron, etc.), and synthetic polymers. Preferably, magnetic beads are used.

[0051] In one embodiment, the identifying agents (e.g., antibodies) are provided in the form of conjugated or linked particles (e.g., beads). In some aspects, one or more identifying agents are bound to beads. In some aspects, the beads are biocompatible, i.e., composed of materials suitable for biological use. In some aspects, the beads are non-toxic to cultured T cells.

[0052] One or more identifying agents can also be used attached directly or indirectly to a support by a variety of methods known and available in the art. Attachment can be covalent, non-covalent, electrostatic, or hydrophobic, and can be achieved by a variety of attachment means, including, for example, chemical, mechanical, or enzymatic means. In some embodiments, the identifying agent comprises an antibody or an antigen-binding fragment thereof, e.g., a Fab. In some embodiments, the identifying agent (e.g., a biotinylated anti-CD3 antibody) can be indirectly attached to a bead via another biomolecule (e.g., an anti-biotin antibody) that is directly attached to the support.

[0053] In one embodiment of the present invention, the identification substance is present on the surface of beads as a support or is attached thereto. The particle size of the beads varies depending on the material and the affinity capture method used for separation and is set appropriately. In a specific embodiment, the beads have a diameter of more than 10 nm and 100 nm or less, or 90 nm or less, or 80 nm or less, or 70 nm or less, or 60 nm or less, preferably 50 nm or less. If the particle size is too small or too large, the target cells cannot be captured, and separation cannot be performed successfully.

[0054] When the identification substance is provided in the form of beads, the number of beads used to contact the cells is usually 10 cells. ６ Per 3.0 x 10 ７ ~1.0 x 10 １１ , preferably 2.0×10 ８ ~2.0 x 10 ９ T cells 10 ６ When converted to per ７ ~2.0 x 10 １１ , preferably 2.0×10 ８ ~3.0 x 10 ９ If the number of beads is too small compared to the number of cells, the target cells cannot be captured sufficiently, making it difficult to carry out effective separation. If the number of beads is too large compared to the number of cells, the possibility of non-specific capture of non-target cells increases, reducing the accuracy of separation.

[0055] The contact time between the T cell-containing composition and the identifying substance is sufficient as long as the identifying substance can capture the antigen expressed on the surface of the T cells, and is usually 0.01 to 48 hours, preferably 0.1 to 24 hours, and more preferably 0.15 to 0.25 hours.

[0056] In one embodiment, the cells in the T cell-containing composition separated by the T cell surface antigen recognizing substance are 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 20, 30, 40 or 50 x 10 ６At a density of cells / mL, preferably 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 x 10 ６ More preferably, the density of the cells / mL is 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 x 10 ６ In a specific embodiment, T cells separated by a T cell surface antigen-recognizing substance, e.g., T cells in a T cell-containing composition, are cultured at a density of 0.1 to 50 x 10 cells / mL. ６ At a density of cells / mL, preferably 1.0 to 10 x 10 ６ At a density of cells / mL, more preferably 2.0 to 8.0 x 10 ６ at a density of 5.0-8.0 x 10 cells / mL ６ The cells are cultured at a density of 10 cells / mL. The cell density can be appropriately set depending on the proportion of T cells in the T cell-containing composition and the method to be performed.

[0057] The medium used in the present invention for culturing T cells is not particularly limited as long as it allows T cells to proliferate. Media typically used in cell culture, such as RPMI 1640, MEM, X-VIVIO, IMDM, DMEM, DC medium, OptiMEM, and CellGro media, can be used. The medium may be supplemented with serum (human serum, fetal bovine serum, etc.) as per conventional methods, or it may be serum-free. Serum-free media are preferred because they are highly safe for clinical application and are less susceptible to differences in culture efficiency due to differences between serum lots. Examples of serum-free media include TexMACS™ (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific), and ALyS culture medium (Cell Science Institute, Inc.). When serum is used, autologous serum, i.e., serum collected from an individual from which the CAR-expressing immune cells are derived (more specifically, a patient receiving the cell population obtained by the production method of the present disclosure), may be used, or artificial serum may be used. In the present invention, it is preferable to use artificial serum. Plasma may also be used. Blood components such as serum or plasma, albumin, or analogs thereof may be used, and artificial substances may also be used. The basal medium may be one suitable for cell culture, and the above-mentioned TexMACS™ (Miltenyi Biotec), AIM V (registered trademark), or ALyS culture medium (Cell Science Institute, Inc.) may be used. Other culture conditions may be any suitable for cell survival and proliferation, and general conditions may be adopted. For example, the culture may be performed in a CO 2 -containing atmosphere set at 37°C. ２ Incubator (CO ２ Examples of suitable culture methods include culturing in an oxygen-rich atmosphere (oxygen concentration of 0 to 5%), and hypoxic culture (oxygen concentration of 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%). The culture time is the time required to obtain a desired amount of T cells, and varies depending on the seeding concentration, but is usually about 1 to 10 days, 3 to 9 days, 4 to 8 days, or 5 to 7 days, and the medium is appropriately supplemented or replaced as needed.

[0058] To support cell survival and proliferation, additional factors may be added to the medium. Examples of additional factors include type 1 cytokine family members, type 2 cytokine family members, TNF superfamily cytokines, IL-1 family cytokines, and other cytokines (such as TNF-β), specifically IL-1 to IL-41, and preferably IL-1, IL-2, IL-7, IL-15, and IL-21. IL-7 and / or IL-15 may be added to the medium when preparing CAR-T cells. The additional factors can be prepared according to standard methods, or commercially available products can be used. The additional factors may be from animal species other than humans, but are preferably derived from humans (which may be recombinant).

