Method for producing car-t cells

Abstract

The present invention provides a method for producing chimeric antigen receptor T (CAR-T) cells, the method comprising a step for isolating, by means of beads, T cells from a T cell supply source using a specific factor. The specific factor is CD45RA+ or the like. The method enables production of CAR-T cells having high anti-tumor activity, and enables production of highly-functional CAR-T cells easily in a short period of time at a low cost.

Description

Method for producing CAR-T cells

[0001] The present invention relates to a method for producing chimeric antigen receptor T cells, and in particular to a method for producing chimeric antigen receptor T cells with high antitumor activity simply, quickly, and at low cost.

[0002] Recently, chimeric antigen receptor (CAR) T cells (hereinafter simply referred to as CAR-T cells) have been developed as an immunotherapy for cancer patients. The T cell receptor (TCR) of cytotoxic T cells (CTLs) is genetically modified to allow the CTLs to directly and selectively recognize tumor cells, thereby exerting antitumor effects (Non-Patent Document 1). CAR-T cells are artificially engineered T cells obtained by introducing a CAR through genetic manipulation. Because CAR-T cell-based cancer therapy kills cancer cells through a mechanism distinct from conventional anticancer drugs and 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 hematological tumors, such as leukemia and malignant lymphoma.

[0003] Currently, CAR-T cell production uses autologous T cells collected from the patient's peripheral blood by apheresis or other methods to introduce the CAR. The T cells are isolated, stimulated with cytokines or antibodies, and then transduced with the CAR gene using a viral vector or transposon. For clinical use, the transduced T cells must be expanded and cultured to the required scale. Thus, the production of CAR-T cells requires a series of complex manufacturing processes. Complex manufacturing processes lead to higher drug prices. Therefore, there is a need for the development of CAR-T cells with high antitumor efficacy that can be produced using simple, safe, and inexpensive methods.

[0004] CD19-targeted CAR-T cell therapy has shown remarkable success in treating B-cell malignancies, but only half of patients achieve long-term remission. Therefore, it is important to enhance the long-term functionality of CAR-T cells without affecting their antitumor efficacy. The antitumor efficacy of CAR-T cells depends on various host factors, including the disease state and an actively hostile tumor microenvironment. Recent clinical studies of CAR-T cell therapy have demonstrated that the quality of the CAR-T cell product is crucial for the function and antitumor efficacy of CAR-T cell therapy (Non-Patent Documents 2-4). Furthermore, non-viral gene transfer using piggyBac (PB) transposon-based gene modification is an effective strategy for CAR-T cell production (Non-Patent Documents 5, 6).

[0005] The properties of T cells contained in T cell sources, including peripheral blood mononuclear cells (PBMCs), affect the phenotype and function of the final CAR-T cells. To improve the success rate and function of CAR-T cell production, it has been reported that enriching the total T cells by removing monocytes and granulocytes from the starting material improves T cell activation and CAR gene transduction efficiency during the CAR-T cell production process (Non-Patent Documents 7-9). Patent Documents 1 and 2 disclose methods for activating CAR gene-transduced T cells to produce CAR-T cells with high antitumor activity. However, no method is known for using an optimal subpopulation (e.g., a subpopulation of PBMCs) as a starting material for CAR-T cell production. Patent Document 3 discloses a method for producing CAR-T cells by separating a T cell-containing fraction using flow cytometry using specific biomarkers, transfecting the resulting cells, and expanding the resulting cells. However, no method is known for isolating an optimal subpopulation as a starting material for CAR-T cell production using techniques such as bead separation.

[0006] Patent Literature 4 discloses a method for producing CAR-T cells by co-culturing gene-transfected T cells with cells engineered to express an antigen during expansion culture. However, no method for expansion culture in the absence of feeder cells is known.

[0007] International Publication No. 2021 / 020526 International Publication No. 2018 / 110374 U.S. Patent No. 10,316,289 International Publication No. 2021 / 020526

[0008] Eshhar Z. et al., Proc Natl Acad Sci USA, 1993, 90: 720-724. 2020 26(12): 1878-1887.Kubo H. et al., Mol Ther Oncolytics. 2021 Mar 5; 20:646-658.Nakamura K. et al., Mol Ther Methods Clin Dev. 2021 Mar 23;21:315-324.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.

[0009] To implement CAR-T cell therapy, quality (high antitumor activity) and quantity (at least 1 × 10 8 Both the tumor-specific and tumor-specific CAR-T cells are required (it is believed that individual CAR-T cells must be prepared). However, CAR-T cells produced by conventional methods do not have sufficient antitumor activity, and producing large amounts of CAR-T cells takes time and may damage the cells. Therefore, an objective of the present invention is to provide a method that enables the production of CAR-T cells with high antitumor activity, and a method that can produce CAR-T cells simply, quickly, and at low cost.

[0010] In light of the above-mentioned problems, the present inventors focused on a T cell source useful as a starting material for CAR-T cell production, specifically, peripheral blood mononuclear cells (PBMCs), and investigated the correlation between the cell membrane surface antigens of these cells and the quality of the resulting CAR-T cells. As a result, they found that superior CAR-T cells can be obtained by using a subpopulation of PBMCs with a specific expression pattern of cell membrane surface antigens as a starting material. Specifically, they found that higher-quality CAR-T cells can be obtained by performing a cell sorting process (cell sorting step) prior to preparing T cells for CAR gene transduction using specific factors expressed on the cell surface (also known as cell surface markers). Furthermore, they found that CAR-T cell expansion can be performed in the absence of feeder cells, and that this process allows for the specific production of highly functional CAR-T cells in a simple, short, and low-cost manner, thereby completing the present invention. Specifically, the present invention is as follows.

[0011] [1] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of separating T cells from a T cell source using a specific factor, wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, CD45RB-, and CD45RC-; The method for producing the cells, wherein the separating step is carried out by at least one method selected from the group consisting of density gradient separation, immunological cell separation technique, magnetic cell separation technique, nylon wool separation method and adhesion method. [2] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of separating T cells from a T cell source using beads with a specific factor, wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, CD45RB-, and CD45RC-. [3] The method of producing the above-mentioned [1] or [2], wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+ and 4-1BBL+. [4] The method of producing the above-mentioned [1] or [2], wherein the specific factor is at least one selected from the group consisting of CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, CD45RB- and CD45RC-.[5] The method for producing T cells according to any one of [1] to [4] above, wherein the T cell source is peripheral blood mononuclear cells. [6] The method for producing T cells according to any one of [2] to [5] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, and polystyrene. [7] The method for producing T cells according to any one of [2] to [6] above, wherein the beads have a particle size of 0.01 to 500 μm. [8] The method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of activating cells in the absence of feeder cells. [9] The method for producing T cells according to [8] above, wherein the cells are T cells or T cells expressing a chimeric antigen receptor.

[10] The method for producing T cells according to [8] or [9] above, wherein the activation treatment is carried out by contacting the cells with a stimulating substance.

[0012]

[11] The manufacturing method according to

[10] above, wherein the contact with the stimulating substance is contact with a substrate to which the stimulating substance is bound.

[12] The manufacturing method according to

[11] above, wherein the contact with the substrate is co-culture of the cells with the substrate.

[13] The manufacturing method according to

[11] above, wherein the substrate is beads or a gel.

[14] The manufacturing method according to

[13] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, and polystyrene.

[15] The manufacturing method according to

[13] or

[14] above, wherein the particle size of the beads is 0.01 to 500 μm.

[16] The manufacturing method according to

[10] above, wherein the contact with the stimulating substance is contact with vesicles encapsulating the stimulating substance.

[17] The manufacturing method according to

[16] above, wherein the vesicles are liposomes, exosomes, microvesicles, or apoptotic bodies.

[18] The manufacturing method according to any one of [8] to

[10] above, wherein the activation treatment is carried out by culturing the cells in a medium containing a stimulating substance.

[19] The manufacturing method according to

[18] above, wherein the activation treatment is carried out by co-culturing the cells with a substrate to which the stimulating substance is bound in a medium containing the stimulating substance.

[20] The manufacturing method according to

[19] above, wherein the substrate to which the stimulating substance is bound is a bead to which an antibody is bound.

[0013]

[21] The method according to any one of

[10] to

[20] above, wherein the stimulating substance is at least one selected from the group consisting of proteins, peptide fragments, and sugar chains.

[22] The manufacturing method according to any one of

[10] to

[21] above, wherein the stimulator provides at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, PPAR-, statin-, CD45RB-, and CD45RC-.

[23] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of treating cells for antigen recognition in the absence of feeder cells.

[24] The method according to

[23] above, wherein the cells are T cells or T cells expressing a chimeric antigen receptor.

[25] The method according to

[23] or

[24] above, wherein the antigen recognition treatment is carried out by contacting the cells with a recognition substance.

[26] The method according to

[25] above, wherein the contact with the recognition substance is contact with a substrate to which a recognition substance is bound.

[27] The method according to

[26] above, wherein the contact with the substrate is co-culture of the cells with the substrate.

[28] The method according to

[26] above, wherein the substrate is beads or a gel.

[29] The method according to

[28] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, and polystyrene.

[30] The manufacturing method according to

[28] or

[29] above, wherein the particle size of the beads is 0.01 to 500 μm.

[0014]

[31] The manufacturing method according to

[25] above, wherein the contact with the recognition substance is contact with vesicles encapsulating the recognition substance.

[32] The manufacturing method according to

[31] above, wherein the vesicles are liposomes, exosomes, microvesicles, or apoptotic bodies.

[33] The manufacturing method according to any one of

[23] to

[25] above, wherein the antigen recognition treatment is carried out by culturing the cells in a medium containing the recognition substance.

[34] The manufacturing method according to

[33] above, wherein the antigen recognition treatment is carried out by co-culturing the cells with a substrate to which a recognition substance is bound in a medium to which a recognition substance is bound.

[35] The manufacturing method according to

[34] above, wherein the substrate to which a recognition substance is bound is a bead to which an antibody is bound.

[36] The manufacturing method according to any one of

[25] to

[35] above, wherein the recognition substance is at least one selected from the group consisting of proteins, peptide fragments, and sugar chains.

[37] The method for producing chimeric antigen receptor T (CAR-T) cells according to any one of

[25] to

[36] above, wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19.

[38] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing cells after CAR gene introduction with beads to which a stimulating substance and a recognition substance are bound in a medium containing a stimulating substance and a recognition substance, the stimulatory substance provides at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, PPAR-, statin-, CD45RB-, and CD45RC-; The method according to any one of [8] to

[37] above, wherein the cognitive substance is at least one selected from the group consisting of EPHB4, HER2 and CD19.

[39] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the steps of: separating T cells from a T cell source using a specific factor; introducing a CAR gene into the separated cells; and expanding the CAR gene-introduced cells, The method for producing CD45R1, CD45R2, CD45R3, CD45R4, CD45R5, CD45R6, CD45R7, CD45R8, CD45R9, CD45R10, CD45R11, CD45R12, CD45R13, CD45R14, CD45R15, CD45R16, CD45R17, CD45R18, CD45R19, CD45R19, CD45R19, CD45R19, CD45R20, CD45R21, CD45R22, CD45R24, CD45R25, CD45R26, CD45R27, CD45R28, CD45R29, CD45R19, CD45R26, CD45R28, CD45R29, CD45R19, CD45R20, CD45R21, CD45R25, CD45R26, CD45R28, CD45R29, CD45R19, CD45R19, CD45R20, CD45R21, CD45R22, CD45R25, CD45R26, CD45R27, CD45R28, CD45R29, CD45R25, CD45R26, CD45R28, CD45R29 ...

[40] The manufacturing method described in

[39] above, wherein the separation step is performed by at least one method selected from the group consisting of density gradient separation, immunological cell separation technology, magnetic cell separation technology, nylon wool separation, and adhesion method.

[0015]

[41] The method of producing according to the above-mentioned

[39] , wherein the separating step is bead separation.

[42] The method of producing according to any of the above-mentioned

[39] to

[41] , wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+ and 4-1BBL+.

[43] The method according to any one of

[39] to

[41] above, wherein the specific factor is at least one selected from the group consisting of CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, CD45RB-, and CD45RC-.

[44] The method according to any one of

[39] to

[43] above, wherein the T cell source is peripheral blood mononuclear cells.

[45] The method according to any one of

[41] to

[44] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, Sepharose, nitrocellulose, and polystyrene.

[46] The method according to any one of

[41] to

[45] above, wherein the particle diameter of the beads is 0.01 to 500 μm.

[47] The method according to

[39] above, wherein the expansion culture includes an activation treatment and an antigen recognition treatment.

[48] ​​The manufacturing method according to

[47] above, wherein the activation treatment is carried out by contacting the cells with a stimulating substance.

[49] The manufacturing method according to

[48] above, wherein the contact with the stimulating substance is contact with a substrate to which a stimulating substance is bound.

[50] The manufacturing method according to

[49] above, wherein the contact with the substrate is co-culture of the cells with the substrate.

[0016]

[51] The manufacturing method according to

[48] or

[49] above, wherein the substrate is beads or a gel.

[52] The manufacturing method according to

[51] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, and polystyrene.

[53] The manufacturing method according to

[51] or

[52] above, wherein the particle size of the beads is 0.01 to 500 μm.

[54] The manufacturing method according to

[48] above, wherein the contact with the stimulating substance is contact with vesicles encapsulating the stimulating substance.

[55] The manufacturing method according to

[54] above, wherein the vesicles are liposomes, exosomes, microvesicles, or apoptotic bodies.

[56] The manufacturing method according to

[47] or

[48] above, wherein the activation treatment is carried out by culturing the cells in a medium containing the stimulating substance.

[57] The manufacturing method according to

[56] above, wherein the activation treatment is carried out by co-culturing the cells with a substrate to which a stimulating substance is bound in a medium containing the stimulating substance.

