Method for culturing t cells

Abstract

The present invention relates to a method for culturing T cells, the method comprising a step for bringing a composition containing T cells into contact with a stimulant, wherein the stimulant activates (i) one or more intracellular signaling domains of one or more components of a TCR complex and (ii) one or more intracellular signaling domains of one or more co-stimulatory molecules. This method makes it possible to maintain and proliferate T cells in a composition containing the T cells into a cell group suitable for gene transfer.

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 in a composition containing T cells that serves 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. Known methods include using tumor-infiltrating lymphocytes (TILs) (Non-Patent Document 1) and introducing a cancer antigen-specific T cell receptor (TCR) gene into T cells in the patient's peripheral blood (Non-Patent Document 2). Recently, a new therapy using chimeric antigen receptor (CAR)-T cells (hereinafter simply referred to as CAR-T cells) has been developed. This involves genetically modifying the T cell receptor (TCR) of cytotoxic T cells (CTLs) to allow the CTLs to directly and selectively recognize tumor cells and exert an antitumor effect (Non-Patent Document 3). That is, CAR-T cells are artificially produced T cells obtained by introducing a CAR through genetic manipulation. Cancer therapy using CAR-T cells kills cancer cells through a mechanism different from that of conventional anticancer drugs or radiation therapy, and is therefore 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, 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, it can be difficult to secure a large number of cells depending on the patient. Even if a sufficient number of cells can be secured, it can be difficult to produce a stable final product depending on the properties of the cells.

[0005] Naive T cells collected from the peripheral blood of a patient that have not received antigen stimulation are activated and mature into "effector T cells" and "memory T cells." To produce a good T cell preparation, it is preferable to maintain the naive / memory state (Non-Patent Document 7).

[0006] The properties of T cells contained in T cell sources, including peripheral blood mononuclear cells (PBMCs), affect the phenotype and function of the final T cell preparation. For example, it has been reported that, in order to improve the success rate and function of CAR-T cell production, removing monocytes and granulocytes from the starting material and concentrating all T cells improves T cell activation and CAR gene transduction efficiency during the CAR-T cell production process (Non-Patent Documents 4 to 6). Patent Documents 1 and 2 disclose methods for activating CAR gene-transduced T cells to produce CAR-T cells with high antitumor activity. Patent Document 3 discloses a method for producing CAR-T cells by separating a T cell-containing fraction using a specific biomarker by flow cytometry, transducing the gene, and expanding the resulting cells. Patent Document 4 discloses a method for producing CAR-T cells by co-culturing transduced T cells with cells engineered to express an antigen during expansion. Patent Documents 5 and 6 describe methods for activating T cells with a stimulant during the production of CAR-T cells. However, none of the documents describe or suggest a method for controlling T cell differentiation and ensuring a constant amount of a homogenous cell population that is rich in naive and memory T cells.

[0007] International Publication No. 2021 / 020526 International Publication No. 2018 / 110374 U.S. Patent No. 10,316,289 Specification International Publication No. 2021 / 020526 Special Publication No. 2021-533746 Special Publication No. 2021-534783

[0008] 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.Noaks E. et al., Mol Ther Methods Clin Dev. 2021 20: 675-687.Stroncek DF. et al., Cytotherapy. 2016 18(7): 893-901.Stroncek DF. et al., J Transl Med. 2017 15(1): 59.Arcangeli S. et al., J Clin Invest. 2022 Jun 15;132(12):e150807.

[0009] When performing gene transfer, it is necessary to secure a required number of T cells with certain properties, but because autologous T cells collected from a 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 a patient into a cell population suitable for gene transfer.

[0010] In view of the above problems, the present inventors have conducted extensive research and found that naive and / or memory T cells can be maintained and proliferated by contacting T cells with a certain amount of a stimulant when culturing them, specifically by contacting a composition containing T cells with a stimulant that activates (i) one or more intracellular signaling domains of one or more components of the TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules.The present inventors have confirmed that this method of culturing T cells is useful as a pre-culture method for a composition containing T cells that is a raw material for producing a T cell preparation, and have thus completed the present invention. That is, the present invention is as follows.