[0059] A composition containing the selected T cells is obtained. The selected T cells can be, and preferably are, used as a starting material for T cell immunization or the production of CAR-T cells. Accordingly, in one embodiment, the present invention provides a method for producing chimeric antigen receptor (CAR-T) cells (hereinafter also referred to as the "method for producing CAR-T cells of the present invention"), comprising the following steps: (1) a step of culturing T cells by the culture method of the present invention described above (pre-culture step), (2) a step of introducing a CAR gene into the pre-cultured T cells (CAR introduction step), and, optionally, (3) a step of expanding the cells into which the CAR gene has been introduced (expansion culture step). Each term will be explained.

[0060] CAR CAR is a structure comprising, from the N-terminus to the C-terminus of a protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signal domain for the effector function of immune cells, and the CAR gene is a gene encoding this receptor. The extracellular domain comprises an antigen recognition site that exhibits specific binding to the target. The transmembrane domain is located between the extracellular domain and the intracellular signal domain. The intracellular signal domain transmits a signal required for the immune cell to exert its effector function. In other words, an intracellular signal domain is used that can transmit a signal required for activating immune cells when the extracellular domain binds to a target antigen.

[0061] There have been several reports of experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20 (R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pulé MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119 :2709-2720, 2012; Porter DL, et al. N Engl J Med 365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5:177 ra38, 2013), and a CAR can be constructed with reference to these reports.

[0062] Introduction of a CAR Gene into T Cells CAR-T cells are prepared by introducing a CAR gene into T cells using a CAR expression vector. Here, the T cells to be introduced with the CAR gene are preferably T cells obtained by the above-described culture method of the present invention. A CAR expression vector refers to a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into T cells. It can be DNA or RNA, and there are no particular limitations on its form or origin; various types of vectors can be used. The vector can be a viral or non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, vaccinia viral vectors, pox viral vectors, and phages. Among these, retroviral vectors, lentiviral vectors, and adeno-associated viral vectors integrate the target gene into the host chromosome, allowing for stable and long-term expression. Each viral vector can be prepared according to standard methods or using commercially available dedicated kits. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, P.L., Gadek, T.R., Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, artificial chromosome vectors, and cosmid vectors.

[0063] A CAR expression vector contains an expression unit for expressing the CAR gene, and the expression unit typically includes a promoter, a CAR gene, and a poly(A) addition signal. The expression unit may be derived from various organisms or viruses or may consist of any sequence, including sequences similar to or modified from these. Examples of promoters that can be used for the CAR expression cassette include the CAG promoter, CMV-IE (cytomegalovirus early gene-derived promoter), SV40 ori, retrovirus LTRSRα, EF1α, and β-actin promoters. Examples of poly(A) addition signal sequences include the SV40 poly(A) addition sequence, the bovine growth hormone gene poly(A) addition sequence, and the globulin poly(A) addition sequence. To control the expression of the CAR gene by the promoter, the CAR gene is typically linked to the 3' end of the promoter directly or via another sequence, and a poly(A) addition signal sequence is located downstream of the CAR gene. Such an expression unit transcribes the CAR gene into messenger RNA (mRNA), and the CAR is translated from the mRNA and presented on the cell surface. The expression unit may contain a detection gene (e.g., a reporter gene, a cell- or tissue-specific gene, a selection marker gene) for detecting gene expression, an enhancer sequence for improving expression efficiency, a WRPE sequence, etc. The detection gene is used for determining the success or efficiency of introduction of the CAR expression vector, detecting CAR gene expression or determining expression efficiency, selecting or sorting cells in which the CAR gene is expressed, etc.Examples of detection genes include the neo gene, which confers resistance to neomycin, the kmr gene and nptII gene, which confers resistance to kanamycin and the like (Bernd Reiss et al. EMBO J. 3 (1984), 3317-3322), the hph gene, which confers resistance to hygromycin (Blochlinger & Diggelmann, Mol Cell Bio 4:2929-2931), and the DHFR gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7)) (all of which are marker genes); and the luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β-glucuronidase (GUS) gene, genes for fluorescent proteins such as GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) or modified forms thereof (e.g., EGFP and d2EGFP) (all of these are reporter genes); and genes such as the epidermal growth factor receptor (EGFR) gene lacking an intracellular domain can be used. The detection gene may be linked to the CAR gene via, for example, a bicistronic regulatory sequence (e.g., an internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is the 2A peptide (T2A) derived from Thosea asigna virus. Other self-cleaving peptides include, but are not limited to, 2A peptides derived from picornaviruses (F2A), 2A peptides derived from foot disease virus (FMDV), 2A peptides derived from equine rhinitis A virus (ERAV), 2A peptides derived from porcine teschovirus (PTV-1), and 2A peptides derived from rotaviruses, insect viruses, aphthoviruses, or trypanosoma viruses. Examples of similar sequences and partially modified sequences are also included.

[0064] A CAR gene expression vector prepared for gene transfer is introduced into T cells by a conventional method. In the case of a viral vector, the vector is introduced into cells by viral infection. In the case of a non-viral vector such as a plasmid, conventional methods such as electroporation, liposome, calcium phosphate, nucleofection, laser, cationic immunoprecipitation, microinjection, and sonoporation can be used for cell introduction, and electroporation is preferred.