[58] The manufacturing method according to

[57] above, wherein the substrate to which a stimulating substance is bound is a bead to which an antibody is bound.

[59] The manufacturing method according to any of

[48] to

[58] above, wherein the stimulating substance is at least one selected from the group consisting of a protein, a peptide fragment, and a sugar chain.

[60] The manufacturing method according to any one of

[48] to

[59] above, wherein the stimulator provides at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, PPAR-, statin-, CD45RB-, and CD45RC-.

[0017]

[61] The manufacturing method according to

[47] above, wherein the antigen recognition treatment is carried out by contacting the cells with a recognition substance.

[62] The manufacturing method according to

[61] above, wherein the contact with the recognition substance is contact with a substrate to which the recognition substance is bound.

[63] The manufacturing method according to

[62] above, wherein the contact with the substrate is co-culture of the cells with the substrate.

[64] The manufacturing method according to

[62] or

[63] above, wherein the substrate is beads or gel.

[65] The manufacturing method according to

[64] above, wherein the material of the beads is selected from the group consisting of magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, and polystyrene.

[66] The manufacturing method according to

[64] or

[65] above, wherein the particle diameter of the beads is 0.01 to 500 μm.

[67] The manufacturing method according to

[61] above, wherein the contact with the recognition substance is contact with vesicles encapsulating the recognition substance.

[68] The manufacturing method according to

[67] above, wherein the vesicles are liposomes, exosomes, microvesicles, or apoptotic bodies.

[69] The manufacturing method according to

[47] or

[61] above, wherein the antigen recognition treatment is carried out by culturing the cells in a medium containing a recognition substance.

[70] The manufacturing method according to

[69] above, wherein the antigen recognition treatment is carried out by co-culturing the cells with a substrate to which a recognition substance is bound, in a medium containing the recognition substance.

[0018]

[71] The manufacturing method according to

[70] above, wherein the substrate to which the recognition substance is bound is a bead to which an antibody is bound.

[72] The manufacturing method according to any of

[61] to

[71] above, wherein the recognition substance is at least one selected from the group consisting of a protein, a peptide fragment, and a sugar chain.

[73] The manufacturing method according to

[72] above, wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19.

[74] A method for manufacturing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing cells after CAR gene introduction with beads to which a stimulating substance and a recognition substance are bound in a medium containing a stimulating substance and a recognition substance, the stimulatory substance provides at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD45RO+, CD27+, CD31+, IL-7R+, CCR7+, CD62L+, CD95+, CD160+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD3-, CD4-, CD8-, CD45RO-, CD57-, CD95-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, PPAR-, statin-, CD45RB-, and CD45RC-; The method according to any one of

[47] to

[73] above, wherein the cognitive substance is at least one selected from the group consisting of EPHB4, HER2 and CD19.

[0019] In another aspect, the present invention is as follows: [1'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of separating T cells from a T cell source using a specific factor, wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD27+, IL-7R+, CCR7+, CD62L+, CD95+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD44+, CD45RO-, CD57-LAG3-, CXCR3-, and IL-2Rβ-, and the separation step is performed using at least one method selected from the group consisting of density gradient separation, immunological cell separation technology, magnetic cell separation technology, nylon wool separation, and adhesion method. [2'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of separating T cells from a T cell source using beads with a specific factor, wherein the specific factor is at least one selected from the group consisting of CD3+, CD4+, CD8+, CD45RA+, CD27+, IL-7R+, CCR7+, CD62L+, CD95+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD44+, CD45RO-, CD57-, LAG3-, CXCR3-, and IL-2Rβ-. [3'] The manufacturing method according to [1'] or [2'], wherein the specific factor is at least one selected from the group consisting of CD3+, CD45RA+, IL-7R+, CD62L+, and CD28+. [4'] The manufacturing method according to any of [1'] to [3'], wherein the T cell source is peripheral blood mononuclear cells. [5'] The manufacturing method according to [5'], wherein the cells are T cells or T cells expressing a chimeric antigen receptor. [6'] The manufacturing method according to [5'], wherein the activation treatment is carried out by contacting the cells with a stimulating substance. [8'] The manufacturing method according to [7'], wherein the contact with the stimulating substance is contact with a substrate to which the stimulating substance is bound. [9'] The manufacturing method according to [8'], wherein the contact with the substrate is co-culture of the cells with the substrate.[10'] The manufacturing method according to any one of [5'] to [7'], wherein the activation treatment is carried out by culturing the cells in a medium containing a stimulating substance. [11'] The manufacturing method according to [10'], wherein the activation treatment is carried out by co-culturing the cells with a substrate to which the stimulating substance is bound in a medium containing the stimulating substance. [12'] The manufacturing method according to [11'], wherein the substrate to which the stimulating substance is bound is a bead to which a protein is bound. [13'] The manufacturing method according to any one of [7'] to [12'], wherein the stimulating substance provides at least one selected from the group consisting of CD4+, CD8+, CD45RA+, CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, CCL19+, CD45RB+, CD3-, CD45RO-, CD57-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, MEK1-, MEK2-, mTOR-, PPAR-, and statin-. [14'] The production method according to [13'], wherein the stimulatory substance provides at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, rapamycin-, simvastatin-, GW9662-, rosiglitazone-, and GW6471-. [15'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising a step of treating cells for antigen recognition in the absence of feeder cells. [16'] The production method according to [15'], wherein the cells are T cells or T cells expressing a chimeric antigen receptor. [17'] The production method according to [15'] or [16'], wherein the antigen recognition treatment is carried out by contacting the cells with a recognition substance. [18'] The manufacturing method according to [17'], wherein the contact with the recognition factor is contact with a substrate to which a recognition substance is bound. [19'] The manufacturing method according to [18'], wherein the contact with the substrate is co-culture of the cells with the substrate. [20'] The manufacturing method according to any of [15'] to [17'], wherein the antigen recognition treatment is carried out by culturing the cells in a medium containing a recognition substance. [21'] The manufacturing method according to [20'], wherein the antigen recognition treatment is carried out by co-culturing the cells with a substrate to which a recognition substance is bound, in a medium containing the recognition substance.[22'] The manufacturing method according to [21'], wherein the substrate to which the recognition substance is bound is a bead to which a protein of the recognition substance is bound. [23'] The manufacturing method according to any one of [17'] to [22'], wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2 and CD19. [24'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing cells after CAR gene introduction with beads to which the stimulating substance and the recognizing substance are bound in a medium containing a stimulating substance and a recognizing substance, wherein the stimulating substance is at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, rapamycin-, simvastatin-, GW9662-, Rosiglitazone-, and GW6471-, and the recognizing substance is at least one selected from the group consisting of EPHB4, HER2, and CD19. [25'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the steps of: separating T cells from a T cell source using a specific factor; introducing a CAR gene into the separated cells; and expanding the CAR gene-introduced cells, wherein the specific factor is at least one selected from the group consisting of CD3+, CD45RA+, CD27+, IL-7R+, CD62L+, CD28+, CD44+, CD45RO-, and CD57-. [26'] The method according to [25'], wherein the separating step is bead separation. [27'] The method according to [25'] or [26'], wherein the specific factor is at least one selected from the group consisting of CD3+, CD45RA+, IL-7R+, CD62L+, and CD28+. [28'] The method according to [25'], wherein the expansion includes an activation treatment and an antigen recognition treatment. [29'] The manufacturing method according to [28'], wherein the activation treatment is carried out by contacting the cells with a stimulating substance. [30'] The manufacturing method according to [29'], wherein the contact with the stimulating substance is contact with a substrate to which a stimulating substance is bound. [31'] The manufacturing method according to [30'], wherein the contact with the substrate is co-culture of the cells with the substrate.[32'] The manufacturing method according to [28'] or [29'], wherein the activation treatment is carried out by culturing the cells in a medium containing a stimulating substance. [33'] The manufacturing method according to [32'], wherein the activation treatment is carried out by co-culturing the cells with a substrate to which the stimulating substance is bound in a medium containing the stimulating substance. [34'] The manufacturing method according to [33'], wherein the substrate to which the stimulating substance is bound is a protein-bound bead. [35'] The manufacturing method according to any of [29'] to [34'], wherein the stimulating substance provides at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, rapamycin-, simvastatin-, GW9662-, Rosiglitazone-, and GW6471-. [36'] The manufacturing method according to [28'], wherein the antigen recognition treatment is carried out by contacting the cells with a recognition substance. [37'] The manufacturing method according to [36'], wherein the contact with the recognition substance is contact with a substrate to which a recognition substance is bound. [38'] The manufacturing method according to [37'], wherein the contact with the substrate is co-culture of the cells with the substrate. [39'] The manufacturing method according to [28'] or [36'], wherein the antigen recognition treatment is carried out by culturing the cells in a medium containing a recognition substance. [40'] The manufacturing method according to [39'], wherein the antigen recognition treatment is carried out by co-culturing the cells with a substrate to which a recognition substance is bound in a medium to which a recognition substance is bound. [41'] The manufacturing method according to [40'], wherein the substrate to which a recognition substance is bound is a bead to which a protein is bound. [42'] The manufacturing method according to [39'], wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19.[43'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing cells after CAR gene introduction with beads to which a stimulating substance and a recognizing substance are bound in a medium containing a stimulating substance and a recognizing substance, wherein the stimulating substance is at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, rapamycin-, simvastatin-, GW9662-, Rosiglitazone-, and GW6471-, and the recognizing substance is at least one selected from the group consisting of EPHB4, HER2, and CD19. [44'] A method for producing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing CAR gene-transfected cells with beads bound to a costimulatory substance and a recognition substance in a medium containing a stimulatory substance in the absence of feeder cells, wherein the stimulatory substance is at least one selected from the group consisting of an anti-CCR7 antibody, an anti-CD62L antibody, an anti-CXCR5 antibody, an anti-CCL5 antibody, an anti-CD327 antibody, an anti-CD73 antibody, SB431542, PD0325901, rapamycin, simvastatin, GW9662, rosiglitazone, and GW6471, and the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19, The method for producing a CAR gene-transduced cell, wherein the costimulatory substance is at least one selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113. [45'] The method for producing a CAR gene-transduced cell, wherein the cell is derived from a T cell separated from a T cell source using a specific factor, and the specific factor is at least one selected from the group consisting of CD3+, CD45RA+, IL-7R+, CD62L+, and CD28+.

[0020] According to the method of the present invention, higher quality CAR-T cells can be obtained, and highly functional CAR-T cells can be produced simply, in a short time, and at low cost.

[0021] Figure 1 shows the isolation process of CD45RA+ PBMCs and CD45RA- PBMCs from total PBMCs. Figure 2 shows the construction of transposon plasmids for CAR-T cells and antigen-expressing feeder cells. ITR: Internal Tandem Repeat, TM: transmembrane domain, cyto: cytoplasmic domain. Figure 3A shows the results of examining CAR transgene expression 24 hours after transfection into CD45RA+ cells (RA+) or CD45RA- cells (RA-) (n = 3, 3 donors). The percentage of cells expressing the CAR transgene is shown. **P < 0.01. Figure 3B shows the results of examining the extent of CAR-positive T cell proliferation 14 days after transfection into CD45RA+ cells (RA+) or CD45RA- cells (RA-) (n = 5, 3 donors). The extent of CAR-positive T cell proliferation is shown as fold increase. Figure 3C shows the representative expression and phenotype of CD19 CAR-T cells derived from CD45RA+ cells or CD45RA- cells, and the results of flow cytometry analysis of exhaustion marker expression in both CAR-T cells. Figure 3D shows the phenotype and exhaustion marker expression in CD45RA+ cell (RA+)-derived CD19 CAR-T cells or CD45RA- cell (RA-)-derived CD19 CAR-T cells (n = 3-6, 3 donors). All data are expressed as mean ± standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. Figure 4A is a volcano plot showing differentially expressed genes with a log2 fold change of >0.9 (29 genes) or <-0.9 (78 genes) and an adjusted FDR <0.05 in CD45RA+ CAR-T cells compared with CD45RA- CAR-T cells. Figure 4B shows the results of reactome pathway analysis. Several gene pathways were significantly downregulated in CD45RA+ CAR-T cells compared with CD45RA- CAR-T cells.Figure 5A shows the results of an in vitro sequential tumor challenge assay to analyze the proportion of CAR-T cells and REH cells when they were serially co-cultured. Representative dot plots are shown. Figure 5B shows the results of an in vitro sequential tumor challenge assay to analyze CAR function. Relative CAR mean fluorescence intensity (MFI) of CAR-T cells during serial co-culture (MFI = 1, before co-culture) (n = 3). *P < 0.05. Figure 5C shows the results of an in vitro sequential tumor challenge assay to analyze CAR function. PD-1, TIM-3, and LAG-3 expression on CAR-T cells during serial co-culture is shown (n = 3). All data are shown as mean ± standard deviation. *P < 0.05. PD-1: programmed cell death protein-1, TIM-3: T cell immunoglobulin mucin-3, LAG-3: lymphocyte-activation gene 3. Figure 6A shows bioluminescence images of 5 NSG mice per group after intravenous infusion of each CAR-T cell. Tumor volume for each mouse, measured as total flux (p / s), is shown. The CD45RA+ CAR-T group demonstrated statistically significant tumor reduction, measured as mean total flux on day 28, compared with the CD45RA- CAR-T group. *P <0.05. Figure 6C shows a Kaplan-Meier plot of overall survival (n = 5 per group). The CD45RA+ CAR-T group achieved prolonged tumor control compared with the CD45RA- CAR-T group. Log-rank test. *P <0.05. Figure 6D shows the results of bone marrow analysis on day 15 after CAR-T cell infusion. Flow cytometry confirmed CAR-T cells (left), REH cells (middle), and PD-1 expression on CAR-T cells (right). *P <0.05. CAR-T cells and REH cells in the bone marrow of long-lived mice infused with RA+ CAR-T cells on days 15, 25, and 50. Representative dot plot data are shown. Graphs show the results of expanding CAR-T cells and measuring the cell count (top row) and CAR expression rate (bottom row).