[0011] [1] A method for culturing T cells, comprising a step of contacting a composition containing T cells with a stimulatory agent, wherein the stimulatory agent activates (i) one or more intracellular signaling domains of one or more components of a TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules, and the amount of the stimulatory agent added is such that 10 cells / mL of the composition containing T cells are 100%. ６ [2] A method for culturing T cells, comprising the step of contacting a composition containing T cells with a stimulating agent, wherein the stimulating agent activates (i) one or more intracellular signaling domains of one or more components of a TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules, and the amount of the stimulating agent added is 1 to 1500 ng per 10 T cells in the composition containing T cells, and the proportion of T cells contained in the composition is 5% or more of the total number of cells contained in the composition. ６A method characterized in that the amount of T cells is 1 to 1500 ng per cell. [3] The method according to [1] or [2], which comprises a step of separating T cells from a composition containing T cells. [4] The method according to any of [1] to [3], wherein the method for culturing T cells is a method for maintaining and expanding T cells. [5] The method according to any of [1] to [4], wherein the T cells are naive and / or memory T cells. [6] The method according to any of [1] to [5], wherein the stimulating agent comprises an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof. [7] The method according to [6], wherein the ratio of the anti-CD3 antibody or an antigen-binding fragment thereof to the anti-CD28 antibody or an antigen-binding fragment thereof contained in the stimulating agent is 1:0.5 to 1:200. [8] The method according to any of [1] to [7], wherein the stimulating agent is present on a support. [9] The method according to [8], wherein the support is a bead or a nanomatrix.

[10] 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 according to [1] or [2] (preculture step), (2) a step of introducing a CAR gene into the precultured T cells (CAR-T introduction step), and, optionally, (3) a step of expanding the cells into which the CAR gene has been introduced (expansion step).

[11] An additive for T cell culture, comprising an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof, wherein the T cells are naive and / or memory T cells.

[12] A method for producing T cells comprising the steps of: ６ The agent according to

[11] , which is used at a dose of 1 to 1500 ng per unit.

[0012] According to the methods of the present invention, young T cells (e.g., naive and / or memory T cells) can be expanded while maintaining their young state, thereby providing a sufficient amount of T cells suitable for T cell therapy or CAR-T cell therapy.

[0013] FIG. 10 shows the results of flow cytometry of cell populations after 7 days of pre-culture (pre-culture Day 7) in the presence of stimulatory agents (anti-CD3 antibody and anti-CD28 antibody).

[0014] The present invention is described below. Terms used herein have the meanings commonly 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 includes a step of contacting a composition containing T cells with a stimulatory agent, wherein the stimulatory agent activates (i) one or more intracellular signaling domains of one or more components of the TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules.

[0015] Composition Comprising T Cells The T cells in the "composition comprising T cells" of the present invention (hereinafter also referred to as the "T cell-containing composition" of the present invention) may be either autologous or allogeneic. 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. T cells in the T cell-containing composition include CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, CD4-positive CD8-positive T cells, CD4-negative CD8-negative T cells, αβ-T cells, γδ-T cells, Treg cells, NK-like T cells, NKT cells, and the like. The T cells may be subsets 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, mesenchymal stem cells, etc. 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, for example, naive and / or memory T cells.

[0016] 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 highly 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 produce large numbers of more differentiated Tcm and Tems.

[0017] In one embodiment, the percentage of naive T cells in the T cell-containing composition is greater than 60%, 65%, 70%, 80%, 90%, or 95% of the total T cells in the T cell-containing composition. In a particular embodiment, 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 a particular embodiment, the naive T cells comprise CD27+CCR7+ T cells. Culturing T cells using the methods of the present invention does not promote T cell differentiation, allowing naive T cells to proliferate while maintaining their characteristics. Therefore, by culturing a T cell-containing composition using the culture method of the present invention, the percentage of naive T cells in the T cell-containing composition can be increased, so the percentage of naive T cells in the T cell-containing composition to which the method of the present invention is applied may be less than 60%.

[0018] In one embodiment, the percentage of memory T cells in the T cell-containing composition is greater than 60%, 65%, 70%, 80%, 90%, or 95% of the total T cells in the T cell-containing composition. In particular embodiments, memory T cells comprise CCR7-positive CD45RA-positive (CCR7+CD45RA+), CCR7-positive CD45RA-negative (CCR7+CD45RA-), CCR7-negative CD45RA-negative (CCR7-CD45RA-), or CCR7-negative CD45RA-positive (CCR7-CD45RA+) T cells. In some embodiments, memory T cells comprise CCR7+CD45RA+ or CCR7+CD45RA- T cells. In some embodiments, memory T cells comprise CCR7+CD45RA+ T cells. Culturing T cells using the methods of the present invention prevents differentiation of T cells and allows memory T cells to proliferate while maintaining their characteristics. Therefore, by culturing a T cell-containing composition using the culture method of the present invention, the percentage of memory T cells in the T cell-containing composition can be increased, so the percentage of memory T cells in a T cell-containing composition to which the method of the present invention is applied may be less than 60%.

[0019] In one embodiment, in the T cell-containing composition, the percentage of naive T cells and / or memory T cells is greater than 60% of the total T cells in the T cell-containing composition.

[0020] The proportion of T cells in a T cell-containing composition is not particularly limited as long as the stimulating agent 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 can 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 (so-called patient) with a specific disease or condition, in need of cell therapy, or to whom cell therapy is applied, the proportion of T cells in the T cell-containing composition can be less than 5%, or can be as low as a few percent. In such cases, T cells in the composition can be concentrated appropriately using various methods known per se.