[0065] In order to improve the efficiency of integration into the host chromosome, it is preferable to carry out gene introduction using the transposon method. The transposon method is a non-viral gene introduction method that utilizes a pair of a gene enzyme (transposase) and its specific recognition sequence to cause gene transposition, thereby allowing any gene to be integrated into the host chromosome. As a transposon method, for example, the piggyBac transposon method can be used. The piggyBac transposon method utilizes a transposon isolated from an insect (Fraser MJ et al., Insect Mol Biol. 1996 May; 5(2): 141-51; Wilson MH et al., Mol THER 2007 Jan; 15(1): 139-45), and enables highly efficient integration into mammalian chromosomes. The piggyBac transposon method has actually been used to introduce genes (see, for example, Nakazawa Y, et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013, etc.).

[0066] The transposon method is not limited to the use of piggyBac, and may be, for example, Sleeping Beauty (Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Cell 91: 501-510), Frog Prince (Miskey C, Izsvak Z, Plasterk RH, Ivics Z (2003) Nucleic Acids Res 31: 6873-6881), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. Mol Gen Genet. 1995 Dec 2004), or the like. 10;249(4):400-5. ; Koga A, Shimada A, Kuroki T, Hori H, Kusumi J, Kyono-Hamaguchi Y, Hamaguchi S. J Hum Genet. 2007;52(7):628-35. Epub 2007 Jun 7. ), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11. ; Johnson-Hamlett MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K, Mead PE (2006) Genesis 44: 438-445.; Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, Toh WC, Korzh V (2006) BMC Dev Biol 6: 5.) or the like may also be used.

[0067] Gene introduction using the transposon method can be performed by a conventional method. For example, for the piggyBac transposon method, a vector carrying a gene encoding the piggyBac transposase (transposase plasmid) and a vector having a structure in which a CAR gene expression unit is sandwiched between piggyBac inverted repeat sequences (transposon plasmid) are prepared, and these vectors can be introduced into target cells by various methods such as electroporation, nucleofection, lipofection, and the calcium phosphate method.

[0068] Antigen Recognition Treatment of CAR-T Cells After CAR Gene Transfer As a treatment after CAR gene transfer, an antigen recognition treatment is further performed to confer antitumor properties to the CAR-T cells. The method of the antigen recognition treatment is not particularly limited as long as the desired effect on the CAR-T cells is obtained, and for example, the antigen recognition treatment can be performed by contacting the CAR-T cells with a recognition substance that is the antigen targeted by the CAR. By performing the antigen recognition treatment, the CAR-T cells are able to specifically recognize the antigen. Furthermore, the CAR-T cells become CAR-T cells that have antitumor properties against the recognized antigen. The recognition substance may be a tumor-associated antigen or a tumor-specific antigen targeted by CAR, such as EPHA2, HER2, EPHB2, EPHB4, EGFR, GD2, Glypican-3, HER2, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, κ light chain, CD 30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD116, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, lambda, Lewis Y, MCSP, mesothelin, MUC1, MUC4, MUC6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGR receptor 2. Examples of the target protein include carcinoembryonic antigen, HMW-MAA, VEGF receptor, fibronectin, tenascin, factors that enhance migration to cancer (e.g., chemokines), antigens present in the extracellular matrix such as carcinoembryonic antigen (CEA) in necrotic regions of tumors, or proteins containing mutations identified by genomic analysis and / or differential expression studies of tumors, among which EPHB4, GD2, HER2, and CD19 are preferred, and EPHB4, HER2, and CD19 are more preferred.

[0069] In some embodiments, culturing in the presence of a T cell activating ligand is possible and is preferred. The T cell activating ligand used in the present invention is not particularly limited, as long as it interacts with a T cell surface molecule and promotes T cell activation and / or proliferation. Examples of such a ligand include CD3, which couples with the TCR and mediates TCR-mediated signal transduction, and molecules that specifically bind to surface molecules known as costimulators of T cell activation, such as CD28, CD80, CD80 / CD86, CD86, 4-1BBL, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L, and function to transmit activation / proliferation signals or co-signals into T cells or antigen-presenting cells. Such molecules may be physiological ligands (or receptors) for the above-mentioned T cell surface molecules, or non-physiological ligands (or receptors) with agonistic activity. A preferred example of a non-physiological ligand is an agonist antibody.

[0070] Tumor-associated or tumor-specific antigens targeted by CAR can be used as cells expressing them. That is, CAR-T cells can be expanded by co-culturing them as feeder cells. Feeder cells are cells engineered to express part or all of a target antigen on their surface so that the CAR introduced into CAR-T cells can bind to the target antigen. Examples of target antigens include the tumor-associated or tumor-specific antigens targeted by the CAR described above. Feeder cells can be prepared by introducing a gene encoding the target antigen into cells using a vector having an expression unit for expressing the target antigen gene, as described above for CAR-T cells. Alternatively, feeder cells can be prepared by producing mRNA of the target antigen gene and directly introducing the mRNA into cells. Alternatively, CAR-T cells can be expanded under feeder-free conditions by co-culturing the aforementioned recognition substance and / or costimulatory factor, for example, while bound to a support.