[0022] 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 producing chimeric antigen receptor T (CAR-T) cells. The method for producing CAR-T cells of the present invention includes the steps of introducing a cancer-specific gene (a so-called CAR gene) into T cells derived from a T cell source and expanding the resulting cells.

[0023] T Cells and T Cell Sources In the present invention, a CAR gene is introduced into T cells. T cells in the present invention include CD4+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, and NKT cells. T cells may be subsets such as naive T cells, effector T cells, or memory T cells. T cells may be cells isolated from a human, or cells obtained by differentiation from cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells. Furthermore, T cells may be either autologous or allogeneic. In the present invention, "autologous cells" refers to cells obtained from a subject receiving the cell population (i.e., CAR-T cells) produced by the method of the present invention, or cells derived from the obtained cells. "Allogeneic cells" refers to cells other than the aforementioned "autologous cells." Preferably, T cells are autologous. CAR-T cells can be obtained by gene transfer into a cell population containing T cells or their precursor cells, such as hematopoietic stem cells. For example, CAR-T cells may be obtained by differentiating cells, such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells, transfected with a CAR gene, or by differentiating cells converted into iPS cells after transfection with a CAR-T gene. CAR-T cells are prepared by transfecting T cells with a CAR gene. In one embodiment, the T cells to be transfected with the CAR gene are T cells separated from a T cell source using beads with a specific factor. The T cell source is not particularly limited as long as it contains T cells that exhibit a specific expression pattern of a specific cell membrane surface antigen (i.e., have a specific cell membrane surface marker). Typically, the T cell source is blood cells or a blood-derived sample, or is a product of or derived from apheresis or leukapheresis. The T cell source in the present invention may also be frozen cells.

[0024] In the present invention, the term "blood cells" refers to cells that constitute blood, and can refer to either a single cell or a cell population containing multiple cells, or to a cell population consisting of one type of cell or a cell population containing multiple types of cells. Blood cells are preferably blood cells excluding red blood cells and platelets, including immune cells such as lymphocytes and monocytes. Blood cells may be cells isolated from a human or cells obtained by differentiation from cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells. They may be either autologous or allogeneic, but are preferably autologous. In a further embodiment, CAR-T cells are prepared by introducing a CAR gene into peripheral blood mononuclear cells (PBMCs). The PBMCs are preferably autologous PBMCs (i.e., PBMCs collected from a subject receiving the cell population produced by the method of the present invention). 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.

[0025] Chimeric antigen receptors (also referred to herein as CARs) are structures that, from the N-terminus to the C-terminus of a protein, comprise a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for immune cell effector function. The CAR gene encodes this receptor. The extracellular domain contains an antigen recognition site that exhibits target-specific binding. The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain transmits signals necessary for immune cell effector function. In other words, when the extracellular domain binds to a target antigen, an intracellular signaling domain is used that can transmit signals necessary for immune cell activation.

[0026] 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 5:177 ra38, 2013), the CAR of the present invention can be constructed with reference to these reports.

[0027] One embodiment of the present invention is characterized by including a step of separating T cells from a T cell source using a specific factor before introducing a CAR gene. In particular, in the present invention, it is preferable to separate T cells from a T cell source using a specific factor using beads before introducing a CAR gene. The step of separating T cells from a T cell source using a specific factor will now be described. This step involves separating T cells expressing a specific cell membrane surface antigen from T cells that do not express that antigen, or separating T cells that do not express that specific cell membrane surface antigen from T cells that do express that antigen, before introducing a CAR gene, to a T cell source to select T cells to be introduced into the CAR gene. Specifically, the specific factor refers to the expression of a specific cell membrane surface antigen (positive, +) or the absence of expression of a specific cell membrane surface antigen (negative, -). The specific factor is not particularly limited as long as it can be used to select T cells to be introduced into the T cell source into the CAR gene, and may be the expression profile (positive or negative) of any cell membrane surface antigen. Examples of cell membrane surface antigens are listed in Tables 1-1 to 1-5, but are not limited thereto.

[0028]

[0029]

[0030]

[0031]

[0032]

[0033] Positive (+) specific factors include: CD3+, CD4+, CD8+, CD45RA+, CD27+, IL-7R+, CCR7+, CD62L+, CD95+, ICOS+, HVEM+, OX40+, CD44+, 4-1BB+, CD40L+, GITR+, IL-15R+, PTGER2+, ICOSLG+, CD38+, CXCR5+, IFNGR2+, BTLA+, CD2 8+, PD-L1+, CCL5+, TNFSF4+, XCL1+, CX3CR1+, CCL3L3+, CCL4+, Notch1+, Notch2+, CD248+, CD 327+, CD79a+, CD35+, CD73+, CD318+, CD305+, CD242+, CD158d+, CD42d+, CD56+, CD257+, CD49 f+, CD201+, CD7+, CD328+, CD300c+, CD11d+, CD228+, CD336+, CD289+, CD172b+, CD167a+, CD3 53+, CD355+, CD217+, CD172g+, CD183+, CD11c+, CD159a+, CD69+, CD366+, CD258+, CD254+, CC L19+, CXCR1+, CXCR2+, CXCR4+, CCR2+, CCR4+, CCR5+, CCR6+, CD6+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD40+, CD52+, CD70+, CD49+, CD51+, CD103+, CD11b+, ITGA+, CD18+, CD19+ and ITGB+. Preferred factors include CD3+, CD4+, CD8+, CD45RA+, CD27+, IL-7R+, CCR7+, CD62L+, CD95+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, CD44+, and 4-1BBL+. Negative (-) specific factors include the following:CD45RO-, CD57-, CD58-, KLRG1-, TIM3-, LAG3-, PD-1-, CD11a-, CD26-, CD122-, CXCR3-, IL-2Rβ-, HLA-DR-, CD86-, 4-1BBL- , CXCL10-, ID2-, CCL4L2-, CTLA4-, CD160-, CCL3-, CD28-, CD249-, CD150-, CD283-, CD131-, 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 are CD45RO-, CD57-, LAG3-, CXCR3-, and IL-2Rβ-. Preferred specific factors include CD3+, CD4+, CD8+, CD45RA+, CD27+, IL-7R+, CCR7+, CD62L+, CD95+, CXCR5+, CD28+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, CD44+, CD45RO-, CD57-, LAG3-, CXCR3-, and IL-2Rβ-, more preferably CD45RA+, IL-7R+, CD62L+, CD3+, and CD28+, and even more preferably CD45RA+ and CD62L+. When CD45RA+ is used as the specific factor, it is preferred because a large number of CAR-positive cells can be obtained. On the other hand, using CD62L+ as a specific factor is preferable because it can increase the proportion of + naive or young memory T cells such as Tscm among T cells or CAR-T cells.

[0034] The process of separating T cells from a T cell source using specific factors can be performed using commonly used methods for separating cells based on the expression status of cell membrane surface antigens. However, preferred embodiments of the present invention 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, with the density at the bottom of the container. Magnetic cell sorting, also known as magnetic cell sorting (MACS), is a method in which a cell suspension is prepared from tissue containing a mixture of various cells, and specific cells within the suspension are magnetically labeled. This allows for the magnetic separation and collection of magnetically labeled and non-magnetically labeled cells. 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. Adhesion utilizes differences in the ability of cells to adhere to substrates (e.g., culture flasks). Specifically, when a T cell source is placed in a culture flask, adhesive monocytes and other cells adhere to the bottom of the flask, while non-adhesive lymphocytes such as T cells float in the culture medium. These cells are then separated into two cell populations using an adhesion method. 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 specified period of time, only the floating cells are collected and used as the T cell source, ignoring the adherent cells. 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 (e.g., collagen, fibronectin, polylysine, plasma treatment, or electrochemical treatment).

[0035] These separation methods and techniques may be used alone or in combination with two or more other techniques. 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 target cells with an antibody or ligand specific to their surface and then separating them using the label as an index. One type of immunomagnetic cell separation technique is magnetic bead separation, which uses magnetic beads (hereinafter sometimes simply referred to as bead separation). Bead separation is preferred in the present invention.

[0036] Bead Separation Bead separation is a method that uses beads labeled with antibodies or ligands specific to the surface of the cells of interest. The bead slurry is contacted with a cell suspension, and the cells bound to the beads are then separated using an affinity capture method specific to some property of the beads. This format facilitates both positive and negative selection. Bead materials commonly used in the art can be used for bead separation, including, but not limited to, magnetic materials, latex, agarose, glass, cellulose, Sepharose, nitrocellulose, and polystyrene. Magnetic materials are preferred, and therefore, magnetic beads are preferably used for bead separation. The particle size of the beads varies depending on the material and the affinity capture method used for separation and is set appropriately. For magnetic beads, the particle size is typically 10 nm to 500 μm, preferably 10 nm to 100 μm, and more preferably 10 nm to 50 μm. If the particle size is too small or too large, the target cells cannot be captured, and separation cannot be performed successfully. Magnetic beads are generally uniform superparamagnetic beads coated with an affinity tag, such as recombinant streptavidin, which can bind to an affinity tag, such as biotinylated immunoglobulins or other biotinylated molecules, such as peptides / proteins or lectins. Magnetic beads are generally uniform microparticles or nanoparticles of Fe3O4 (magnetic core). In one embodiment, the magnetic beads are coated with an antibody (preferably a monoclonal antibody) against a cell surface antigen expressed or not expressed by the target T cells. The antibody-tagged magnetic beads are then introduced into a cell suspension of a T cell source. The magnetic beads bind to T cells bearing specific factors, or not, and they can be separated by applying a magnetic field. This can be facilitated by passing the suspension through a high-gradient magnetic separation column placed under a strong magnetic field. Magnetically labeled cells remain on the column, while unlabeled cells pass through. When the column is removed from the magnetic field, the magnetically retained cells are eluted. Both the labeled and unlabeled fractions can be completely recovered.The magnetic beads used for bead separation are commercially available, or can be produced by applying a desired coating to available magnetic beads themselves using a known method.

[0037] Introduction of CAR Gene into T Cells (Production of CAR-T Cells) In the present invention, CAR-T cells are prepared by introducing a CAR gene into T cells using a CAR expression vector. The T cells to be introduced with the CAR gene are preferably T cells with a characteristic cell surface marker expression pattern that have been bead-isolated from the above-mentioned T cell source using specific factors. A CAR expression vector refers to a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into T cells. Various types of vectors can be used, regardless of whether they are DNA or RNA, and are not particularly limited in terms of form or origin. 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. 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.

[0038] CAR expression vectors contain an expression unit for expressing the CAR gene, which typically includes a promoter, a CAR gene, and a poly(A) addition signal. Expression units can be derived from various organisms or viruses or consist of any sequence, including analogous sequences and modified units thereof. Promoters that can be used in CAR expression cassettes include the CAG promoter, CMV-IE (cytomegalovirus early gene promoter), SV40 ori, retrovirus LTRSRα, EF1α, and β-actin promoters. 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 CAR gene expression, 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. The CAR gene is transcribed into messenger RNA (mRNA) by such an expression unit, and the CAR is translated from the mRNA and displayed on the cell surface. The expression unit may contain a detection gene (e.g., a reporter gene, a cell- or tissue-specific gene, or a selection marker gene) for detecting gene expression, an enhancer sequence for improving expression efficiency, a WRPE sequence, etc. The detection gene is used to determine the success or efficiency of CAR expression vector introduction, detect CAR gene expression or determine expression efficiency, select or separate cells in which the CAR gene is expressed, etc.Genes for detection 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 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 the GFP (Gerdes, FEBS Lett. 389 (1996), Genes for fluorescent proteins such as CAR (Carboxymethyltransferase 44-47) or their variants (e.g., EGFP and d2EGFP) (collectively referred to as reporter genes); 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. Examples of self-cleaving peptides include the 2A peptide (T2A) derived from Thosea asigna virus. Other self-cleaving peptides include, but are not limited to, the 2A peptide (F2A) derived from picornaviruses, foot-and-foot disease virus (FMDV), equine rhinitis A virus (ERAV), and porcine teschovirus (PTV-1), as well as 2A peptides derived from rotaviruses, insect viruses, aphthoviruses, or trypanosoma viruses. Similar sequences and partially modified sequences are also possible.

[0039] The CAR gene expression vector prepared for gene transfer is introduced into T cells by a conventional method. In the case of a viral vector, it is introduced into cells by viral infection. In the case of a non-viral vector such as a plasmid, it can be introduced into cells by conventional methods such as electroporation, liposome method, calcium phosphate method, nucleofection method, laser method, cationic method, microinjection method, sonoporation, etc., and preferably introduced by electroporation.

[0040] To improve the efficiency of integration into the host chromosome, transposon-based gene transfer is preferred. The transposon method is a non-viral gene transfer technique 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-based methods include the piggyBac transposon method, which 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).

[0041] 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) may also be used.

[0042] Gene introduction using the transposon method can be performed by conventional methods. 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 repeats (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.