[0021] 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 from 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 applied, is used as the T cell-containing composition. Biological samples include, but are not limited to, body 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.

[0022] 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.

[0023] T cell-containing compositions containing an enriched proportion of T cells can be obtained by selecting, isolating, or enriching T cells from a T cell source. For example, selection, isolation, or enrichment can be performed by separating and obtaining T cells from a T cell source or by separating and removing non-T cells from a T cell source based on properties possessed by the T cells and / or non-T cells. As one example, T cells can be separated from a PBMC sample by negative selection for markers expressed on B cells, monocytes, or other white blood cells, e.g., non-T cells, such as CD14. As another example, a CD3+ T cell selection step can be performed to generate a population enriched for CD3-positive (CD3+) T cells from a T cell source, such as a PBMC or leukapheresis sample.

[0024] 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.

[0025] Specific methods for selecting, isolating, or enriching T cells from a T cell source include the following: Separating T cells expressing a specific cell membrane surface antigen from cells that do not express that antigen. This separation process can be performed using commonly used methods for separating cells based on the expression status of cell membrane surface antigens, with density gradient separation, immunological cell separation, magnetic cell separation, nylon wool separation, and adhesion methods being preferred. 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, with the density at the bottom of the container. Magnetic cell sorting (MACS) is a technique that 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 the non-magnetically labeled cells using magnetic force. Immunological cell separation is a method of separating cells using antigen-antibody reactions. The nylon wool separation method utilizes the property that B cells adhere well to nylon wool. The adhesion method is a cell separation method that utilizes differences in the adhesive ability of cells to a substrate (e.g., a culture flask). Specifically, when a T cell source is placed in a culture flask, adhesive monocytes and other cells adhere to the bottom of the culture flask, while non-adhesive lymphocytes such as T cells float in the culture medium. Therefore, the adhesion method is used to separate these two cell groups. To remove cells that adhere to the culture vessel, the T cell source is placed in the culture vessel for a certain period of time (e.g., 1 second to 7 days). After the certain period of time, the adherent cells are not used, and only the floating cells are collected and used as the T cell source. Examples of culture vessels include, but are not limited to, culture flasks, culture chambers, culture bags, culture plates, culture dishes, and bioreactors. Furthermore, the material of the culture vessel is not particularly limited, and examples include polystyrene, TPP, PETG, and glass. 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.).

[0026] 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.

[0027] Stimulating Agent In the present invention, a stimulating agent (hereinafter also referred to as "stimulating agent of the present invention") 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 the cells with the stimulating agent. Contacting with the stimulating agent promotes the survival (maintenance) and proliferation of T cells. It also promotes the survival (maintenance) and proliferation of T cells in a state that is susceptible to gene transfer. Furthermore, it supports the maintenance and / or proliferation of young T cells such as naive T cells and memory T cells, and when CAR-T cells are prepared from such young T cells, it can confer a high antitumor effect to the CAR-T cells. Preferred memory T cells include stem cell memory T cells (Tscm).

[0028] In the present invention, a stimulatory agent activates (i) one or more intracellular signaling domains of one or more components of a TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules. In one embodiment, a stimulatory agent of the present invention comprises a combination of one or more agents (agent 1) capable of activating the intracellular signaling domain of a TCR complex and one or more agents (agent 2) capable of activating one or more intracellular signaling domains of one or more costimulatory molecules. Agent 1 and agent 2 may be the same or different, but are preferably different. The term "agent" as used herein includes both agent 1 and agent 2. Examples of agents include ligands. Agents as contemplated herein can include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other biomolecules with affinity for a desired target. In some aspects, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, 4-1BB (CD137), OX40, or ICOS. T cell costimulatory molecules are molecules that activate T cells by working cooperatively (secondary signaling) with the TCR (primary signaling).

[0029] In some aspects, the stimulatory agent is attached to a support and binds to one of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R, IFN In some embodiments, the support comprises one or more agents (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to one or more of CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO.

[0030] Thus, in a preferred embodiment of the present invention, the substance that activates (i) one or more intracellular signaling domains of one or more components of a TCR complex and (ii) one or more intracellular signaling domains of one or more costimulatory molecules is a combination of (i) a substance that activates one or more intracellular signaling domains of one or more components of a TCR complex (agent 1) and (ii) a substance that activates one or more intracellular signaling domains of one or more costimulatory molecules (agent 2). The ratio of agent 1 to agent 2 is not particularly limited as long as the desired effect is obtained, but typically, agent 1:agent 2 is 1:0.5-200, preferably 1:0.5-100, more preferably 1:0.5-50, even more preferably 1:0.5-10, particularly preferably 1:0.5-2, and most preferably 1:1.