[0071] The medium used for preparing CAR-T cells is not particularly limited, and media used in ordinary cell culture, such as RPMI1640, MEM, X-VIVO, IMDM, DMEM, DC medium, OptiMEM, and CellGro media, can be used. The medium may be a medium supplemented with serum (human serum, fetal bovine serum, etc.) in accordance with conventional methods, or a serum-free medium. It is preferable to use a serum-free medium because it is highly safe for clinical application and is less likely to result in differences in culture efficiency due to differences between serum lots. Examples of serum-free media include TexMACS™ (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific), and ALyS culture medium (Cell Science Institute, Inc.). When serum is used, autologous serum, i.e., serum collected from an individual from which the CAR-expressing immune cells are derived (more specifically, a patient receiving the cell population obtained by the production method of the present disclosure), may be used, or artificial serum may be used. In the present invention, it is preferable to use artificial serum. Plasma may also be used. Blood components such as serum or plasma, albumin, or analogs thereof may be used, and artificial substances may also be used. The basal medium may be one suitable for cell culture, and the above-mentioned TexMACS™ (Miltenyi Biotec), AIM V (registered trademark), or ALyS culture medium (Cell Science Institute, Inc.) may be used. Other culture conditions may be any suitable for cell survival and proliferation, and general conditions may be adopted. For example, the culture may be performed in a CO 2 -containing atmosphere set at 37°C. ２ Incubator (CO ２ Examples of suitable oxygen-rich culture include culturing in an oxygen-rich environment (oxygen concentration of 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%), and low-oxygen culture (oxygen concentration of 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%).

[0072] To support cell survival, proliferation, or activation, additional factors may be added to the medium. Examples of additional factors include type 1 cytokine family members, type 2 cytokine family members, TNF superfamily cytokines, IL-1 family cytokines, and other cytokines (such as TNF-β), specifically IL-1 to IL-41, and preferably IL-1, IL-2, IL-7, IL-15, and IL-21. IL-7 and / or IL-15 may be added to the medium when preparing CAR-T cells. The additional factors can be prepared according to standard methods, or commercially available products can be used. The additional factors may be from animal species other than humans, but are preferably derived from humans (which may be recombinant).

[0073] By using T cells obtained by the culture method of the present invention, a CAR gene can be introduced more efficiently and the survival rate is also high, compared to conventional methods. For example, by using T cells cultured by the culture method of the present invention, a CAR introduction efficiency of 30%, 40%, 50%, 60%, or 70% or more, preferably 80% or more, can be achieved.

[0074] The CAR-T cells thus obtained or a cell population containing the same can be used in the treatment of cancer, particularly in the treatment of cancer that expresses the target antigen of the CAR-expressing immune cells. The cancer may be a solid tumor or a blood tumor. Specific cancers include, but are not limited to, various B-cell lymphomas (follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative neoplasms, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer. In a preferred embodiment, the cancer is a solid tumor. Examples of solid tumors include neuroblastoma, brain tumor, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer.

[0075] CAR-T cells or a cell population containing the same are administered in a therapeutically effective amount that is appropriately determined depending on the age, body weight, body surface area, symptoms, etc. of the subject. The subject in the present disclosure is usually a human, preferably a cancer patient. The cell population containing CAR-T cells produced by the method of the present invention is administered in a dose of, for example, 1 x 10 ４ pieces ~ 1x10 １０ The cell population of the present disclosure may be administered in individual doses. The route of administration is not particularly limited, and may be administered intratumorally, peritumorally, intraventricularly, intravenously, intraarterially, intraportally, intradermally, subcutaneously, intramuscularly, or intraperitoneally. The cell population of the present disclosure may be administered systemically or locally, and local administration may include direct injection into the target tissue, organ, or tissue. The administration schedule is determined appropriately depending on the age, weight, body surface area, symptoms, etc. of the subject, and may be a single administration or multiple continuous or regular administrations.

[0076] A composition comprising CAR-T cells or a cell population comprising the same may contain, in addition to the cell population to be administered to a subject, components such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting the cells, antibiotics for the purpose of preventing bacterial contamination, and various components for the purpose of activating, proliferating, or inducing differentiation of the cells (vitamins, cytokines, minerals, carbon sources, nitrogen sources, trace metals, electrolytes, growth factors, steroids, etc.). The composition can be prepared by a conventional method.

[0077] In the method for producing CAR-T cells of the present invention, all or part of the steps can be automated. The automation is not particularly limited, but examples include the following: (i) recovery / isolation of T cells from a specimen; (ii) introduction of a CAR gene (production of CAR-T cells); (iii) activation of CAR-T cells; (iv) expansion and culture of CAR-T cells; (v) recovery and quality control of CAR-T cells, and tests therefor. (i) Recovery / isolation of T cells from a specimen corresponds to the culture method of the present invention. Automation is expected to reduce time (significantly reducing time compared to manual processes), improve quality (consistent process management improves product quality and safety), and reduce labor (reducing human error and reducing the workload).

[0078] In one embodiment, the present invention provides a preparation for separating T cells. In one embodiment, the present invention provides a preparation for culturing CAR-T cells. In one embodiment, the present invention provides a preparation for producing and / or isolating T cells that serve as a raw material for producing T cells (T cell preparations) effective for use in T cell therapy. Such preparations are additives for T cell culture that can be added during T cell culture. These preparations are also collectively referred to as "preparations of the present invention." The preparations of the present invention contain a T cell surface antigen-recognizing substance and can be added to a medium during T cell culture. The amount of the preparation of the present invention to be used is not particularly limited as long as the desired effect is obtained. For example, the amount can be determined in accordance with the above-mentioned conditions for contacting the T cell-containing composition with the T cell surface antigen-recognizing substance.