[0043] One embodiment of the method for producing CAR-T cells of the present invention is characterized by including a step of activating T cells in the absence of feeder cells. In this invention, T cell activation refers to supporting the maintenance and / or proliferation of T cells, such as naive T cells, young memory T cells, and effector T cells, thereby conferring a high antitumor effect upon the preparation of CAR-T cells. T cell activation in the absence of feeder cells can be performed either before or after CAR gene transduction into T cells (in this case, T cells are referred to as CAR-T cells), but is preferably performed during both processes. Treatment before CAR gene transduction is performed to sufficiently activate T cells, allowing the activated T cells to be maintained and / or expanded in a state that is susceptible to gene transduction. Treatment after CAR gene transduction is performed to sufficiently activate CAR-T cells, allowing the activated CAR-T cells to be maintained and / or expanded in a state that is susceptible to recognizing tumor-associated or tumor-specific antigens targeted by the CAR, thereby enabling the CAR-T cells to be expanded to the required scale. Furthermore, it can support the maintenance and / or proliferation of naive or memory T cells in CAR-T cells, thereby conferring a high antitumor effect to CAR-T cells. For example, it is possible to obtain a sufficient amount of CAR-T cells with high antitumor activity for clinical use. The activation treatment is not particularly limited as long as it achieves the desired effect on T cells or CAR-T cells, and can be performed, for example, by contact with a stimulating substance. The stimulating substance is a signal molecule that controls cell activity by autocrine, paracrine, endocrine, or other methods. It may be a substance that can be secreted by all cells (including cells other than T cells or CAR-T cells) contained in the culture system, or it may be an exogenous substance. Culture supernatant from other cells, such as mesenchymal stem cells, can also be added to activate T cells.

[0044] (Activation Treatment of T Cells Before CAR Gene Transfer) Activation treatment of T cells before CAR gene transfer can be carried out, specifically, by contact with a stimulating substance. The stimulating substance is not particularly limited as long as it specifically stimulates T cells and sufficiently activates them, and examples thereof include proteins, peptide fragments, and sugar chains. Stimulating substances induce or inhibit the expression of stimulating factors. Stimulating factors are broadly classified into those that activate T cells by increasing their expression within cells, or those that activate T cells by promoting the expression of stimulating factors within T cells upon contact with the stimulating substance (positive: stimulating factor (+)), and those that activate T cells by decreasing their expression within cells, or those that activate T cells by suppressing the expression of stimulating factors within T cells upon contact with the stimulating substance (negative: stimulating factor (-)). Stimulating substances that provide stimulating factors (+) include the stimulating factors themselves, and stimulating substances that provide stimulating factors (-) include inhibitors of stimulating factors (e.g., antibodies).The former stimulatory factor (+) cells include CD4+, CD8+, CD45RA+, CCR7+, CD62L+, ICOS+, HVEM+, OX40+, 4-1BB+, CD40L+, GITR+, TCF1+, BCL6+, IL-15R+, PTGER2+, ICOSLG+, NFKB1+, SATB1+, CD38+, BACH2+, TCF7+, LEF1+, ID3+, LEF1-AS1+, SATB1-AS1+, NFATC1+, CXCR 5+, IFNGR2+, BTLA+, PD-L1+, BCL2L2+, CD101+, CCL5+, TNFSF4+, XCL1+, PRDM15+, BCL2L10+, CX3CR1+, CCL3L3+ , CCL4+, carnosine+, vitaminA+, vitaminE+, Ca+, TLAM+, AMPK+, HIF-1+, Dexamethasone+, CD248+, CD327+, CD79a+ , CD35+, CD73+, CD318+, CD305+, CD242+, CD158d+, CD42d+, CD56+, CD257+, CD49f+, CD201+, CD7+, CD328+, CD3 00c+, CD11d+, CD228+, CD336+, CD289+, CD172b+, CD167a+, CD353+, CD355+, CD217+, CD172g+, CD183+, CD11c+, These include CD159a+, CD69+, CD366+, CD258+, CD254+, CCL19+, CXCR1+, CXCR2+, CXCR4+, CCR2+, CCR4+, CCR5+, CCR6+, CD6+, CD45RB+, CD45RC+, CD40+, CD52+, CD70+, CD49+, CD51+, CD103+, CD11b+, ITGA+, CD18+, CD19+, and ITGB+. Preferred stimulatory factors include CD4+, CD8+, CD45RA+, CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, CCL19+, CD45RB+, and CD45RC+, more preferably CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, and CD73+, even more preferably CD62L+, CD327+, and CD73+, and most preferably CD62L+. The stimulatory factors are summarized in Tables 2-1 to 2-4.

[0045]

[0046]

[0047]

[0048]

[0049] The latter stimulatory factor (-) includes CD3-, CD45RO-, CD57-, CD58-, CD44-, KLRG1-, Perforin-, Granzyme A-, Granzyme B-, TIM3-, LAG30-, PD-1-, CD11a-, CD26-, CD122-, CXCR3-, IL-2Rβ-, HLA-DR-, PRDM1-, CD86-, 4-1BBL-, CXC L10-, ID2-, RUNX1-, TIGIT-, CCL4L2-, CTLA4-, CSF1-, BATF-, BCL2-, IFNG-, BATF3-, CCL3-, BC L2L11-, BCL2L14-, BCL2A1-, BCL2L15-, MEK1-, MEK2-, GSK3β-, AKT-, mTOR-, c-Myc-, GLS1-, PP AR-, statin-, PFK1-, IRBIT-, Wnt-, p16-, p21-, CD28-, CD249-, CD150-, CD283-, CD131-, CD66a- , CD253-, CD120b-, CD61-, CD87-, CD280-, CD195-, CD54-, CD265-, CD203a-, CD271-, CD68-, CD 307c-, CD357-, CD196-, CD25-, CD154-, CD203c-, CD134-, CD106-, CD244-, CD71-, CD220-, CD158e-, CD123-, CD16b-, CD364-, CD230-, CD41-, CD361-, CD161-, CCR4-CD292-, and IL-21-. Preferred stimulatory factors include CD3-, CD45RO-, CD57-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, MEK1-, MEK2-, mTOR-, PPAR-statin-, CD28-, and IL-21-, and more preferred are MEK1-, MEK2-, mTOR-, PPAR-, and statin-. Specifically, preferred MEK1- and MEK2- stimulators are SB431542- and PD0325901-, preferred mTOR- stimulators are rapamycin-, preferred PPAR- stimulators are GW9662-, rosiglitazone-, and GW6471-, and preferred statin- stimulators are simvastatin-. The respective stimulatory factors are summarized in Tables 3-1 to 3-4.

[0050]

[0051]

[0052]

[0053]

[0054] This treatment promotes the survival (maintenance) and proliferation of T cells. It also promotes the survival (maintenance) and proliferation of T cells in a state that makes them more susceptible to gene transfer. Furthermore, it supports the maintenance and / or proliferation of T cells, such as naive T cells, memory T cells, and effector T cells, and can confer a strong antitumor effect when CAR-T cells are prepared.

[0055] Activation treatment of T cells before gene transfer is optional, and in some cases the CAR gene can be directly transferred into the T cell source.

[0056] After CAR gene introduction, CAR-T cells are expanded to the required scale. The expansion culture in the CAR-T cell production method of the present invention includes an activation treatment to maintain and / or proliferate CAR-T cells in a state where they are likely to recognize tumor-associated or tumor-specific antigens targeted by the CAR, and an antigen recognition treatment to confer antitumor properties to the CAR-T cells. The CAR-T cell production method of the present invention is characterized in that the expansion culture is performed in the absence of feeder cells.

[0057] (Activation of CAR-T Cells After CAR Gene Transfer) CAR-T cells after CAR gene transfer can be activated by contact with a stimulatory substance. Stimulatory substances are not particularly limited as long as they specifically stimulate CAR-T cells and sufficiently activate them, and examples include proteins, peptide fragments, and sugar chains. Stimulatory substances induce or inhibit the expression of stimulatory factors. Stimulatory factors are broadly classified into those that activate CAR-T cells by increasing their expression within the cells, or those that activate CAR-T cells by promoting their expression within the CAR-T cells upon contact with the stimulatory substance (positive: stimulatory factor (+)), and those that activate CAR-T cells by decreasing their expression within the cells, or those that activate CAR-T cells by suppressing their expression within the CAR-T cells upon contact with the stimulatory substance (negative: stimulatory factor (-)). Stimulatory substances that provide stimulatory factors (+) include the stimulatory factors themselves, and stimulatory substances that provide stimulatory factors (-) include inhibitors of stimulatory factors (e.g., antibodies). The former stimulatory factors (+) are similar to those used in the T cell activation treatment and are summarized in Tables 2-1 to 2-4. Preferred are CD4+, CD8+, CD45RA+, CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, CCL19+, CD45RB+, and CD45RC+, more preferably CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, and CD73+, even more preferably CD62L+, CD327+, and CD73+, and most preferably CD62L+. The latter stimulatory factors (-) are similar to those used in the T cell activation treatment and are summarized in Tables 3-1 to 3-4. Preferred are CD3-, CD45RO-, CD57-, LAG3-, CXCR3-, IL-2Rβ-, BCL2L14-, MEK1-, MEK2-, mTOR-, PPAR- and statin-, and more preferred are MEK1-, MEK2-, mTOR-, PPAR- and statin-.Specifically, MEK1- and MEK2- are preferably SB431542- or PD0325901-, mTOR- are preferably rapamycin-, PPAR- are preferably GW9662-, rosiglitazone-, or GW6471-, and statins are preferably simvastatin-. This treatment promotes the survival (maintenance) and proliferation of CAR-T cells. Furthermore, this treatment promotes the survival (maintenance) and proliferation of CAR-T cells in a state in which they are more likely to recognize tumor-associated antigens or tumor-specific antigens targeted by the CAR, thereby maintaining and / or expanding naive or young memory T cells within the CAR-T cells, thereby enabling the production of a sufficient number of CAR-T cells with high antitumor activity.

[0058] The activation treatment of T cells or CAR-T cells can also be explained as follows. The activation treatment of T cells or CAR-T cells (hereinafter, sometimes referred to as "T cells, etc.") can be carried out by contacting T cells, etc. with a stimulating substance. Stimulating substances can be broadly classified into (1) substances that induce and / or enhance signal transduction, which promotes the activation of T cells, etc., and (2) substances that suppress signal transduction, which inhibits the activation of T cells, etc. In this specification, stimulating substances (1) and (2) may be referred to as stimulating substance (+) and stimulating substance (-), respectively. The induction and / or enhancement or inhibition of signal transduction by stimulating substance (+) or stimulating substance (-) may be achieved at any stage, from upstream to downstream, in signal transduction, as long as the desired effect is obtained.

