T cells with enhanced glucose uptake ability on-demand, cell population, and pharmaceutical composition containing same

Abstract

The present disclosure provides cells that are competitive to cancer cells without starvation and exhaustion even in an environment in which glucose is depleted. More specifically, the present disclosure provides T cells with enhanced glucose uptake ability, wherein the T cells are modified such that the expression of glucose transporters in the T cells is regulated in response to changes in tumor microenvironmental conditions, and / or the T cells comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironmental conditions.

Description

T cells with enhanced glucose uptake on demand, cell population, and pharmaceutical composition containing the same

[0001] The present disclosure relates to T cells, cell populations, and pharmaceutical compositions comprising such cells that have enhanced glucose uptake capacity on demand.

[0002] Cancer cells reprogram their metabolic systems to favor their own cell proliferation, actively utilizing glycolysis, which is inefficient at producing ATP even in the presence of oxygen, to increase glucose uptake and lactate production (Warburg effect).

[0003] In the tumor microenvironment, there are many factors that suppress the metabolic activity and function of antitumor T cells. In addition to immunosuppressive factors, metabolic competition between tumors and T cells contributes to the formation of an immunosuppressive environment. When T cells are stimulated by TCR, Ca ２＋ The intracellular concentration of NFAT increases, activating calcineurin. Activation of calcineurin causes the dephosphorylated transcription factor NFAT to translocate into the nucleus, where it interacts with other transcription factors to promote and activate the transcription of genes such as the IL-2 gene.

[0004] On the other hand, cancer cells consume large amounts of glucose and deplete it, so T cells, which require glucose as an energy source, ｅｆｆ When tumor-specific T cells of this type infiltrate tumors, they receive signals from TCR but do not respond to the Ca ２＋ The concentration of NFAT decreases and nuclear translocation of NFAT is reduced, resulting in T cell dysfunction and suppression of cell proliferation and cytokine production.

[0005] In addition to glucose, cancer cells also consume large amounts of nutrients such as amino acids and fatty acids, which cause T cells to malfunction. Thus, in the tumor microenvironment, the metabolic mechanisms that support the active proliferation of cancer cells inhibit the antitumor effector functions of tumor-specific T cells.

[0006] Therefore, there is a need for cells that can compete with cancer cells without starving or becoming exhausted even in such an environment.

[0007] The present disclosure provides T cells with enhanced glucose uptake capacity that function in a tumor environment without starvation or exhaustion, a cell population containing such cells, and a pharmaceutical composition containing such cells. A representative feature of the present disclosure is that the enhanced glucose uptake capacity is achieved on demand.