[0079] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature. Furthermore, those skilled in the art will understand that any substance having the same effect or action can be substituted.

[0080] Example 1: T Cell Culture and Production of CAR-T Cells (1) T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) using magnetic beads conjugated with anti-CD4 antibodies and magnetic beads conjugated with anti-CD8 antibodies, followed by gene transfer by electroporation to produce CAR-T cells. (Materials and Methods) 1. T Cell Separation (Isolation) Frozen PBMCs (HEM) were thawed in a 37°C water bath and washed with D-PBS (Wako). Isolation was performed using CD4 microbeads and CD8 microbeads (both Miltenyi Biotec) according to the product protocol. To vary the amount of T cell surface antigen-recognizing substance used to separate T cells, the amount of beads conjugated with each antibody was varied. When both CD4 microbeads and CD8 microbeads are used, they are also referred to as "CD4+CD8+ beads." In this example, the recommended amount of beads is the amount recommended by the manufacturer. For example, the recommended amount for "CD4+CD8+ beads" is 8 x 10 ９ The total amount of protein bound to the beads used for the separation of 10 cells corresponds to approximately 55 mg (10 ６ (Approximately 6.875 μg per cell.) The total amount of protein includes the amount of protein that may be present in the solution containing the beads used for T cell separation, and hereafter, the term "total amount of protein" may include not only the protein bound to the beads but also the protein present in the solution containing the beads.

[0081] 2. Gene transfer After T cell isolation, the positive fraction was centrifuged, and the cell pellet was suspended in electroporation buffer (Miltenyi Biotec). Electroporation was performed using a TCA cuvette on a CliniMACS Prodigy (Miltenyi Biotec). The PB transposase plasmid and pIRII-EPHB4-28z plasmid were used as the plasmid DNA.

[0082] 3. Expansion Culture After electroporation, the cells were collected and suspended in a complete culture medium containing ALyS™705 Medium (Cell Science & Technology Institute) supplemented with 5% artificial serum (Animal-free; Cell Science & Technology Institute), IL-7 (10 ng / mL; Miltenyi Biotec), and IL-15 (5 ng / mL; Miltenyi Biotec). The cells were then cultured at 37°C in 5% CO. ２ The cells were cultured for 14 days under incubation. Complete culture medium was added every 2-3 days, and the cells were expanded to a 24-well cell bind plate or a T25 flask (CORNING). During the culture, the cells were stimulated by adding beads to which EPHB4-CD80-4-1BBL was bound.

[0083] 4. CAR-T Cell Evaluation Test The aggregation rate was evaluated on Day 7 and Day 14, and on Day 14, cell count measurement, FCM analysis, and an in vitro anticancer test were performed.

[0084] Because CAR-T cells proliferate to some extent in clumps, they were suspended immediately before measurement and then measured using an NC-3000 (MS Techno Systems). The program Viability and Cell Count Assay was used, which can analyze the number of live cells, the number of dead cells, the viability, the cell diameter, and the aggregation rate.

[0085] 4-2. Confirmation of T cell separation Cells were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies (BioLegend) using a MACSQuant analyzer 10 (Miltenyi Biotec). Antibody staining was performed at 1 x 10 ６ The manufacturer's specified per test amount was added to each cell / 100 μL 4 ° C D-PBS, and the mixture was incubated at 4 ° C for 15 minutes before use. Measurements were performed by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on 7-AAD-negative and CD3-positive cell groups. Analysis of FCM results was performed using MACS Quantify analyzer software.

[0086] 4-3. Flow cytometry (FCM) measurement Cells were stained with anti-Ephrin B2 antibody (R&D), anti-CD3 antibody, anti-CD45RA antibody, and anti-CCR7 antibody (BioLegend) using a MACSQuant analyzer 10 (Miltenyi Biotec). Antibody staining was performed using 1 × 10 ６ The manufacturer's recommended per-test volume was added to each cell / 100 μL 4°C D-PBS and incubated at 4°C for 15 minutes. Anti-Ephrin-B2 antibody was added for primary staining, the secondary antibody for primary staining for secondary staining, and all remaining antibodies for tertiary staining. A wash step with 1 mL of D-PBS was performed between each staining. Measurement of the CAR positivity rate was performed by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative and CD3-positive cell group. Analysis of T subsets was performed by gating on the 7-AAD-negative, CD3-positive, and CAR-positive cell group, with CD45RA on the X-axis and CCR7 on the Y-axis. MACS Quantify analyzer software was used to analyze FCM results.