[0031] In some aspects, one or more agents are antibodies. 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 agent can comprise an antibody fragment (including an antigen-binding fragment), such as a Fab, Fab'-SH, Fv, scFv, or F(ab'). ２ It is recognized that constant regions of any isotype can be used in the antibodies contemplated herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be derived from any human or animal species (e.g., murine species). In some aspects, the agent is an antibody that binds to and / or recognizes one or more components of the T cell receptor.

[0032] Preferred examples of active substance 1 include an anti-CD3 antibody or an antigen-binding fragment thereof, and preferred examples of active substance 2 include an anti-CD28 antibody or an antigen-binding fragment thereof. Particularly preferred examples of active substance 1 include an anti-CD3 antibody, and particularly preferred examples of active substance 2 include an anti-CD28 antibody.

[0033] In some embodiments, the conditions for contacting the T cell-containing composition with a stimulating agent are appropriately set depending on the type of stimulating agent used and the proportion of cells, particularly T cells, in the T cell-containing composition. Generally, the stimulating agent is used to contact 10 cells in the T cell-containing composition. ６ In some embodiments, the conditions for contacting the T cell-containing composition with a stimulating agent are appropriately set depending on the type of stimulating agent used, but typically, 10 to 1500 ng, preferably 1 to 1000 ng, preferably 1 to 500 ng, preferably 1 to 200 ng, preferably 1 to 150 ng, preferably 1 to 100 ng, more preferably 1 to 50 ng, even more preferably 1 to 20 ng, and particularly preferably 1 to 10 ng is used per T cell. ６1 to 1500 ng, preferably 1 to 1000 ng, preferably 1 to 500 ng, preferably 1 to 200 ng, preferably 1 to 150 ng, preferably 1 to 100 ng, more preferably 1 to 50 ng, and particularly preferably 1 to 10 ng is used per particle.

[0034] Although not shown in the above example, an embodiment may be adopted in which, for example, a stimulant is added to the culture medium and simultaneously bound to the support.

[0035] Examples of the support to which the stimulant 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, and collagen.

[0036] In one embodiment, the stimulatory agent comprises particles (e.g., beads) conjugated or linked to one or more agents (e.g., biomolecules) capable of expanding T cells. In some aspects, the one or more agents are bound to the 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 the cultured T cells.

[0037] One or more agents can also be used attached directly or indirectly to the 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 agent is an antibody or an antigen-binding fragment thereof, e.g., a Fab. In some embodiments, a biomolecule (e.g., a biotinylated anti-CD3 antibody) can be indirectly attached to the bead via another biomolecule (e.g., an anti-biotin antibody) that is directly attached to the support.

[0038] In one embodiment of the present invention, the stimulating agent is present on the surface of beads as a support or is attached thereto. In a specific embodiment, the beads comprise a diameter of greater than 1 μm and not greater than 9 μm, or not greater than 8 μm, or not greater than 7 μm, or not greater than 6 μm, or not greater than 5 μm. The bead:cell ratio is 100:1 to 1:100, preferably 60:1 to 1:60, more preferably 20:1 to 1:60, and even more preferably 10:1 to 1:60.

[0039] In one embodiment, the cells in the T cell-containing composition 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 ６ At 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 or 10 x 10 ６ More preferably, at a density of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 x 10 cells / mL ６ In certain aspects, T cells, e.g., T cells in a T cell-containing composition, are cultured at a density of 0.1-50 x 10 cells / mL in the presence of a stimulatory agent. ６ At a density of cells / mL, preferably 1.0 to 10 x 10 ６ At a density of cells / mL, more preferably 5.0 to 8.0 x 10 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.

[0040] The medium used in the present invention for culturing T cells is not particularly limited as long as it can be used in the presence of a stimulant. Media typically used in cell culture, such as RPMI 1640, MEM, X-VIVIO, IMDM, DMEM, DC medium, and OptiMEM, 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 2 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.

[0041] 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 during CAR-T cell preparation (described below). 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).

[0042] The T cell-containing composition thus obtained, expanded in the presence of a stimulant, may have a proportion of naive or young memory T cells of 20%, 30%, 45%, 50%, 55%, or 60% or more, preferably 60% or more. Therefore, the T cell-containing composition expanded in the presence of a stimulant can be, and preferably is, used as a starting material for T cell immunization or the production of CAR-T cells. As an example, the production of CAR-T cells using the T cell-containing composition obtained by the culture method of the present invention will be described.

[0043] Production of CAR-T Cells 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 signaling domain for the effector function of immune cells, and the CAR gene is a gene encoding this receptor. The extracellular domain contains an antigen recognition site that exhibits specific binding to the target. The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain transmits a signal required for the immune cell to exert its effector function. In other words, an intracellular signaling domain is used that can transmit a signal required for activating the immune cell when the extracellular domain binds to a target antigen.

[0044] 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; Pule 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 5:177ra38, 2013), and CARs can be constructed with reference to these reports.