[0008] Accordingly, the present disclosure provides the following. [Item 1] A T cell with enhanced glucose uptake ability, wherein the T cell has been modified to express a glucose transporter on demand and / or the expression of the glucose transporter has been enhanced on demand. [Item 2] The T cell according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 2A] The T cell according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item 3] The T cell according to any one of the above items, wherein the T cell has effector function. [Item 4] The T cell is an effector T cell (T ｅｆｆ) precursor cells. [Item 5] The T cell of any one of the above items, wherein the on-demand expression and / or enhancement is achieved specifically in a tumor environment. [Item 6] The T cell of any one of the above items, wherein the CAR is expressed in the T cell. [Item 7] The T cell of any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item 8] The T cell of any one of the above items, wherein the glucose transporter is GLUT3. [Item 9] The T cell of any one of the above items, wherein the T cell is a human T cell. [Item 10] A cell population comprising T cells with enhanced glucose uptake ability according to demand (on-demand). [Item 11] The cell population of any one of the above items, wherein the T cells include cells that have the property of having effector function when introduced into a body. [Item 12] The cell population according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 12A] The cell population according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 13] The cell population according to any one of the above items, wherein the T cells have been modified to express a glucose transporter on demand (on demand) and / or the expression of a glucose transporter has been enhanced on demand (on demand). [Item 14] The cell population according to any one of the above items, wherein the T cells have effector function. [Item 15] The cell population, wherein the T cells are ｅｆｆ [Item 16] The cell population according to any one of the above items, comprising T ｅｆｆ [Item 16A] The cell population according to any one of the above items, comprising a progenitor cell of T ｅｆｆ[Item 17] The cell population according to any one of the above items, wherein the T cells (progenitor cells of T cell type 1 or 2) comprise at least one cell type selected from the group consisting of Tnv, Tscm, Tcm, and Temra. [Item 17] The cell population according to any one of the above items, wherein the CAR is expressed in the T cells. [Item 18] The cell population according to any one of the above items, wherein the glucose transporters comprise GLUT1, GLUT2, GLUT3, and GLUT4. [Item 19] The cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 20] The cell population according to any one of the above items, wherein the T cells are human T cells. [Item 21] A pharmaceutical composition comprising the T cells according to any one of the above items or the cell population according to any one of the above items. [Item 22] The pharmaceutical composition according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 22A] The pharmaceutical composition according to any one of the above items, which is for the prevention or treatment of cancer. [Item 23] The pharmaceutical composition according to any one of the above items, which is used to cure cancer. [Item 24] The pharmaceutical composition according to any one of the above items, which is used to prevent metastasis or recurrence of cancer. [Item 25] The pharmaceutical composition according to any one of the above items, which is used to prevent or treat cancer so that it does not recur. [Item 26] A pharmaceutical composition for preventing or treating a disease in a subject, the pharmaceutical composition comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population comprising said T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population comprising said T cells to the subject in an appropriate dosage and administration. [Item 26A] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 26B] The pharmaceutical composition according to any one of the preceding items, wherein the T cells comprise a chimeric antigen receptor (CAR).[Item 26C] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item 26D] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer. [Item 27] ​​The T cells with enhanced glucose uptake ability have not been modified to express a glucose transporter on demand, and the expression of the glucose transporter has not been enhanced on demand. ｅｆｆand a T cell or cell population modified to express a glucose transporter on demand and / or whose expression is enhanced on demand. [Item 28] The composition of any one of the above items, wherein the glucose transporter is GLUT3. [Item 29] The T cell or cell population of any one of the above items for use as a medicament. [Item 30] The cell or cell population of any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 30A] The cell or cell population of any one of the above items, which is for the prevention or treatment of cancer. [Item 31] The cell or cell population of any one of the above items, which is for the cure of cancer. [Item 32] The cell or cell population of any one of the above items, which is for the prevention of cancer metastasis or recurrence. [Item 33] The cell or cell population of any one of the above items, which is for the prevention or treatment of cancer to prevent recurrence. [Item 34] A cell or cell population for preventing or treating a disease in a subject, the cell or cell population being characterized by: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell, which has an appropriate expression characteristic according to the index; and (C) administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject in an appropriate manner and dosage. [Item 34A] The cell or cell population according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 34B] The cell or cell population according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item 34C] The cell or cell population according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item 34D] The cell or cell population according to any one of the preceding items, wherein the disease includes cancer.[Item 35] The T cells with enhanced glucose uptake ability have not been modified to express a glucose transporter on demand, and the expression of the glucose transporter is not enhanced on demand. ｅｆｆand the cell or cell population of any one of the above items, wherein the cell or cell population is modified to express a glucose transporter on demand and / or wherein expression of the glucose transporter is enhanced on demand. [Item 36] The cell or cell population of any one of the above items, wherein the glucose transporter is GLUT3. [Item 37] A method for treating or preventing a subject in need thereof, comprising administering to the subject an effective amount of the T cell or cell population of any one of the above items. [Item 38] The method of any one of the above items, wherein the treatment or prevention in the subject is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 38A] The method of any one of the above items, wherein the treatment or prevention in the subject includes the prevention or treatment of cancer. [Item 39] The method of any one of the above items, wherein the treatment or prevention in the subject includes curing cancer. [Item 40] The method of any one of the above items, wherein the treatment or prevention in the subject includes prevention of cancer metastasis or recurrence. [Item 41] The method of any one of the above items, wherein the treatment or prevention of the subject comprises preventing or treating cancer so that it does not recur. [Item 42] A method for preventing or treating a disease in a subject, the method comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate dosage and administration. [Item 42A] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 42B] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 42C] The method of any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease.[Item 42D] The method according to any one of the above items, wherein the disease includes cancer. [Item 43] The T cells with enhanced glucose uptake ability are T cells that have not been modified to express a glucose transporter on demand, and the expression of the glucose transporter is not enhanced on demand. ｅｆｆand wherein the T cell or cell population of the above items is modified to express a glucose transporter on demand and / or the expression of the glucose transporter is enhanced on demand. [Item 44] The method of any one of the above items, wherein the glucose transporter is GLUT3. [Item 45] Use of the T cell or cell population of the above items for manufacturing a medicament comprising the cell or cell population. [Item 46] The use of any one of the above items, wherein the medicament is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 46A] The use of any one of the above items, wherein the medicament is for the prevention or treatment of cancer. [Item 47] The use of any one of the above items, wherein the medicament is for the cure of cancer. [Item 48] The use of any one of the above items, wherein the medicament is for the prevention of cancer metastasis or recurrence. [Item 49] The use of any one of the above items, wherein the medicament is for the prevention or treatment of cancer so that it does not recur. [Item 50] The medicament is the use of any one of the above items for the prevention or treatment of a disease in a subject, the use comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate manner and dosage. [Item 50A] The use of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 50B] The use of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 50C] The use of any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item 50D] The use of any one of the above items, wherein the disease comprises cancer.[Item 51] The T cells with enhanced glucose uptake ability are not modified to express a glucose transporter on demand, and the expression of the glucose transporter is not enhanced on demand. ｅｆｆ and a gene encoding a glucose transporter that is modified to express the glucose transporter on demand and / or has enhanced expression of the glucose transporter on demand. [Item 52] The use of any one of the above items, wherein the glucose transporter is GLUT3.