[0059] In one embodiment, the stimulator (+) can be, but is not limited to: CD4+ → anti-CD4 antibody CD8+ → anti-CD8 antibody CD45RA+ → anti-CD45RA antibody, anti-CD62L antibody, anti-CXCR5 antibody, anti-CCL5 antibody, anti-CCR7 antibody, anti-CD73 antibody, anti-CD327 antibody, anti-CD4 antibody, anti-TIM3 antibody, anti-CD45RO antibody, anti-CCL19 antibody, anti-4-1BB antibody, anti-CD8 antibody, anti-CD45RB antibody, anti-CD44RO antibody, anti-CD79a antibody, anti-CD80 antibody, anti-CD248 antibody, GW9662, Rosiglitazone, GW6471, simvastatin, SB431542, rapamycin, PD0325901, catechin CCR7+ → Anti-CCR7 antibody, anti-CD62L antibody, anti-CXCR5 antibody, anti-CCL5 antibody, anti-CD73 antibody, anti-CD327 antibody, anti-CD4 antibody, anti-TIM3 antibody, anti-CD45RO antibody, anti-CCL19 antibody, anti-4-1BB antibody, anti-CD8 antibody, anti-CD45RB antibody, anti- CD44RO antibody, anti-CD79a antibody, anti-CD80 antibody, anti-CD248 antibody, GW9662, Rosiglitazone, GW6471, simvastatin, SB431542, Rapamycin, PD0325901, catechin CD62L+ → Anti-CD62L antibody, GlyCAM-1 protein, CD34 protein, PSGL-1 protein ICOS+ → Anti-ICOS antibody HVEM+ → Anti-HVEM antibody, CD258 protein OX40+ → anti-OX40 antibody 4-1BB+ → anti-4-1BB antibody CD40L+ → anti-CD40L antibody, CD40 protein GITR+ → anti-GITR antibody TCF1+ → agents that increase the expression of the transcription factor TCF1, PPAR inhibitors (GW6471, rosiglitazone, GW9662, pemafibrate, ezetimibe, fenofibrate, TPST-1120, fenofibrate, simvastatin, gemfibrozil, fenofibrate, aleglitazar, NS-220, fenofibrate) acid, rosuvastatin, tesaglitazar, fenofibrate, pravastatin,pirinixic acid, choline fenofibrate, docosahexaenoic acid, clofibrate, atorvastatin, choline fenofibrate, treprostinil, bocidelpar, REN001, icosapent, GW501516, T3D-959, bezafibrate, peroxisome proliferator-activated receptor gamma agonist, glimepiride, rosiglitazone, clopidogrel, telmisartan, pioglitazone, metformin , rosiglitazone, sulfonylurea, balsalazide, metformin, pioglitazone, sulfonylurea, PPAR gamma inhibitor, inolitazone, telmisartan, INS, metformin, pioglit azone, exenatide, INS, pioglitazone, rosiglitazone, aloglipti n, metformin, pioglitazone, exenatide, INS, metformin, piogli tazone, amlodipine, telmisartan, mesalamine, INS, pioglitazon e, pioglitazone, sulfonylurea, grimepiride, pioglitazone, me tformin, pioglitazone, hydrochlorothiazide, telmisartan, ico sapent, aspirin, dipyridamole, telmisartan, troglitazone, alo gliptin, pioglitazone, farglitazar, sulfasalazine, nicotinic acid, pioglitazone, rosiglitazone-metformin combination, aleglitazar, GED-0507-34-levo, catechin, which blocks its upstream activity, is also effective), Ro41-5253, guggulsterone BCL6+ → Increases the expression of the transcription factor BCL6, PPAR inhibitors (GW6471,Rosiglitazone、GW9662、pemafibrate、ezetimibe、fenofibrate、TPST-1120、fenofibrate、simvastatin、gemfibrozil、fenofibrate、aleglitazar、NS-220、fenofibric acid、rosuvastatin、tesaglitazar、fenofibrate、pravastatin、pirinixic acid、choline fenofibrate、docosahexaenoic acid、clofibrate、atorvastatin、choline fenofibrate、treprostinil、bocidelpar、REN001、icosapent、GW501516、T3D-959、bezafibrate、peroxisome proliferator-activated receptor gamma agonist、glimepiride、rosiglitazone、clopidogrel、telmisartan、pioglitazone、metformin、rosiglitazone、sulfonylurea、balsalazide、metformin、pioglitazone、sulfonylurea、PPAR gamma inhibitor、inolitazone、telmisartan、INS、metformin、pioglitazone、exenatide、INS、pioglitazone、rosiglitazone、alogliptin、metformin、pioglitazone、exenatide、INS、metformin、pioglitazone、amlodipine、telmisartan、mesalamine、INS、pioglitazone、pioglitazone、sulfonylurea、glimepiride、pioglitazone、metformin、pioglitazone、hydrochlorothiazide、telmisartan、icosapent、aspirin、dipyridamole、telmisartan、troglitazone、alogliptin、pioglitazone、Farglitazar, sulfasalazine, nicotinic acid, pioglitazone, rosiglitazone-metformin combination, aleglitazar, GED-0507-34-levo, catechin, which blocks upstream activity, is also effective), Ro41-5253, guggulsterone IL-15R+ → anti-IL-15R antibody, IL-15 PTGER2+ → anti-PTGER2 antibody ICOSLG+ → anti-ICOSLG antibody, ICOS protein NFKB1+ → Substances that increase the expression of the transcription factor NFKB1, LTα1α2, BAFF, growth factors such as FGF, IGF, EGF, PDGF, HGF, and BDNF, as well as TNF-α, IL-1, Ca, DTA-1, and catechin. SATB1+ → Substances that increase the expression of the transcription factor SATB1, including TGFβR inhibitors SB431542, YL-13027, galunisertib, SB-505124, SH3051, vactosertib, IN1233, PF-06952229, SJN2511, SB-525334, SM1-71, GFH018, and IL-23. CD38+ → Anti-CD38 antibodies. BACH2+ → Substances that increase the expression of the transcription factor BACH2, including IL-1β and IFNγ. TCF7+ → drugs that increase the expression of the transcription factor TCF7, PPAR inhibitors (GW6471, rosiglitazone, GW9662, pemafibrate, ezetimibe, fenofibrate, TPST-1120, fenofibrate, simvastatin, gemfibrozil, fenofibrate, aleglitazar, NS-220, fenofibric acid, rosuvastatin, tesaglitazar, fenofibrate, pravastatin, pirinixic acid, choline fenofibrate, docosahexaenoic acid, clofibrate, atorvastatin, choline fenofibrate, treprostinil, bocidelpar, REN001, icosapent, GW501516, T3D-959, bezafibrate,peroxisome proliferator-activated receptor gamma agonist, glimepiride, rosiglitazone, clopidogrel, telmisartan, pioglitazone, metformin , rosiglitazone, sulfonylurea, balsalazide, metformin, pioglitazone, sulfonylurea, PPAR gamma inhibitor, inolitazone, telmisartan, INS, metformin, pioglit azone, exenatide, INS, pioglitazone, rosiglitazone, aloglipti n, metformin, pioglitazone, exenatide, INS, metformin, piogli tazone, amlodipine, telmisartan, mesalamine, INS, pioglitazon e, pioglitazone, sulfonylurea, grimepiride, pioglitazone, me tformin, pioglitazone, hydrochlorothiazide, telmisartan, ico sapent, aspirin, dipyridamole, telmisartan, troglitazone, alo gliptin, pioglitazone, farglitazar, sulfasalazine, nicotinic acid, pioglitazone, rosiglitazone-metformin combination, aleglitazar, GED-0507-34-levo, catechin, which blocks upstream activity, is also effective), Ro41-5253, guggulsterone LEF1+ → Increases expression of the transcription factor LEF1, PPAR inhibitors (GW6471, rosiglitazone, GW9662, pemafibrate, ezetimibe, fenofibrate, TPST-1120, fenofibrate, simvastatin, gemfibrozil, fenofibrate, aleglitazar, NS-220, fenofibric acid, rosuvastatin,tesaglitazar, fenofibrate, pravastatin, pirinixic acid, choline fenofibrate, docosahexaenoic acid, clofibrate, atorvastatin, choline fenofibrate, treprostinil, bocidelpar, REN001, icosa pent, GW501516, T3D-959, bezafibrate, peroxisome proliferator-activated receptor gamma agonist, glimepiride, rosiglitazone, clopidogrel, telmisartan, pioglitazone, metformin, rosiglitazone, sulfonylurea, balsalazide, metformin, pioglitazone, sulfonylurea, PPAR gamma inhibitor, inolizone, telmisartan, INS, metformin, pioglitazone, exenatide, INS, pioglitazone, rosiglitazone, alogliptin, metformin, pioglitazone, exenatide, INS, metformin, pioglitazone, amlodipine, telmisartan, mesalamine, INS, pioglitazone, pioglitazone, sulfonylurea, glimepiride, pioglitazone, metformin, pioglitazone, hydrochlorothiazide, telmisartan, icosa pent, aspirin, dipyridamole, telmisartan, troglitazone, alogliptin, pioglitazone, faraglitazar, sulfasalazine, nicotinic acid, pioglitazone, rosiglitazone-metformin combination, aleglitazar, GED-0507-34-lev, catechin that stops its upstream also has an effect), Ro41-5253,Guggulsterone ID3+ → TGFβ LEF1-AS1+ → LEF1-AS1 SATB1-AS1+ → SATB1-AS1 NFATC1+ → Increases expression of transcription factor NFATC1, RANKL, NFKB CXCR5+ → Anti-CXCR5 antibody IFNGR2+ → Anti-IFNGR2 antibody BTLA+ → Anti-BTLA antibody, HVEM protein PD-L1+ → Anti-PD-L1 antibody BCL2L2+ → Increases expression of anti-apoptotic protein BCL2L2, dolastatin 15 CD101+ → Anti-CD101 antibody CCL5+ → Anti-CCL5 antibody TNFSF4+ → Anti-TNFSF4 antibody XCL1+ → Anti-XCL1 antibody PRDM15+ → Increases the expression of the transcription factor PRDM15 BCL2L10+ → Increases the expression of the anti-apoptotic protein BCL2L10, dolastatin 15 CX3CR1+ → Anti-CX3CR1 antibody CCL3L3+ → Anti-CCL3L3 antibody CCL4+ → Anti-CCL4 antibody Carnosine+ → Carnosine Rapamycin+ → Rapamycin vitamin A+ → Vitamin A vitamin E+ → Vitamin E Ca+ → Ca, calcium ion, anti-CD20 antibody TLAM+ → TLAM AMPK+ → AMPK HIF-1+ → Increases the expression of the transcription factor HIF-1 CD248+ → Anti-CD248 antibody CD327+ → Anti-CD327 antibody CD79a+ → Anti-CD79a antibody CD35+ → Anti-CD35 antibody CD73+ → Anti-CD73 antibody CD318+ → Anti-CD318 antibody CD305+ → Anti-CD305 antibody CD242+ → Anti-CD242 antibody CD158+ → Anti-CD158 antibody CD42d+ → anti-CD42d antibody CD56+ → anti-CD56 antibody CD257+ → anti-CD257 antibody CD49f+ → anti-CD49f antibody, anti-CD29 antibody CD201+ → anti-CD201 antibody,プロテインC CD7+ → Anti-CD7 antibody CD328+ → Anti-CD328 antibody CD300c+→ Anti-CD300c antibody CD11d+ → Anti-CD11d antibody CD228+ → Anti-CD228 antibody CD336+ → Anti-CD336 antibody CD289+ → Anti-CD289 antibody CD172b+ → Anti-CD172b antibody CD167a+ → Anti-CD167a antibody CD353+ → Anti-CD353 antibody CD355+ → Anti-CD355 antibody CD217+ → Anti-CD217 antibody, IL-17 CD172g+→ Anti-CD172g antibody CD11c+ → Anti-CD11c antibody CD159a+→ Anti-CD159a antibody, anti-CD94 antibody CD69+ → Anti-CD69 antibody CD258+ → Anti-CD258 antibody, HVEMTAP CD254+ → Anti-CD254 antibody CCL19+ → Anti-CCL19 antibody CXCR1+ → Anti-CXCR1 antibody CXCR2+ → Anti-CXCR2 antibody CXCR4+ → Anti-CXCR4 antibody CCR2+ → Anti-CCR2 antibody CCR4+ → Anti-CCR4 antibody CCR5+ → Anti-CCR5 antibody CCR6+ → Anti-CCR6 antibody CD6+ → Anti-CD6 antibody CD45RB+ → Anti-CD45RB antibody CD45RC+ → Anti-CD45RC antibody CD40+ → Anti-CD40 antibody CD52+ → Anti-CD52 antibody CD70+ → Anti-CD70 antibody, CD27 inhibitor CD49+ → Anti-CD49 antibody, anti-CD29 antibody, lambda glutamine, flavonoids CD51+ → Anti-CD51 antibody, flavonoids CD103+ → Anti-CD103 antibody CD11b+ → Anti-CD11b antibody ITGA+ → Anti-ITGA antibody CD18+ → Anti-CD18 antibody CD19+ → Anti-CD19 antibody ITGB+ → Anti-ITGB antibody TIIM3+ → Anti-TIM3 antibody, Ceacam-1, PtdSer, HMGB1 CD45RO+→ Anti-CD45RO antibody CD44+→ Anti-CD44 antibody, Hiralonic acid Dexamethasone+→ Dexamethasone,

[0060]

[0061] For example, when the stimulating substance is an antibody, stimulation with an anti-CD62L antibody or an anti-CD327 antibody can be achieved by adding an anti-CD62L antibody alone, an anti-CD327 antibody alone, or a combination of an anti-CD62L antibody and an anti-CD327 antibody to a medium and culturing for 1 second to 20 days, preferably 1 second to 14 days, and more preferably 1 second to 10 days. Alternatively, stimulation with an anti-CD62L antibody or an anti-CD327 antibody can be achieved by culturing for 1 second to 20 days, preferably 1 second to 14 days, and more preferably 1 second to 10 days, in a culture vessel (e.g., a culture dish) coated with an anti-CD62L antibody alone, an anti-CD327 antibody alone, or a combination of an anti-CD62L antibody and an anti-CD327 antibody. For example, when the stimulatory substance is an inhibitor, stimulation with rapamycin or simvastatin can be imparted by adding rapamycin alone, simvastatin alone, or a combination of rapamycin and simvastatin to the medium and culturing for 1 second to 20 days, preferably 1 second to 14 days, and more preferably 1 second to 10 days.

[0062] (Antigen Recognition Treatment of CAR-T Cells After CAR Gene Transfer) As a further treatment after CAR gene transfer, an antigen recognition treatment is performed to confer antitumor properties to the CAR-T cells. The method of antigen recognition treatment is not particularly limited as long as the desired effect on the CAR-T cells is obtained, but for example, it can be performed by contacting the CAR 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 the 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, CD30, 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 Examples of the target protein include HLA-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, carcinoembryonic antigen, HMW-MAA, VEGF receptor, fibronectin, tenascin, factors that enhance migration to cancer (e.g., chemokines) or antigens present in the extracellular matrix such as carcinoembryonic antigen (CEA) in necrotic areas 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.

[0063] Furthermore, from the viewpoint of the maintenance / proliferation of T cells and / or CAR-T cells, a costimulatory substance can be added. The costimulatory substance is not limited as long as it stimulates costimulatory molecules in T cells or CAR-T cells, and examples thereof include CD80, CD86, 4-1BBL, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113.

[0064] Tumor-associated or tumor-specific antigens targeted by CARs can be used as cells expressing them. That is, CAR-T cells 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 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. Feeder cells can be prepared by introducing a gene encoding the target antigen into cells using a vector carrying 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 for the target antigen gene and directly introducing the mRNA into cells. Alternatively, the target antigen, costimulatory substance, or stimulatory factor can be expressed on the surface of feeder cells by introducing genes for the target antigen gene and a costimulatory substance or stimulatory factor (here, stimulatory factor (+)) into feeder cells. That is, in one embodiment, the target antigen-expressing cells contain genes for one or more exogenous costimulatory substances or stimulatory factors. Methods for introducing genes for costimulatory substances or stimulatory factors include gene transfer using an expression vector containing genes for the costimulatory substances or stimulatory factors and genes for the target antigen; and gene transfer using an expression vector or mRNA for a stimulatory factor other than the expression vector or mRNA for the target antigen, either simultaneously with or separately from the expression vector or mRNA for the target antigen. Expansion culture using feeder cells can be performed according to Patent Document 1.

[0065] In another embodiment of the present invention, the T cell activation treatment before CAR gene introduction and the expansion culture after CAR gene introduction (including the activation treatment of CAR-T cells after CAR gene introduction and the antigen recognition treatment of CAR-T cells after CAR gene introduction) are performed in the absence of feeder cells. The activation treatment in the absence of feeder cells is performed, for example, by contact with a stimulating substance (described above), specifically by contact with a substrate to which the stimulating substance is bound, contact with vesicles containing the stimulating substance, or culture in a medium containing the stimulating substance. Furthermore, the antigen recognition treatment in the absence of feeder cells is performed, for example, by contact with a recognition substance (described above), specifically by contact with a substrate to which the recognition substance is bound, contact with vesicles containing the recognition substance, or culture in a medium containing the recognition substance. Furthermore, when a costimulatory substance (described above) is used in the expansion culture after CAR gene introduction, the treatment is performed by contact with a substrate to which the costimulatory substance is bound, contact with vesicles containing the costimulatory substance, or culture in a medium containing the stimulatory substance.