[0045] 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 in a T cell-containing composition expanded by the culture method of the present invention. A CAR expression vector refers to a molecule capable of transporting a nucleic acid molecule encoding a CAR gene into T cells. The nucleic acid molecule may be DNA or RNA, and there are no particular limitations on its form or origin; various types of vectors can be used. The vector may 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. Non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, artificial chromosome vectors, and cosmid vectors.

[0046] 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 that confers resistance to neomycin, the kmr gene and nptII gene that confers resistance to kanamycin and the like (Bernd Reiss et al. EMBO J. 3 (1984), 3317-3322), the hph gene that confers resistance to hygromycin (Blochlinger & Diggelmann, Mol Cell Bio 4:2929-2931), and the DHFR gene that confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7)) (all of these are marker genes); the luciferase gene (Giacomin, P1. Sci. 116(1996), 59-72; Scikantha, J. Bact. 178(1996), 121), the β-glucuronidase (GUS) gene, and GFP (Gerdes, FEBS Lett. 389(1996), 44-47) or its variants (e.g., EGFP and d2EGFP) (all of these are reporter genes); or 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, 2A peptides (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptides (E2A) derived from equine rhinitis A virus (ERAV), 2A peptides (P2A) 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.

[0047] 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.

[0048] To improve the efficiency of integration into the host chromosome, gene introduction using the transposon method is preferred. 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 induce gene transposition, allowing any gene to be integrated into the host chromosome. Examples of transposon methods that can be used include the piggyBac transposon method. 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), enabling 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.).

[0049] Transposon methods are not limited to those using piggyBac; 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 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 Hamlet 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.

[0050] 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.

[0051] Antigen Recognition Treatment and Expansion of CAR-T Cells After CAR Gene Transfer As a treatment after CAR gene transfer, an antigen recognition treatment can be further performed to confer antitumor properties to CAR-T cells. The method of antigen recognition treatment is not particularly limited as long as the desired effect on CAR-T cells is obtained, and can be performed, for example, by contacting the CAR-T cells with a recognition substance that is the antigen targeted by CAR. By performing the antigen recognition treatment, the CAR-T cells can 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.The contact between the CAR-T cells and a recognition substance, which is an antigen targeted by the CAR, can be, for example, contact between the CAR-T cells and a support to which the recognition substance is bound.

[0052] Examples of the support to which the recognition substance is bound include the supports described above in the "Stimulating Agent" section. A beaded support (with a particle size of 10 nm to 500 μm, preferably 10 nm to 100 μm, more preferably 10 nm to 50 μm) or a gel support is preferred. In addition to the recognition substance, a costimulatory factor may be bound to the support, and such binding is a preferred embodiment. Examples of costimulatory factors include CD40, CD80, 4-1BBL, OX40, OX40L, CD52, CD54, CD70, CD58, CD86, CD95, CD252, CD275, etc., and ligands of the integrin family (CD49a to CD49h, CD51, CD103, CD41, CD11a to CD11c, ITGA9 to ITGA11, CD18, CD19, CD61, ITGB4 to ITGB8, etc.). In one embodiment, the costimulatory factor is one or more costimulatory factors selected from CD40, CD80, 4-1BBL, and OX40L, preferably CD80 and / or 4-1BBL, more preferably CD80 and 4-1BBL. In a preferred embodiment, beads bound to EPHB4-CD80-4-1BBL are used for antigen recognition treatment of CAR-T cells after CAR gene transfer.

[0053] Furthermore, tumor-associated or tumor-specific antigens targeted by CAR can also be used as cells expressing them. That is, CAR-T cells with antitumor activity can be expanded by co-culturing them as feeder cells. These feeder cells are cells engineered to express part or all of the target antigen on their surface, so that the CAR introduced into the 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 expressing a target antigen 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 preparing mRNA of the target antigen gene and directly introducing the mRNA into cells. In certain embodiments, the target antigen and costimulatory factor may be expressed on the surface of target antigen-expressing cells by introducing a costimulatory factor gene into cells together with a tumor-associated or tumor-specific antigen gene.

[0054] The obtained CAR-T cells can be expanded ex vivo until a sufficient number is obtained. The medium used for preparing and expanding CAR-T cells is not particularly limited, and media used in conventional cell culture, such as RPMI1640, MEM, X-VIVIO, IMDM, DMEM, DC medium, and OptiMEM, can be used. The medium may be a medium supplemented with serum (human serum, fetal bovine serum, etc.) according to 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%).

[0055] 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).

[0056] 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 / CD86, ICOS, 4-1BB (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.