[0087] 4-4. Anticancer test was carried out using an in vitro method. The cancer cells used were Rh30 cells (ATCC), a human rhabdomyosarcoma cell line. 2 x 10 cancer cells were used per condition. ５The cells were prepared in a complete medium at 1 mL per well, seeded onto a 24-well cell bind plate (Corning), and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 2 to 24 hours as adherent cells. After that, CAR-T cells were seeded at a ratio of CAR-T cells to cancer cells of 4:1 or 1:1, and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 3 days. The medium used during co-culture was the complete medium for Rh30 cells (RPMI1640-10% FBS medium), with a total volume of 2 mL per well. On day 3 of culture, the cells in the supernatant were collected in a 15 mL tube, and the adherent cells were dispersed with Trypsin-EDTA (0.25%) (Thermo Fisher Scientific). A 5-fold volume of complete medium was added to inactivate the trypsin, and the cells were then collected in the same 15 mL tube (the same tube used for collecting the supernatant). Cell counts were measured using 100 μL of the collected medium (see "4-1. Cell Count Measurement"). FCM measurements were performed to determine the ratio of CAR-T cells to cancer cells and the properties of the CAR-T cells. The properties of the CAR-T cells were examined using the same method as in "4-2. FCM Measurement." The CAR-positive rate was measured by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative and CD3-positive cell group. Analysis of T subsets was performed by gating on the 7-AAD-negative, CD3-positive, and CAR-positive cell group, with CD45RA on the X-axis and CCR7 on the Y-axis. The FCM results were analyzed using MACSQuantify analyzer software. The ratio was calculated by staining CAR-T cells with an anti-CD3 antibody and cancer cells with an anti-CD221 antibody (BioLegend) using MACSQuant analyzer 10 (Miltenyi Biotec). Antibody staining was performed at 1 x 10 ６The manufacturer's specified per test amount was added to each cell / 100 μL 4 ° C D-PBS and allowed to react at 4 ° C for 15 minutes. Dead cells were stained with 7-AAD (Miltenyi Biotec), and the cells gated on 7-AAD negative were plotted with CD3 on the X axis and CD221 on the Y axis. FCM results were analyzed using MACS Quantify analyzer software.

[0088] (Results, etc.) The evaluation results of the positive fraction after cell separation using CD4+CD8+ beads (evaluation immediately after bead separation) are shown in Table 1. For the CD4+CD8+ bead-separated sample, when fewer beads were used ((2) and (3) in Table 1) than the recommended amount ((1) in Table 1), the recovery rate of T cells decreased, but the purity was good.

[0089]

[0090] For sample (2) in Table 1, which showed favorable results, the amount of each antibody, which is the T cell surface antigen-recognizing portion, was quantified out of the total protein amount. The results are shown in Tables 2 and 3 as the optimal protein amount. After magnetically recovering the beads, the proteins were reduced and alkylated, and then digested with trypsin. The peptides were then extracted using a solid-phase column, and the antibody concentration was quantified by LC / MS. The total protein amount of CD45RA microbeads (CD45RA+) (Miltenyi Biotec) and the amount of protein derived from the antibody, which is the T cell surface antigen-recognizing portion, were also quantified. The results are shown in Tables 2 and 3.

[0091]

[0092]

[0093] The same effect was obtained when beads with a low amount of antibody bound to them were used to change the amount of T cell surface antigen-recognizing substance used to separate T cells. Separation using beads with a low amount of identifying substance bound to them resulted in less aggregation during CAR-T cell production, making production easier.

[0094] After gene transfer following bead separation, the aggregation rates on day 7 (Day 7) and day 14 (Day 14) of culture were compared, and the results are shown in Table 4. ６ The amount of protein bound to each bead represents the total protein amount, which corresponds to the values ​​based on the complex in this specification. As shown in Table 2, the amounts of antibody-derived protein were CD4+ (0.0013 μg, 0.0084 pmol), CD8+ (0.0005 μg, 0.0034 pmol), and CD4+CD8+ (0.0018 μg, 0.0118 pmol), which correspond to the values ​​based on the antigen-recognition moiety (recognition site only) in this specification. Regarding the cell count and agglutination rate on Day 7 and Day 14, when the recommended amount of beads was used, the agglutination rate was high on both Day 7 and Day 14.

[0095]

[0096] The results of the induction evaluation and anti-cancer evaluation against Rh30 cancer cells on Day 1 when CAR-T cells were produced from the positive fraction after bead separation are shown in Table 5. ６ The amount of protein bound to each bead represents the total protein amount, and corresponds to the values ​​on a complex basis in this specification. As shown in Table 2, the amounts of antibody-derived protein were CD4+ (0.0013 μg, 0.0084 pmol), CD8+ (0.0005 μg, 0.0034 pmol), and CD4+CD8+ (0.0018 μg, 0.0118 pmol), which correspond to the values ​​for the antigen recognition portion (recognition site only) in this specification. Samples containing a high amount of protein in beads during bead separation (samples using beads in the amount recommended by the manufacturer) showed reduced anticancer activity against Rh30 cancer cells, and the proportion of young cells (Naive / Tscm) was also reduced.

[0097]

[0098] Example 2: Culturing T Cells and Production of CAR-T Cells (2) T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) using magnetic beads conjugated with anti-CD45RA antibodies, and then transfected with genes by electroporation to produce CAR-T cells. (Materials and Methods) 1. Separation of T Cells Frozen PBMCs (HEM) were thawed in a 37°C water bath and washed with D-PBS (Wako). CD45RA microbeads (Miltenyi Biotec) were used for separation according to the product protocol. In this example, the amount of beads conjugated with the identification substance was varied to vary the amount of T cell surface antigen identification substance used to separate T cells. In this example, the recommended amount of beads is the amount recommended by the manufacturer, e.g., 1 x 10 ９ The total amount of protein used for the separation of 10 cells corresponds to approximately 70 mg (10 ６ (Approximately 70 μg per cell).

[0099] 2. Gene transfer The gene transfer was carried out in the same manner as in Example 1.

[0100] 3. Expansion culture was carried out in the same manner as in Example 1.

[0101] 4. CAR-T Cell Evaluation Test This was carried out in the same manner as in Example 1. To confirm T cell separation, an anti-CD45RA antibody (BioLegend) was used instead of the anti-CD4 antibody and anti-CD8 antibody.