[0066] There are no particular limitations on the combination of embodiments of the activation treatment, antigen recognition treatment, and costimulatory substance treatment of T cells in the absence of feeder cells, as long as the desired effect is obtained. For example, various treatments of T cells in the absence of feeder cells can be performed using the following combinations, but are not limited to these: (Embodiment 1) Added to the culture medium: stimulatory substance, recognition substance, costimulatory substance Binding to the substrate and / or encapsulated in vesicles: None (Embodiment 2) Added to the culture medium: stimulatory substance, recognition substance Binding to the substrate and / or encapsulated in vesicles: costimulatory substance (Embodiment 3) Added to the culture medium: stimulatory substance, costimulatory substance Binding to the substrate and / or encapsulated in vesicles: recognition substance (Embodiment 4) Added to the culture medium: recognition substance, costimulatory substance Binding to the substrate and / or encapsulated in vesicles: stimulatory substance (Embodiment 5) Added to the culture medium: stimulatory substance Binding to the substrate and / or encapsulated in vesicles: recognition substance, costimulatory substance (Embodiment 6) Added to the culture medium: recognition substance Binding to the substrate and / or encapsulated in vesicles: stimulatory substance, costimulatory substance (Embodiment 7) Added to the culture medium: costimulatory substance Binding to the substrate and / or encapsulated in vesicles: stimulatory substance, recognition substance (Embodiment 8) Addition to medium: None Binding to substrate and / or encapsulation in vesicles: Stimulatory substances, recognition substances, costimulatory substances

[0067] Although not shown in the above example, an embodiment may be adopted in which a stimulating substance is added to the culture medium and simultaneously bound to the substrate, etc. When adopting such an embodiment, for example, the stimulating substance added to the culture medium and the stimulating substance bound to the substrate may be the same or different.

[0068] In addition, the substance added to the culture medium or the substance bound to the substrate / encapsulated in the vesicles may be of one or more types. For example, when a costimulatory substance is bound to the substrate, the costimulatory substance may be of one or more types.

[0069] Examples of substrates to which stimulatory substances, recognition substances, and / or costimulatory substances (hereinafter sometimes referred to as "stimulatory substances, etc.") are bound include films, sponges, fibers, rods, beads, gels, etc., 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. Examples of materials include magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, polystyrene, retronectin, collagen, etc. Examples of vesicles encapsulating stimulatory substances, etc. include liposomes, exosomes, microvesicles, and apoptotic bodies. Contact with stimulatory substances, etc. is preferably achieved by culturing in a medium containing a stimulatory substance, etc. (e.g., a stimulatory substance, recognition substance, or costimulatory substance bound to a substrate, or a stimulatory substance, recognition substance, or costimulatory substance encapsulated in vesicles), i.e., by co-culturing CAR-T cells with the stimulatory substance, etc.

[0070] Co-culture: Co-culturing CAR-T cells with stimulatory substances and the like allows the CAR-T cells to proliferate efficiently through stimulation by the stimulatory substances and the like. Preferably, CAR-T cells are cultured for, for example, 1 second to 2 weeks after CAR gene transduction to allow for cell recovery and stable expression of the transgene. Because CAR-T cells may become exhausted after prolonged culture, they are more preferably used for co-culture within 1 second to 1 week, 1 second to 72 hours, or 1 second to 48 hours after CAR gene transduction. The co-culture period is not limited to, but is 1 second to 21 days, preferably 1 second to 14 days. When target antigen-expressing cells (feeder cells) are used as the recognition substance, the ratio of CAR-T cells to feeder cells at the start of co-culture (CAR-T cells / feeder cells) is not particularly limited, but is, for example, 0.05 to 20, preferably 0.1 to 10, and more preferably 0.5 to 5, in terms of the total number of cells engineered to express the CAR and target antigen, respectively. The cell density during co-culture is, for example, 1 x 10 6 pieces / mL ~ 100x10 6 The concentration is 1 / mL. When activation treatment, antigen recognition treatment, and / or costimulatory substance treatment are performed in the absence of feeder cells, the stimulatory substance is subjected to each treatment while bound to a substrate, encapsulated in vesicles, or contained in the medium. The amount of the stimulatory substance administered is not particularly limited as long as it is an amount that can sufficiently activate and proliferate CAR-T cells, and is set appropriately depending on the type of stimulatory substance. An excess amount is usually added.

[0071] The medium used for co-culture and for preparing CAR-T cells and / or feeder 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 supplemented with serum (e.g., 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 variations 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 the individual from whom 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, artificial serum is preferred. Plasma may also be used. Blood components such as serum or plasma, albumin, or their analogs may be used, or artificial media may also be used. The basal medium may be any suitable medium for cell culture, such as the aforementioned TexMACS™ (Miltenyi Biotec), AIM V (registered trademark), or ALyS medium (Cell Science Institute, Inc.). Other culture conditions may be any suitable for cell survival and proliferation, and general conditions may be used. Examples include culturing in a CO2 incubator (CO2 concentration 5%) set at 37°C, or culturing in low oxygen (oxygen concentration 0-20%, preferably 0-10%, more preferably 1-5%).

[0072] Additional factors may be added to the culture medium to support cell survival and proliferation. Examples of such factors include type 1 cytokine family members, type 2 cytokine family members, TNF superfamily cytokines, IL-1 family cytokines, and other cytokines (e.g., 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 also be added to the culture medium during CAR-T cell preparation. Supplementary factors can be prepared according to standard methods, or commercially available products can be used. Supplementary factors may be derived from animals other than humans, but are preferably derived from humans (even recombinant).

[0073] By expanding CAR-T cells (co-culturing with stimulatory substances, recognition substances, and / or costimulatory substances), a cell population containing CAR-T cells of sufficient quantity and quality for clinical use can be obtained. The method of the present invention allows for the efficient production of CAR-T cell populations with high efficacy compared to conventional methods. For example, the CAR-T cell population obtained by the method of the present disclosure may have a CAR-T cell percentage of 20%, 30%, or 40% or more, preferably 40% or more. Chronically activated T cells highly express multiple immune checkpoint molecules, resulting in their inability to proliferate and attack target cells. This phenomenon is called exhaustion. Even if T cells are returned to the body, their ability to proliferate and attack cancer is weakened, making them unlikely to provide a high therapeutic effect. The cell population obtained by the method of the present invention exhibits suppressed exhaustion. The degree of exhaustion can be assessed by techniques commonly used in the art, such as 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, particularly PD-1. Expression of exhaustion markers can be detected using antibodies against these markers. CAR-T cell populations obtained by the methods of the present disclosure have low expression of the exhaustion marker PD-1; for example, the percentage of PD-1-expressing cells among CAR-T cells may be less than 10%, preferably less than 5%, and more preferably less than 1%. Furthermore, the CAR-T cell population obtained by the methods of the present disclosure may have a proportion of naive or young memory T cells among the CAR-T cells of 20%, 30%, 45%, 50%, 55%, or 60% or more, preferably 60% or more.

[0074] After expansion, the CAR-T cells are recovered. The recovery procedure may be performed by a conventional method. For example, recovery may be performed by pipetting, using a liquid transfer tube, centrifugation, or the like. In a preferred embodiment, prior to the recovery procedure, a step of culturing the cells after expansion in the presence of a stimulating substance is performed. This step enables efficient expansion and also has the advantage of increasing the cell viability. If desired, the CAR-T cell population after expansion may be subjected to a cell separation step such as bead separation. By performing the cell separation step, the purity of CAR-T cells with higher efficacy can be increased.

[0075] The cell population containing CAR-T cells produced by the method of the present invention can be used to treat cancer, particularly cancers that express the target antigen of the CAR-expressing immune cells. The cancer may be a solid tumor or a hematologic tumor. Specific cancers include, but are not limited to, various B-cell lymphomas (e.g., follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma), 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, such as 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.

[0076] The cell population containing CAR-T cells produced by the method of the present invention is administered at a therapeutically effective dose that is determined appropriately depending on the age, 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 104 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 subject's age, weight, body surface area, symptoms, etc., and may be a single administration or multiple continuous or regular administrations.

[0077] Compositions containing cell populations containing CAR-T cells produced by the methods of the present invention may contain, in addition to the cell population to be administered to a subject, various components, such as dimethyl sulfoxide (DMSO) or serum albumin for cell protection, antibiotics for preventing bacterial contamination, and various components for cell activation, proliferation, or differentiation induction (vitamins, cytokines, minerals, carbon sources, nitrogen sources, trace metals, electrolytes, growth factors, steroids, etc.). The compositions can be prepared by conventional methods. The present invention is 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. Those skilled in the art will understand that alternatives with similar effects and actions are also possible.

[0078] Materials and Methods 1. Ethical Approval and Consent to Participate This study was approved by the Kyoto Prefectural University of Medicine Institutional Review Board, and the recombinant DNA experiments were approved by the Kyoto Prefectural University of Medicine Recombinant DNA Experiment Safety Committee. All experiments involving human participants were conducted in accordance with the guidelines of the Declaration of Helsinki. All animal experiments and procedures were approved by the Kyoto Prefectural University of Medicine Institutional Review Board.

[0079] 2. Blood Donors and Cell Lines. PBMCs were isolated from whole blood samples by density gradient centrifugation using Lymphocyte Separation Medium 1077 (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) followed by repeated washing with Dulbecco's phosphate-buffered saline (D-PBS, Nacalai Tesque, Kyoto, Japan). Viable cell counts were determined using standard trypan blue staining and an automated cell counter model R1 (Olympus, Tokyo, Japan). Human lymphoblastic leukemia (REH) cell line was purchased from the American Type Culture Collection (Manassas, VA). REH expressing firefly luciferase (FFLuc) and green fluorescent protein (GFP) (REH-FFLuc-GFP) was obtained by transfecting REH cells with piggyBac transposon (PB)-based pIRII-FFLuc-puroR-GFP (Non-Patent Document 6) followed by fluorescence-activated cell sorting (FACS). REH cells and REH-FFLuc-GFP cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA) and maintained in a humidified incubator at 37°C under a 5% CO2 atmosphere.

[0080] 3. PB-Based CAR-T Cell Production: CD45RA+ and CD45RA- PBMCs were magnetically isolated from total PBMCs using CD45RA MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach, Germany) (Figure 1). The CD19-CAR transgene was then introduced into these cells using the PB transposon system, as previously described (Non-Patent Documents 5 and 6). Briefly, the PB transposase plasmid (7.5 μg per 100 μL of electroporation buffer) and pIRII-CD19-28z (7.5 μg / 100 μL) (Figure 2) were transfected into P3 Primary Cell 4D-Nucleofector tubes. TMApproximately 4 × 10 cells were transfected using the X kit (Lonza, Program; FI-115) or MaxCyte ATX® (MaxCyte Inc.) using a protocol optimized for transfection of DNA plasmids into resting T cells (Protocol; RTC 14-3). 6 At the same time, approximately 1 × 10 CD45RA+ or CD45RA- PBMCs were transfected with an antigen-presenting feeder plasmid (pIRII-tCD19-CD80-41BBL; 15 μg per 100 μL) (Figure 2) by electroporation. 6 After electroporation, CAR-T cells and feeder cells were transduced into whole PBMCs. TM The cells were cultured in complete culture medium containing 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). Feeder cells were inactivated by UV irradiation 24 hours after electroporation and cocultured with CAR-T cells for 14 days (Non-Patent Documents 5 and 6). EPHB4 receptor-redirected CAR-T cells were generated using the PB transposon system as previously described for use as control CAR-T cells in in vivo stress tests (Non-Patent Document 5) (Figure 2).

[0081] 4. Flow cytometry. Expression of CD19-CAR molecules on the T cell surface was measured by flow cytometry using recombinant human CD19 Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-human immunoglobulin (Ig)-G Fc fragment-specific antibody (Merck Millipore, Burlington, MA). To characterize the phenotype of CAR-T cells, allophycocyanin (APC)-conjugated anti-CD3 antibody, APC-conjugated anti-CD8 antibody, phycoerythrin (PE)-conjugated anti-CD4 antibody, PE-conjugated anti-CD45RA antibody, and APC-conjugated anti-CCR7 antibody (all BioLegend, San Diego, CA, USA) were used. As markers of CAR-T cell exhaustion and senescence, we used APC-conjugated anti-programmed cell death protein-1 (PD-1) antibody, APC-conjugated anti-T cell immunoglobulin mucin-3 (TIM-3) antibody, Alexa Fluor 647-conjugated anti-CD223 (LAG-3) antibody, and Peridinin-Chlorophyll-Protein (PerCP) / Cyanine 5.5-conjugated anti-CD57 antibody (all BioLegend). To determine the phenotype of REH cells, we used FITC-conjugated anti-CD19 antibody (BioLegend). All flow cytometry data were analyzed using BD Accurio. TM C6 Plus or BD FACSCalibur TM (BD Biosciences, Franklin Lakes, NJ) and FlowJo TM Analysis was performed using software (BD Biosciences).

[0082] 5. Analysis of exhaustion markers expressed on the T cell surface after electroporation. Electroporation was performed using the same protocol as for CAR-T production, and GFP plasmid was introduced into all PBMCs. After electroporation, GFP-transfected PBMCs were cultured in a complete culture medium consisting of the same components as used for CAR-T production. Non-electroporated PBMCs were cultured in the same medium and used as a control. After 48 hours of culture, GFP-positive T cells and CD3-positive T cells were gated, and the expression of PD-1, TIM-3, and LAG-3 on the T cell surface was analyzed by flow cytometry.