[0057] By using a T cell-containing composition expanded using the T cell culture method of the present invention, a CAR-T cell population that is expected to be more effective than conventional methods can be efficiently produced. For example, a CAR-T cell population obtained using a T cell-containing composition expanded using the T cell culture method of the present invention may have a CAR-T cell percentage of 20%, 30%, or 40% or more, preferably 40% or more. When T cells are chronically activated, they highly express numerous immune checkpoint molecules, making them unable to proliferate or attack target cells. This phenomenon is called exhaustion, and T cells that have fallen into an exhausted state have reduced proliferation and cancer attack capabilities even when returned to the body, making it difficult to expect a high therapeutic effect. Exhaustion of T cells in the T cell-containing composition obtained by the T cell culture method of the present invention is suppressed. The degree of exhaustion can be assessed by methods commonly used in the art, for example, by examining the expression status of exhaustion markers, which is a simple and easy method. Exhaustion markers include programmed death 1 (PD-1), T-cell immunoglobulin mucin-3 (Tim-3), lymphocyte activation gene 3 (LAG3), adenosine A2a receptor (A2aR), cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), and in particular PD-1. Expression of exhaustion markers can be detected using antibodies against these markers. CAR-T cells obtained using a T cell-containing composition expanded by the T cell culture method of the present invention have low expression of PD-1, an exhaustion marker, and for example, the proportion of PD-1-expressing cells among CAR-T cells may be less than 10%, preferably less than 5%, and more preferably less than 1%.

[0058] The cell population containing CAR-T cells thus obtained can be used for the treatment of cancer, particularly for 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.

[0059] The cell population containing CAR-T cells is administered at a therapeutically effective dose 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 at a therapeutically effective 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.

[0060] A composition containing a cell population containing CAR-T cells 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 conventional methods. The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless particularly limited, the reagents and materials used are commercially available or can be prepared according to known literature, etc. Furthermore, those skilled in the art will understand that substitutes can be used as long as they have similar effects and actions.

[0061] Example 1: Cultivation of T cells and production of CAR-T cells Cryopreserved peripheral blood mononuclear cells (PBMCs) were used to pre-culture T cells, followed by production of CAR-T cells. (Materials and Methods) 1. Cell Preparation MaxiBeads (Miltenyi Biotec) 1 x 10 ８ 7.5 μg each of anti-CD3 antibody and anti-CD28 antibody (BioLegend) was added to each PBMC and allowed to bind for 2 hours at 4°C (antibody-bound beads). PBMC (HEM) were thawed in a 37°C water bath and resuspended in complete 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). 7.5 × 10 cells were collected and analyzed. ６ Cells were seeded in a 48-well plate (Corning) at 1 mL / well, and 10 ng of the antibody-bound beads prepared above, diluted with complete medium, was added thereto. The cells were incubated at 37°C, 5% CO ２The cells were incubated for 7 days. Antibody-conjugated beads were used, with 10 ng of anti-CD3 antibody and 10 ng of anti-CD28 antibody. The cells were cultured every 2-3 days, with complete medium being added, and expanded to a 24-well plate or a T25 flask (Corning). On Day 7 (preculture Day 7), cell counts and FCM analysis were performed.

[0062] 2. Gene Transfer After pre-culture, the cell suspension was centrifuged at 300 × g for 5 minutes at room temperature, and the resulting cell pellet was suspended in 300 μL / sample of room-temperature electroporation buffer (Miltenyi Biotec). Plasmid DNA (5 μg / μL in physiological saline) was added to the cell suspension as follows: PB transposase plasmid: 4 μL per 300 μL of electroporation buffer; pIRII-EPHB4-28z plasmid: 4 μL per 300 μL of electroporation buffer. The cell and DNA suspension was placed in an electroporation cuvette (Mirus), and approximately 7.5 × 10 of each plasmid DNA was added using a Miltenyi Biotec Prodigy electroporator unit. ６ PBMCs were transfected by electroporation.

[0063] After gene transfer, the cells were transferred to complete medium and cultured for approximately 14 days (CAR-T cell Day 14). On days 1 and 8 after electroporation, 3 x 10 beads bound to EPHB4-CD80-4-1BBL were added. ６ The cells were seeded into a 48-well plate (Corning) and then scaled up according to the cell growth. On day 14, cell counting, FCM analysis, and an anti-cancer test were performed.