[0102] (Results, etc.) After cell separation using CD45RA beads, it was confirmed that CD45RA+ cells accounted for approximately 80% or more of the positive fraction, and that cells were recovered with high purity.

[0103] When the aggregation rates on the 7th day of culture were compared, it was confirmed that, as in Example 1, when the recommended amount of beads was used, the aggregation rate was high.

[0104] The results of the induction evaluation and anti-cancer evaluation against Rh30 cancer cells on Day 1 when CAR-T cells were produced from the positive fraction after bead separation are shown in Table 6. ６The amount of protein bound per bead represents the total protein amount, which corresponds to the value on a complex basis herein. As shown in Table 2, the amount of antibody-derived protein was CD45RA+ (0.0106 μg, 0.0700 pmol), which corresponds to the value on an antigen-recognition moiety basis herein. For CD45RA beads, the amount of beads used ((2) in Table 4) less than the recommended amount ((1) in Table 6) had a higher anticancer activity against Rh30 cancer cells. The proportion of young cells (Naive / Tscm) after the anticancer test was also higher in (2), demonstrating that the production of CAR-T cells after separating T cells using a small amount of beads promotes sustained anticancer activity.

[0105]

[0106] Example 3: Culturing T cells and producing CAR-T cells by viral method T cells were separated from cryopreserved peripheral blood mononuclear cells (PBMCs) using magnetic beads bound to anti-CD4 antibodies and magnetic beads bound to anti-CD8 antibodies, and then gene transfer was carried out by viral method to produce CAR-T cells. (Materials and Methods) 1. T cell sorting (separation) and cell preparation T cells were sorted in the same manner as in Example 1, and the positive fraction was collected and resuspended in TexMACS Medium (Miltenyi Biotec) supplemented with 5% artificial serum (Animal-free; Cell Science & Technology Institute), IL-7 (100 U / mL; Miltenyi Biotec), IL-15 (200 U / mL; Miltenyi Biotec), T cell TransAct (10 μL / 1 × 10 ６ The cells were suspended in a complete medium containing 100% ethanol (Miltenyi Biotec) and cultured overnight.

[0107] 2. Gene transduction Cells cultured overnight were infected with CD19-CAR lentivirus.

[0108] 3. Expansion culture: The cells were cultured at 37°C in 5% CO while adding a complete culture medium prepared by adding 5% artificial serum (Animal-free; Cell Science & Technology Institute), IL-7 (100 U / mL; Miltenyi Biotec), and IL-15 (200 U / mL; Miltenyi Biotec) to TexMACS Medium (Miltenyi Biotec) as needed. ２ The cells were cultured in an incubator for 5 days.

[0109] 4. CAR-T cell evaluation test The aggregation rate was evaluated on Day 1, and on Day 6, cell count measurement, FCM analysis, and an in vitro anticancer test were performed.

[0110] Because CAR-T cells proliferate to some extent in clumps, they were suspended immediately before measurement and then measured using an NC-3000 (MS Techno Systems). The program Viability and Cell Count Assay was used, which can analyze the number of live cells, the number of dead cells, the viability, the cell diameter, and the aggregation rate.

[0111] 4-2. Confirmation of T cell separation This was carried out in the same manner as in Example 1.

[0112] 4-3. Flow cytometry (FCM) measurement Cells were stained with anti-CD19 antibody (R&D), anti-CD3 antibody, anti-CD45RA antibody, and anti-CCR7 antibody (BioLegend) and analyzed using a MACSQuant analyzer 10 (Miltenyi Biotec). Antibody staining was performed using 1 × 10 ６The manufacturer's recommended per-test volume was added to each cell / 100 μL 4°C D-PBS and incubated at 4°C for 15 minutes. Primary staining involved the addition of anti-CD19 antibody, secondary staining involved the addition of the secondary antibody, and tertiary staining involved the addition of all remaining antibodies. A wash step with 1 mL of D-PBS was performed between each staining. Measurement of the CAR positivity rate was performed by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative and CD3-positive cell group. Analysis of T subsets was performed by gating on the 7-AAD-negative, CD3-positive, and CAR-positive cell group, with CD45RA on the X-axis and CCR7 on the Y-axis. MACS Quantify analyzer software was used to analyze FCM results.

[0113] 4-4. Anticancer test was carried out by in vitro method. NALM6 cells (ATCC), a human lymphoblastic leukemia cell line, were used as cancer cells. 1 × 10 cancer cells were used per condition. ５ The cells were prepared in a complete medium at 1 mL per well, seeded onto a 48-well plate (Corning), and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 2 to 24 hours as adherent cells. After that, CAR-T cells were seeded at a ratio of 1:1 between CAR-T cells and cancer cells, and the cells were incubated at 37°C and 5% CO ２The cells were cultured in an incubator for 3 days. The medium used during co-culture was the complete medium for NALM6 cells (RPMI1640-10% FBS medium), with a total volume of 1 mL per well. On day 3 of culture, the cells were harvested, and 100 μL of the collected medium was used to measure cell counts (see "4-1. Cell Count Measurement"). FCM measurements were performed to determine the ratio of CAR-T cells to cancer cells and the properties of CAR-T cells. The properties of CAR-T cells were investigated using the same method as in "4-2. FCM Measurement"). The CAR positivity rate was measured by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative and CD3-positive cell population. Analysis of T subsets was performed by gating on 7-AAD-negative, CD3-positive, and CAR-positive cell groups, with CD45RA on the X-axis and CCR7 on the Y-axis. MACSQuantify analyzer software was used to analyze the FCM results. The ratio was calculated by staining CAR-T cells with an anti-CD3 antibody and cancer cells with an anti-CD19 antibody (BioLegend), and the analysis was performed using a MACSQuant analyzer 10 (Miltenyi Biotec). Antibody staining was performed at 1 x 10 ６ The manufacturer's specified per test amount was added to each cell / 100 μL 4 ° C D-PBS and allowed to react at 4 ° C for 15 minutes. Dead cells were stained with 7-AAD (Miltenyi Biotec), and the cells gated on 7-AAD negative were plotted with CD3 on the X axis and CD19 on the Y axis. FCM results were analyzed using MACS Quantify analyzer software.