[0083] 6. Sequential killing assay 1 x 10 5 REH cells and 1 × 10 cells derived from CD45RA+ PBMCs or CD45RA- PBMCs 5 CD19 CAR-T cells (RA+ CAR or RA- CAR, respectively) were co-cultured in 24-well cell culture plates. After 3 days, the CD19 CAR-T cells were harvested, counted, and treated with fresh REH cells at a 1:1 ratio for reconstitution. Cell counting and treatment with fresh REH cells were repeated every 3 days for a total of 3 replicates. The killing effect of these CD19 CAR-T cells was assessed by counting the number of remaining REH cells using flow cytometry, and the CAR mean fluorescence intensity (MFI) and exhaustion markers of these CAR-T cells were analyzed by flow cytometry.

[0084] 7. RNA Sequencing and Bioinformatics Analysis. RA+ and RA- CAR-T cells were cocultured with REH cells at a 1:1 effector:target ratio for 3 days (RA+ / Stimulation+, RA- / Stimulation+, RA+ / Stimulation-, and RA- / Stimulation-, respectively) with or without antigen stimulation. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Total RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific). Library preparation and high-throughput sequencing were performed by Eurofins Genomics (Ebersberg, Germany). mRNA was enriched and strand-specific libraries were prepared, as needed. Sequencing was performed using the NextSeq 500 / 550 System (Illumina, San Diego, CA, USA) and the NextSeq 500 / 550 Mid Output Kit v2.5 150 cycles (Illumina). Adapter sequences and low-quality reads were removed using fastp version 0.21.0. Filtered reads were aligned to the human reference genome (GRCh38.p13) using STAR version 2.7.6a. Counts per gene and transcripts per million were calculated using RSEM version 1.3.3. Counts per million calculations and differential expression analysis were performed using the edge R version 3.32.0 R package and the R v4.0.3 environment (https: / / www.R-project.org / ). Pathway analysis was performed using the R package for the Reactome Pathway Analysis.We also analyzed and visualized the differential gene expression profiles between RA+ and RA- CARs using Morpheus (https: / / software.broadinstitute.org / morpheus) and specific gene signatures related to T cell activation, exhaustion, and differentiation.

[0085] 8. In vivo stress test using a systemic tumor model mouse. Female 8-week-old NOD.Cg-Prkdc scid II2rg tm1Wjl / SzJ (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed at Kyoto Prefectural University of Medicine for at least one week before the start of the experiment. Food and water were available ad libitum. REH-FFLuc-GFP cells (5 × 10 5 ) was suspended in D-PBS and injected into mice via the tail vein. Six days later, 1 × 10 5 RA+ CAR-Ts, RA- CAR-Ts, or unrelated EPHB4 receptor-redirected CAR-T cells as a control were injected via the tail vein, and tumor burden was monitored using an IVIS Lumina Series III system (PerkinElmer, Inc.). Regions of interest on displayed images were quantified in photons per second (ph / s) using Living Image v2 (PerkinElmer, Inc.) as previously described (Non-Patent Document 5). Bone marrow (BM) cells were serially aspirated from the tibia at several time points. These BM cells were stained with PE-conjugated anti-human CD3 antibody and APC-conjugated anti-human PD-1 antibody (BioLegend), and long-term persistence of human T cells was assessed by flow cytometry.

[0086] 9. Statistics Statistical comparisons between two groups were determined by two-tailed parametric or nonparametric tests (Mann-Whitney U-test) for unpaired data and by two-tailed paired Student's t-test for matched samples. All data are presented as mean ± standard deviation. The log-rank test was used to compare survival curves obtained by the Kaplan-Meier method. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 9 software.

[0087] Results Example 1: Using magnetic separation targeting human CD45RA, CD45RA+ or CD45RA- subpopulations were isolated from total PBMCs. As a result, two peaks, CD45RA high and negative, were observed in the lymphocyte fraction, while CD45RA positive cells were evenly distributed from high to low in the monocyte fraction (Figure 1). Magnetic bead sorting efficiently separated CD45RA+ and CD45RA- PBMCs, with over 98% CD45RA-positive cells in the CD45RA+ fraction and over 93% CD45RA-negative cells in the CD45RA- fraction (Figure 1). Next, the efficiency of transient transduction of the CD19 CAR transgene into CD45RA+ or CD45RA- PBMCs was evaluated 24 hours after electroporation. When unstimulated, magnetically sorted CD45RA+ or CD45RA- PBMCs were electroporated with a CD19 CAR transgene, the CD45RA+ PBMC subpopulation showed higher transduction efficiency than the CD45RA- PBMC subpopulation at 24 hours post-electroporation (CD45RA+ PBMC: 77.5% ± 9.8% vs. CD45RA- PBMC: 39.7% ± 3.8%) (Fig. 3A). Furthermore, CD19 CAR-T cells derived from CD45RA+ PBMCs (RA+ CAR-T) showed higher expansion potential after 14 days of culture compared with CD19 CAR-T cells derived from CD45RA- PBMCs (RA- CAR-T) (RA+ CAR-T: 32.5 ± 9.3-fold vs. RA- CAR-T: 11.0 ± 5.4-fold) (Fig. 3B). After 14 days of expansion, these CAR-T cells were measured for CAR positivity, phenotype, and expression of the exhaustion marker PD-1 by flow cytometry.Compared with RA-CAR-T, RA+CAR-T cells had a higher CAR-positive rate (RA+CAR-T: 68.3% ± 3.8% vs. RA-CAR-T: 46.6% ± 7.3%), predominant CD8 expression (RA+CAR-T: 84.0% ± 3.4% vs. RA-CAR-T: 34.1% ± 10.6%), lower expression of the exhaustion marker PD-1 (RA+CAR-T: 3.1% ± 2.5% vs. RA-CAR-T: 19.2% ± 6.4%) and reduced expression of the T cell senescence marker CD57 (RA+CAR-T: 6.8% ± 3.6% vs. RA-CAR-T: 20.2% ± 6.9%), and a higher proportion of naive / stem cell memory (CD45RA+ / CCR7+ fraction; RA+CAR-T: The expression of TIM-3 and LAG-3 in PBMCs was significantly higher in both CAR-T and PBMCs (71.9% ± 9.7% vs. 8.0% ± 5.3%) than in RA-CAR-T (Figure 3C, Figure 3D). Meanwhile, other activation / exhaustion markers, such as TIM-3 and LAG-3, were highly expressed in both types of CAR-T cells (Figure 3D). This finding was consistent with previous studies on PB-CAR-T cells. Because most PB-CAR-T cells were previously generated by electroporation, we hypothesized that the high expression of TIM-3 and LAG-3 might be induced by electroporation. However, PBMCs 48 hours after electroporation barely expressed PD-1, TIM-3, or LAG-3 on T cells (data not shown). Therefore, it is likely that the expression of TIM-3 and LAG-3, but not PD-1, on CAR-T cells is induced during the culture period, rather than by electroporation.

[0088] Example 2: To investigate the effects of RA+ or RA- CAR-T cells at the molecular level and identify pathways involved in the favorable phenotype of RA+ CAR-T cells, genome-wide transcriptional profiling focusing on immunogenic gene signatures was performed. A total of 29 genes were overexpressed and 78 genes were underexpressed in RA+ CAR-T cells compared with CD45RA- CAR-T cells (Figure 4A). Reactome pathway analysis revealed that the gene expression differences observed in RA+ CAR-T cells were related to costimulation via the noncanonical NF-κB pathway and the CD28 family pathway, while the PD-1 signaling pathway was significantly downregulated in RA+ CAR-T cells (Figure 4B). Transcriptome profiling also demonstrated that RA+ CAR-T cells exhibited an activated, but not exhausted, profile characterized by upregulation of T cell activation markers, including transcription factor 7, lymphoid enhancer-binding factor 1, CCR7.R, and IL7R. Furthermore, even after antigen stimulation, RA+ CAR-T cells displayed an activated but not exhausted profile, with reduced expression of T cell activation markers such as PD-1, LAG3, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and eomesodermin (data not shown). These results suggest that RA+ CAR-T cells are enriched for naive / memory phenotypes and are resistant to T cell exhaustion induced by antigen stimulation.

[0089] Example 3 To evaluate the antileukemic activity of RA+ CAR-T and RA- CAR-T cells, tumor rechallenge assays were performed by adding fresh REH cells to CAR-T cells every 3 days. Both RA+ and RA- CAR-T cells were able to completely kill REH cells even after multiple tumor rechallenges (Figure 5A). After antigen stimulation, RA+ CAR-T cells showed lower cell surface CAR molecule expression than RA- CAR-T cells (Figure 5B). PD-1 expression in RA+ CAR-T cells was lower than that in RA- CAR-T cells during multiple antigen stimulations (Figure 5C). Meanwhile, LAG-3 expression was comparable between RA+ and RA- CAR-T cells, whereas TIM-3 expression was higher in RA+ CAR-T cells than in RA- CAR-T cells. These expressions gradually decreased in RA+ and RA- CAR-T cells during multiple antigen stimulations, and the relatively high expression of TIM-3 and LAG-3 did not impair the killing effect of CAR-T cells (Fig. 5C).

[0090] Example 4 To evaluate the in vivo antitumor effects of RA+ and RA- CAR-T cells, an in vivo stress test was performed in which the dose of CAR-T cells was reduced to the functional limit, and these CAR-T cells were maintained and expanded in vivo to achieve antitumor effects. NSG mice were treated with 5 × 10 5 Six days later, 1 × 10 REH-FFLuc cells were injected into the tail vein of each mouse. 5Mice were infused with RA+ CAR-T, RA- CAR-T, or control (EPHB4) CAR-T positive cells. RA+ CAR-T cells induced greater tumor regression and prolonged median survival compared with RA- CAR-T cells (Figure 6A-C). At day 15, the bone marrow of the RA+ CAR-T cell group contained abundant human CD3-positive T cells with lower PD-1 expression and relatively fewer REH cells than the RA-CAR-T cell group (Figure 6D). Furthermore, serial bone marrow examinations confirmed the proliferation of human CD3-positive T cells in two of the long-lived mice in the RA+ CAR-T group even 50 days after administration (Figure 6E). This indicates that RA+ CAR-T cells are antigen-driven and function long-term in vivo.

[0091] Example 5: CAR-T cell expansion culture was performed in the presence or absence of feeder cells, and the effects were examined. In this example, a mixture of three types of stimulatory beads was used, but one or two types can also be used. Four or more types can also be used. When using two or more types of stimulatory beads, the combination is not particularly limited. (Materials and Methods) 1. Protein Reconstitution (i) CD19 protein (Acro BIOSYSTEMS) (ii) CD80 protein (Acro BIOSYSTEMS) (iii) 4-1BBL protein (Acro BIOSYSTEMS)

[0092] 2. Preparation of stimulation beads Miltenyi MACSiBeads are reacted with the above 1. and used for CAR-T stimulation.

[0093] 3. CAR-T cell production Day 0: Blood was collected, and CD19CAR / PB transposase and antigen-presenting feeders (AP feeders) were electroporated (EP). Culture was initiated under the following four conditions: 1) AP 2) 2.5M beads 3) 5M beads 4) 10M beads Day 1: A portion of the AP feeder was UV-irradiated and added to 1). 2) to 4) contained 2.5M, 5M, and 10M beads, respectively. Day 3: Expansion culture was performed for each group. Day 8: Cell count and CAR expression rate were measured. The remaining cells were split into two groups. 1) AP 2) AP (same as 1) 3) 2.5M Beads 4) 2.5M + 2.5M Beads 5) 5M Beads 6) 5M + 5M Beads 7) 10M Beads 8) 10M + 10M Beads 4), 6), and 8) were re-added with 2.5M, 5M, and 10M beads, respectively. Culture medium was added and the cells were further cultured. On Day 13, the cell number and CAR expression rate were measured and graphed.

[0094] The results are shown in Figure 7. A higher proliferation rate was observed when cells were expanded using beads coupled with stimulatory factors compared to when cells were expanded using feeder cells.

[0095] Example 6: The above examples demonstrate that CAR-T cells produced from CD45RA+ cells have high gene transfer efficiency, high cell proliferation capacity, strong anti-cancer effects, and low expression rates of exhaustion markers. Therefore, we investigated candidate specific factors other than CD45RA+. Omics analysis was performed on four types of cells: "CD45RA+ with and without stimulation" and "CD45RA- with and without stimulation." As a result of the analysis, several specific factors were identified (see Table 1).

[0096] Example 7: EPHB4-CAR-T cells were produced from cryopreserved PBMCs. Frozen PBMCs (CTL) were thawed in a 37°C water bath, diluted with Dulbecco's phosphate-buffered saline (D-PBS, Wako), and counted using an NC-3000 (MS Techno Systems). The thawed PBMCs were centrifuged, and the cell pellet was suspended in electroporation buffer (Miltenyi Biotec). The PB transposase plasmid and pIRII-EPHB4-28z were added to the resulting cell suspension, and then transfected into PBMCs via electroporation. After transfection, the cells were transferred to a complete culture medium containing ALySTM705 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) and cultured for 14 days. At that time, beads conjugated with a recognition substance and a costimulatory substance (EPHB4-CD80-4-1BBL) were added on days 1 and 8 after electroporation, and the cells were expanded. The CAR-T cells obtained after the culture were subjected to cell counting, FCM analysis, and anti-cancer tests.

[0097] The method of producing CAR-T cells from frozen PBMCs also demonstrated high cell proliferation (13.8-fold), similar to that achieved using fresh blood, with a high CAR positivity rate of 74.7%. FCM analysis of the resulting CAR-T cells revealed that the proportion of naive and stem cell memory T cells (Tscm) was 25%, with almost no expression of the exhaustion marker PD-1, confirming their high anti-cancer efficacy. These results suggest that CAR-T cells can be produced from frozen PBMCs, just as they are from fresh blood. Similar results were also obtained when CD19 was used as the CAR gene (data not shown).