[0064] (Analysis and Results) 1. Measurement of Cell Count The number of CAR-T cells was measured using NC-3000 (MS Techno Systems). The program Viability and Cell Count Assay was used, which can analyze the number of live cells, number of dead cells, viability, cell diameter, and aggregation rate. 2. Flow cytometry (FCM) measurement Cells were stained using anti-Ephrin B2 antibody (R&D), anti-CD3 antibody, anti-CD45RA antibody, anti-CCR7 antibody, and anti-PD-1 antibody (BioLegend), and the measurement was performed using a MACSQuant (registered trademark) analyzer 10 (Miltenyi Biotec). Antibody staining was performed using 1 x 10 ６ The manufacturer-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. 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 washing step with 1 mL of D-PBS was inserted between each staining. To measure the CAR positivity rate and PD-1, dead cells were stained with 7-AAD (Miltenyi Biotec), and 7-AAD-negative and CD3-positive cell groups were analyzed by FCM. The results were analyzed using MACSQuantify TM 3. Anticancer test: This was carried out in vitro. The cancer cells used were Rh30 cells (ATCC), a human rhabdomyosarcoma cell line. 2 x 10 cancer cells were used per condition. ５ The medium was adjusted to give 1 mL of cells per well, and the cells were seeded onto a 24-well cell bind plate (Corning). The plate was then 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 CAR-T cells to cancer cells of 4:1, and the cells were incubated at 37°C and 5% CO ２The cells were cultured in an incubator for 3 days (co-culture Day 3). The medium used during co-culture was the same as that used 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). Five volumes of medium were added to inactivate the trypsin, and the cells were collected in the same 15 mL tube (the tube used for collecting the supernatant). 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 ("2. FCM Measurement"). The CAR positivity rate and PD-1 were measured by staining dead cells with 7-AAD (Miltenyi Biotec), and analyzing the 7-AAD-negative and CD3-positive cell groups using FCM. The FCM results were analyzed using MACSQuantify. TM The ratio was determined by staining the CAR-T cells with an anti-CD3 antibody and the cancer cells with an anti-CD221 antibody (BioLegend), using a MACSQuant (registered trademark) analyzer 10 (Miltenyi Biotec). Antibody staining was performed at 1 x 10 ６ The per test amount specified by the manufacturer 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 analyzed. FCM results were analyzed using MACSQuantify TM analyzer software was used.

[0065] CAR-T cells were prepared and analyzed under the same conditions as in "1. Cell preparation", except that the amounts of anti-CD3 antibody and anti-CD28 antibody added to PBMCs were changed to 75 ng, 300 ng, and 1000 ng, respectively.

[0066] 4. Results, etc. The results of flow cytometry on pre-culture Day 7, CAR-T cell Day 14, and co-culture Day 3 are shown in Table 1. The results of flow cytometry of the cell population on pre-culture Day 7 are shown in Figure 1. Stimulators (agent 1 - factor 1, anti-CD3 antibody; agent 2 - factor 2, anti-CD28 antibody). Tscm: stem cell memory T cells (both CD45RA and CCR7 positive) Tcm: central memory T cells (CD45RA negative and CCR7 positive)

[0067]

[0068] The FCM results revealed that a high stimulation dose (a large amount of stimulant added) reduced the proportion of young cells (naive / Tscm and Tcm) in the resulting CAR-T cells after anti-cancer testing (post-coculture). This suggests that even with a high stimulation dose, CAR-T cells can be produced, but they are potentially exhausted.

[0069] CAR-T cells were prepared and analyzed under the same conditions as in "1. Cell preparation," except that the stimulating agent added to PBMCs was changed to 1, 3, or 30 μL of TransAct (Miltenyi Biotec), and similar results were obtained.

[0070] In "1. Cell preparation," CAR-T cells were prepared and analyzed under the same conditions except that the percentage of T cells contained in the PBMCs was changed to 30%, 50%, or 80%, and similar results were obtained.

[0071] Example 2: Culture after T Cell Sorting and Production of CAR-T Cells Cryopreserved peripheral blood mononuclear cells (PBMCs) were used to separate T cells, which were then pre-cultured to produce CAR-T cells. (Materials and Methods) 1. Cell Preparation Anti-CD3 antibody and anti-CD28 antibody (BioLegend) were diluted with D-PBS and seeded onto a 48-well plate (Corning) at 10 ng and 1000 ng, respectively, and coated at 37°C for 1 hour. PBMCs (HEM) were thawed in a 37°C water bath, and T cells were isolated using CD4 and CD8 magnetic beads according to the manufacturer's recommended protocol. The subsequent procedures were the same as in Example 1, except that the cells were seeded onto antibody-coated 48-well plates.

[0072] 2. Gene transfer The amount of PB transposase plasmid and pIRII-EPHB4-28z plasmid was 2 μL per 300 μL, and the amount of PBMCs used for transfecting the plasmid DNA by electroporation was approximately 1.5 × 10 ７ The procedure was the same as in Example 1, except that the number of beads added was 0.25 × 10. 3. Expansion culture Beads were added on the 1st and 7th days after electroporation. ６ The procedure was the same as in Example 1, except that the number of pieces was changed to 1.

[0073] (Analysis and Results) 1. Measurement of cell number, 2. Flow cytometry (FCM) measurement, and 3. Anticancer test were performed in the same manner as in Example 1.

[0074] 4. Results, etc. The results of flow cytometry on Day 7 of pre-culture, Day 14 of CAR-T cells, and Day 3 of co-culture are shown in Table 2.

[0075]

[0076] The FCM results revealed that a high stimulation dose (a large amount of stimulant added) reduced the anti-cancer activity of the resulting CAR-T cells, and also reduced the proportion of young cells (naive / Tscm and Tcm) after the anti-cancer test (after co-culture). This suggests that even when the stimulation dose is high, CAR-T cells can be produced, but they have potentially exhausted properties.