[0114] (Results, etc.) As in Example 1, it was confirmed that CD4+ and CD8+ T cells were successfully isolated.

[0115] When the aggregation rates on the first day of culture were compared, it was confirmed that, as in Example 1, when the recommended amount of beads was used, the aggregation rate was high.

[0116] CAR-T cells were produced from the positive fraction after bead separation, and the results of evaluation on Day 6 and anti-cancer evaluation against NALM6 cancer cells are shown in Table 7. Cells 10 of sample (2) in Table 7 ６ The amount of protein bound per bead represents the total protein amount, which corresponds to the value on a complex basis herein. As shown in Table 2, the amount of antibody-derived protein was CD4+ (0.0013 μg, 0.0084 pmol), CD8+ (0.0005 μg, 0.0034 pmol), and CD4+CD8+ (0.0018 μg, 0.0118 pmol), which corresponds to the value on an antigen-recognition moiety basis (recognition site only) herein. For CD4+CD8+ beads, the CAR-positive rate and anti-cancer activity against NALM6 cancer cells were higher when fewer beads (Table 7, (2)) were used than the recommended amount (Table 7, (1)). The proportion of young cells (Naive / Tscm) after the anti-cancer test was also higher in (2), demonstrating that producing CAR-T cells after separating T cells using a smaller amount of beads promotes sustained anti-cancer activity.

[0117]

[0118] According to the method of the present invention, it is possible to obtain T cells with high gene transfer efficiency and high survival rate. As a result, it is possible to obtain sufficient amounts of T cells suitable for T cell therapy and CAR-T cell therapy. Furthermore, it is possible to prevent aggregation of beads and / or cells when separating T cells using beads. This application is based on Patent Application No. 2024-109170 filed in Japan (filing date: July 5, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for culturing T cells, comprising the step of contacting a composition containing T cells with a substance that identifies a T cell surface antigen, wherein the amount of the substance that identifies a T cell surface antigen added is such that the number of cells in the composition containing T cells is 10 ６ A method in which the amount of the complex per individual is 0.1 to 7.0 μg.

2. A method for culturing T cells, comprising the step of contacting a composition containing T cells with a T cell surface antigen identifying substance, wherein the amount of the T cell surface antigen identifying substance added is such that 10 T cells are identified in the composition containing T cells. ６ The amount of the antigen-recognizing moiety per antibody is 0.0001 to 2.0 μg and / or 0.0001 to 20 pmol.

3. The method according to claim 1 or 2, characterized in that the proportion of T cells contained in the composition containing said T cells is 5% or more of the total number of cells contained in the composition.

4. The method of claim 1 or 2, wherein the T cell surface antigen-recognizing substance comprises an antibody and / or an antigen-binding fragment thereof.

5. The method of claim 4, wherein the antibody is an antibody against at least one selected from the group consisting of CD4, CD8, CD45, CD45RA, CD45RO, CD62L, CD127, CD28, CD3, CD27, CD44, CD57, CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a.

6. The method according to claim 1 or 2, wherein the T cell surface antigen recognizing substance is present on a support.

7. The method of claim 6, wherein the support is a bead or a nanomatrix.

8. The method according to claim 1 or 2, wherein the method for culturing T cells is a method for isolating T cells.

9. The method of claim 8, wherein said separation is by magnetic separation.

10. The method according to claim 1 or 2, wherein the method for culturing T cells is a method for maintaining and expanding T cells.

11. The method of claim 1 or 2, wherein the T cells comprise naive and / or memory T cells.

12. The method of claim 1 or 2, wherein the T cells comprise 60% or more naive and / or memory T cells.

13. A method comprising expressing a chimeric antigen receptor in T cells obtained by the culture method of claim 1 or 2.

14. An additive for T cell culture containing a T cell surface antigen-recognizing substance, comprising: 10 cells; ６ A formulation characterized by using a T cell surface antigen-recognizing substance at a dose of 0.1 to 7.0 μg per individual on a complex basis, wherein the T cell surface antigen-recognizing substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody.

15. An additive for T cell culture containing a T cell surface antigen-recognizing substance, which is used to culture 10 T cells in a composition containing T cells. ６ A formulation characterized by using a T cell surface antigen-recognizing substance at a dose of 0.0001 to 2.0 μg and / or 0.0001 to 20 pmol per cell based on the antigen recognition portion, wherein the T cell surface antigen-recognizing substance comprises an anti-CD4 antibody and / or an anti-CD8 antibody.

16. The preparation according to claim 14 or 15, which is for producing T cells.

17. A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the following steps: (1) culturing T cells by the method of claim 1 or 2 (pre-culture step), (2) introducing a CAR gene into the pre-cultured T cells (CAR introduction step), and, optionally, (3) expanding the cells into which the CAR gene has been introduced (expansion step).

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