[0098] Example 8: Flow cytometry and bead separation were compared as methods for separating T cell subpopulations. PBMCs were isolated from whole blood samples by density gradient centrifugation using Lymphocyte Separation Medium 1077 (Fujifilm Wako Pure Chemical Industries, Ltd.) and repeated washing with Dulbecco's phosphate-buffered saline (Nacalai Tesque). CD45RA+ and CD45RA- PBMCs were separated from total PBMCs by magnetic separation using CD45RA MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach, Germany). When separating total PBMCs by flow cytometry, total PBMCs were stained with anti-CD45RA antibody and separated using a cell sorter. Table 4 summarizes the time required for flow cytometry and bead separation depending on the cell volume.

[0099]

[0100] Compared to flow cytometry, the bead separation method can be performed in a closed system, allowing for the easy and rapid isolation of T cell subpopulations regardless of the amount of cells sorted. Based on these results, the bead separation method, which places less strain on cells and is also suitable for clinical applications, is preferred.

[0101] Example 9: The separation of T cell subpopulations using specific factors was investigated. Frozen PBMCs (CTL) were thawed in a 37°C water bath and diluted with Dulbecco's phosphate-buffered saline (D-PBS, Wako). Cell counts were then measured using an NC-3000 (MS Techno Systems). The thawed PBMCs were centrifuged, and the cell pellet was suspended in MACS buffer (0.5% BSA, 2 mM EDTA in D-PBS). Cells expressing CD45RA on the cell membrane surface (CD45RA+) were isolated using CD45RA MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach, Germany). After separation, the cells were counted, and the required amount of cell suspension was centrifuged. The cell pellet was suspended in electroporation buffer. The PB transposase plasmid and pIRII-EPHB4-28z were added to the resulting cell suspension, followed by electroporation. After transfection, the cells were transferred to a complete culture medium containing ALySTM705 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) for 14 days. On days 1 and 8 after electroporation, beads conjugated with a recognition substance and a costimulator (EPHB4-CD80-4-1BBL) were added and cultured. For isolation of specific factors other than CD45RA+, the same procedure was followed, except that isolation was performed using CD62L+, CD28+, IL-7R+, CD3+, CD27+, CD44+, CD44RO-, or CD57-, respectively. The resulting CAR-T cells were subjected to cell counting, FCM analysis, and anticancer testing. The results are shown in Table 5. In the table, "-" indicates a T cell population obtained without using any specific factor.

[0102]

[0103] CAR-T cells prepared from T cell subpopulations separated by specific factors showed a high number of CAR-positive cells, and FCM analysis showed a high proportion of naive or young memory T cells (especially Tscm / naive and Tcm), with almost no expression of the exhaustion marker PD-1. Furthermore, they also had a high anti-cancer effect, and the increase or decrease in the CAR-positive rate and the proportion of naive or young memory T cells after anti-cancer testing suggested a sustained anti-cancer effect.

[0104] Example 10: CAR-T cells were produced by activating them with stimulatory substances in the absence of feeder cells. The CAR-T cells prepared in Example 7 were cultured for 5–7 days in culture medium supplemented with various stimulatory substances. The stimulatory substances used were as follows: anti-CD62L antibody, PD0325901 (MEK inhibitor), SB431542 (TGF-βIR inhibitor), rapamycin (mTOR inhibitor), simvastatin (HMG-CoA reductase inhibitor), GW9662, rosiglitazone (PPARγ inhibitor), GW6471 (PPARα inhibitor), anti-CD327 antibody, anti-CD73 antibody, anti-CXCR5 antibody, anti-CCR7 antibody, and anti-CCL5 antibody. The CAR-T cells obtained after culture were subjected to cell counting, FCM analysis, and anti-cancer testing. The results are shown in Table 6 below. Each value in the table, except for "cancer cell survival rate" after "anticancer test," is a relative value to the control value.

[0105]

[0106] As shown in Table 6, the addition of various stimuli resulted in a higher proportion of young, non-exhausted T cells (especially Tscm / naive and Tcm) even after anti-cancer testing, compared with the control group (i.e., without the addition of stimuli). At the same time, the anti-cancer activity was comparable or even higher with the addition of stimuli compared with the control group. These results demonstrate that the use of specific stimuli can produce high-quality CAR-T cell populations with sustained and potent anti-tumor effects.

[0107] Example 11: The cell preparation and expansion methods for gene transfer were compared between conventional and novel methods. 1. Generation of EPHB4-CAR-T Cells from PBMCs. EPHB4-CAR-T cells were produced from cryopreserved PBMCs. Frozen PBMCs (CTL) were thawed in a 37°C water bath and diluted with Dulbecco's phosphate-buffered saline (D-PBS, Wako). The cell count was measured using an NC-3000 (MS Techno Systems). The thawed PBMCs were centrifuged, and the cell pellet was suspended in electroporation buffer (Miltenyi Biotec). The PB transposase plasmid and pIRII-EPHB4-28z were added to the resulting cell suspension, and the PBMCs were transfected by electroporation. After transfection, the cells were transferred to a complete culture medium containing ALySTM705 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) for 14 days. On days 1 and 8 after electroporation, beads conjugated with a recognition substance and a costimulator (EPHB4-CD80-4-1BBL) were added and expanded. On day 1, samples containing 25 ng / mL rapamycin or feeder cells were also prepared.

[0108] Preparation of feeder cells. EPHB4-CAR-T cells were produced from cryopreserved PBMCs. Frozen PBMCs (CTL) were thawed in a 37°C water bath, diluted with Dulbecco's phosphate-buffered saline (D-PBS, Wako), and counted using an NC-3000 (MS Techno Systems). Thawed PBMCs were centrifuged, and the cell pellet was suspended in electroporation buffer (Miltenyi Biotec). The resulting cell suspension was supplemented with the feeder plasmid pIRII-dEPHB4-CD80-BBL, which was then electroporated into PBMCs. After transfection, the cells were transferred to a complete culture medium containing ALySTM705 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), and cultured for 1 day. The cells were then irradiated with UV light and added in equal amounts as feeders to the CAR-transfected cells.

[0109] 2. Generation of EPHB4-CAR-T cells from cell populations separated with CD62L beads. Frozen PBMCs (CTL) were thawed in a 37°C water bath and diluted with Dulbecco's phosphate-buffered saline (D-PBS, Wako). The cell count was measured using an NC-3000 (MS Techno Systems). The thawed PBMCs were centrifuged, and the cell pellet was suspended in MACS buffer (0.5% BSA, 2 mM EDTA in D-PBS). Cells expressing CD62L on the cell membrane surface (CD62L + ) were isolated using CD62L MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach, Germany). After isolation, the cell count was measured, and the required amount of cell suspension was centrifuged. The cell pellet was then suspended in electroporation buffer. Gene transfer and expansion were then performed as described in "1" above. The CAR-T cells obtained after culture were subjected to cell counting, FCM analysis, and anti-cancer testing. The results are shown in Table 7 below. Note that each value in the table, except for "cancer cell viability" after "anti-cancer testing," is a relative value to the value of a T cell population prepared by using "co-stimulatory beads" as a stimulation method during expansion of a cell population obtained without "bead separation" ("-"). Note that the costimulatory beads used were MACSiBeads conjugated with EPHB4, CD80, and 4-1BBL.

[0110]

[0111] Comparison before anti-cancer test When comparing the feeder method and the feeder-free method, the feeder method had a higher Tscm / Naive ratio, but a lower number of CAR-positive cells. With the feeder-free method, although the Tscm / Naive ratio was lower, it remained at approximately 30% or higher under all conditions, resulting in a higher number of CAR-positive cells. In CAR-T cells generated from PBMCs using the feeder-free method, the Tscm / Naive ratio was higher when rapamycin was added than when costimulatory beads were used alone. Furthermore, the Tscm / Naive ratio was increased even in CAR-T cells generated after CD62L bead separation.

[0112] Comparison after anti-cancer test CAR-T cells that had a good Tscm / Naive ratio before the anti-cancer test tended to maintain the CAR positivity rate and Tscm / Naive ratio even after the anti-cancer test, suggesting a sustained anti-cancer effect.

[0113] According to the method of the present invention, higher quality CAR-T cells can be obtained, and highly functional CAR-T cells can be produced simply, in a short time, and at low cost.

[0114] This application is based on patent application No. 2021-179855 filed in Japan (filing date: November 2, 2021), the contents of which are incorporated in their entirety into this specification.

Claims

1. A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the step of isolating T cells from a T cell source using a specific factor, The aforementioned specific factor is at least one selected from the group consisting of CD45RA+, CD62L+, IL-7R+, CD28+, CD3+, CD27+, CD44+, CD57-, CD45RO-, CD4+, CD8+, CCR7+, CD95+, CXCR5+, CCL5+, CCL19+, CD45RB+, CD45RC+, CD80+, 4-1BBL+, LAG3-, CXCR3-, and IL-2Rβ-. A manufacturing method wherein the separation step is performed by at least one selected from the group consisting of density gradient separation, immunological cell separation technique, magnetic cell separation technique, nylon wool separation method, and adhesion method.

2. The manufacturing method according to claim 1, wherein the specific factor is at least one selected from the group consisting of CD45RA+, CD62L+, IL-7R+, CD28+, and CD3+.

3. The method for producing a T cell according to claim 1, wherein the T cell source is peripheral blood mononuclear cells.

4. The manufacturing method according to claim 1, further comprising the step of carrying out the expansion culture of the isolated T cells in the absence of feeder cells.

5. A process of separating T cells from a T cell source using a specific factor. The process of introducing the CAR gene into isolated cells, and A method for producing chimeric antigen receptor T (CAR-T) cells, comprising the step of expanding the culture of cells into which the CAR gene has been introduced, A manufacturing method characterized in that the specified factor is at least one selected from the group consisting of CD45RA+, CD62L+, IL-7R+, CD28+, CD3+, CD27+, CD44+, CD57-, and CD45RO-.

6. The manufacturing method according to claim 5, wherein the separation step is bead separation.

7. The manufacturing method according to claim 5 or 6, wherein the specific factor is at least one selected from the group consisting of CD45RA+, CD62L+, IL-7R+, CD28+, and CD3+.

8. The manufacturing method according to claim 5, wherein the expanded culture includes an activation treatment and an antigen recognition treatment.

9. The manufacturing method according to claim 8, wherein the activation treatment is carried out by contact between cells and a stimulating substance.

10. The manufacturing method according to claim 9, wherein the contact with the irritant is contact with a substrate to which the irritant is bound.

11. The manufacturing method according to claim 10, wherein the contact with the substrate is a co-culture of the cells and the substrate.

12. The manufacturing method according to claim 8 or 9, wherein the activation treatment is carried out by culturing the cells in a culture medium containing a stimulating substance.

13. The manufacturing method according to claim 12, wherein the activation treatment is carried out by co-culturing the cells and a substrate to which the stimulating substance has been bound in a culture medium containing the stimulating substance.

14. The manufacturing method according to claim 13, wherein the substrate to which the stimulating substance is bound is a bead to which a protein is bound.

15. The manufacturing method according to claim 9, wherein the irritant is at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, Rapamycin-, simvastatin-, GW9662-, Rosiglitazone-, and GW6471-.

16. The manufacturing method according to claim 8, wherein the antigen recognition process is carried out by contact between cells and a recognition substance.

17. The manufacturing method according to claim 16, wherein the contact with the recognition substance is contact with a substrate to which the recognition substance is bound.

18. The manufacturing method according to claim 17, wherein the contact with the substrate is a co-culture of the cells and the substrate.

19. The manufacturing method according to claim 8 or 16, wherein the antigen recognition treatment is carried out by culturing the cells in a culture medium containing a recognition substance.

20. The manufacturing method according to claim 19, wherein the antigen recognition treatment is carried out by co-culturing the cells and a substrate to which the recognition substance is bound in a culture medium containing the recognition substance.

21. The manufacturing method according to claim 20, wherein the substrate to which the recognition substance is bound is a bead to which a protein is bound.

22. The manufacturing method according to claim 19, wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19.

23. A method for producing chimeric antigen receptor T (CAR-T) cells, comprising co-culturing cells after CAR gene transfer with beads conjugated to the stimulant and the recognition substance in a culture medium containing the stimulant and the recognition substance, The irritant is provided as at least one selected from the group consisting of CCR7+, CD62L+, CXCR5+, CCL5+, CD327+, CD73+, SB431542-, PD0325901-, Rapamycin-, simvastatin-, GW9662-, Rosiglitazone-, and GW6471-. The manufacturing method according to claim 8, wherein the recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19.

24. A method for producing chimeric antigen receptor T (CAR-T) cells, in the absence of feeder cells, co-cultures cells after CAR gene transfer with beads conjugated with a co-stimulator and a recognition substance in a culture medium containing a stimulating substance. The cells after the introduction of the CAR gene are derived from T cells isolated from a T cell source using a specific factor, The aforementioned specific factor is at least one selected from the group consisting of CD45RA+, CD62L+, IL-7R+, CD28+, and CD3+. The stimulating substance is at least one selected from the group consisting of anti-CCR7 antibody, anti-CD62L antibody, anti-CXCR5 antibody, anti-CCL5 antibody, anti-CD327 antibody, anti-CD73 antibody, SB431542, PD0325901, Rapamycin, simvastatin, GW9662, Rosiglitazone, and GW6471. The recognition substance is at least one selected from the group consisting of EPHB4, HER2, and CD19. A method for producing the aforementioned co-stimulator, wherein the co-stimulator is at least one selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113.