[0077] Example 3: Production of CAR-T cells using a virus CAR-T cells were produced by a viral method using cryopreserved peripheral blood mononuclear cells (PBMCs). (Materials and Methods) 1. Cell preparation The same method as in Example 1 was used, except that 10 μL or 60 μL of TransAct (Miltenyi Biotec) was added instead of antibody-bound beads. 2. Gene transfer The cells were centrifuged in the same manner as in Example 1, and the resulting cell pellet was suspended in ALyS™705 Medium (Cell Science & Technology Institute). The cells were infected with a CD19-CAR lentivirus suspension, and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator. 3. Expansion After gene transfer, the cells were cultured for 7 days with the addition of complete medium (CAR-T cells, Day 7). One day after infection, CD19-CD80-4-1BBL-bound beads were added and the cells were expanded. The culture vessels were prepared in the same manner as in Example 1. On Day 7, cell counts, FCM analysis, and anticancer tests were performed.

[0078] (Analysis and Results) 1. Cell counts were measured using the same method as in Example 1. 2. Flow cytometry (FCM) measurements were performed using the same method as in Example 1, except that anti-CD19 antibodies were used for primary antibody staining. 3. Anticancer test CD19-expressing NALM6 cells (ATCC) were used as cancer cells. For the test, cells were collected on day 3 of co-culture and subjected to analysis. Furthermore, the co-cultured cells were replaced with fresh medium, and then added to the NALM6 cells. The anti-cancer test was repeated six times (for a total of 18 days) by culturing for three days. The medium used during co-culture was the same as that used for NALM6 cells (RPMI 1640-10% FBS medium). The ratios were as follows: CAR-T cells were stained with an anti-CD3 antibody, and cancer cells were stained with an anti-CD19 antibody (BioLegend). Analysis was performed using a MACSQuant® analyzer 10 (Miltenyi Biotec). Other methods were the same as in Example 1.

[0079] 4. Results, etc. The results of flow cytometry on Day 7 of pre-culture, Day 7 of CAR-T cells, and Day 3 of co-culture are shown in Table 3.

[0080]

[0081] FCM results showed that cells with good proliferation and anticancer activity were obtained regardless of whether the amount of TransAct used during pre-culture was 10 μL or 60 μL. Furthermore, even after co-culture, the cells were found to be of high quality, with a naive / Tscm ratio of 20% or higher. The CAR-T cells produced killed 100% of NALM6 cells, even after the second to sixth co-culture cycles, demonstrating the production of persistent cells.

[0082] According to the method of the present invention, naive and / or memory T cells can be maintained and expanded, and a sufficient amount of T cells suitable for T cell therapy or CAR-T cell therapy can be obtained. This application is based on Japanese Patent Application No. 2023-221979 (filing date: December 27, 2023), the contents of which are incorporated in full herein.

Claims

1. A method for culturing T cells, comprising the step of contacting a composition containing T cells with a stimulant, wherein the stimulant activates (i) one or more intracellular signaling domains of one or more components of the TCR complex and (ii) one or more intracellular signaling domains of one or more co-stimulatory molecules, and the addition amount of the stimulant is 1 to 1500 ng per 10 ６ cells in the composition containing T cells, and 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.

2. A method for culturing T cells, comprising the step of contacting a composition containing T cells with a stimulant, wherein the stimulant activates (i) one or more intracellular signaling domains of one or more components of the TCR complex and (ii) one or more intracellular signaling domains of one or more co-stimulatory molecules, and the amount of the stimulant added is 1 to 1500 ng per 10 ６ T cells in the composition containing T cells. A method characterized by this.

3. The method according to claim 1 or 2, comprising the step of separating T cells from a composition containing T cells.

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

5. The method according to claim 1 or 2, wherein the T cells are naive and / or memory T cells.

6. The method according to claim 1 or 2, wherein the stimulant comprises an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof.

7. The method according to claim 6, wherein the ratio of the anti-CD3 antibody or an antigen-binding fragment thereof to the anti-CD28 antibody or an antigen-binding fragment thereof contained in the stimulant is 1:0.5 to 1:

200.

8. The method according to claim 1 or 2, wherein the stimulant is present on a support.

9. The method according to claim 8, wherein the support is beads or a nanomatrix.

10. 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 according to claim 1 or 2 (pre-culture step), (2) a step of introducing a CAR gene into the pre-cultured T cells (CAR-T introduction step), and optionally (3) a step of expanding and culturing the cells into which the CAR gene has been introduced (expansion culture step).

11. An additive for culturing T cells, comprising an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof, wherein the T cells are naive and / or memory T cells.

12. T cell 10 ６ The agent according to claim 11, which is used at a dose of 1 to 1500 ng per ６ .

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