[0009] The present disclosure also provides the following. [Item A1] A T cell with enhanced glucose uptake ability, wherein the expression of a glucose transporter in the T cell is regulated in response to changes in tumor microenvironment conditions. [Item A1A] A T cell with enhanced glucose uptake ability, wherein the T cell is modified so that the expression of a glucose transporter in the T cell is regulated in response to changes in tumor microenvironment conditions. [Item A2] The T cell according to any of the above items, wherein the tumor microenvironment conditions include the degree of transduction of a TCR-associated signal. [Item A3] The T cell according to any one of the above items, wherein the TCR-associated signal is mediated by a molecule selected from the group consisting of a TCR signal or a part thereof, and a chimeric signal of a TCR signal and another antigen (also referred to as a CAR signal). [Item A4] The T cell according to any one of the above items, wherein the TCR-associated signal is mediated by a molecule comprising a TCR signal domain. [Item A5] The T cell of any one of the above items, wherein the change in tumor microenvironment conditions comprises the presentation of tumor antigens and / or costimulatory ligands, and / or a change in cytokine conditions. [Item A6] The T cell of any one of the above items, wherein the degree of TCR-associated signal transmission comprises the degree of TCR stimulation. [Item A7] The T cell of any one of the above items, wherein the degree of TCR-associated signal transmission comprises at least one selected from the group consisting of the degree of TCR stimulation, TCR costimulation, and humoral factors. [Item A8] The T cell of any one of the above items, wherein the degree of TCR-associated signal transmission comprises the degree of TCR stimulation and at least one selected from the group consisting of TCR costimulation and humoral factors. [Item A9] The T cell of any one of the above items, wherein the degree of TCR-associated signal transmission comprises the degree of TCR stimulation, TCR costimulation, and humoral factors. [Item A10] The T cell according to any one of the above items A, wherein the TCR stimulation is performed by a transcription factor selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families, which translocate into the nucleus upon activation of the T cell. [Item A11] The T cell according to any one of the above items, wherein the tumor microenvironment conditions include a degree of oxygen concentration.[Item A12] A T cell comprising a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. [Item A13] The T cell of any one of the above claims, wherein the tumor microenvironment sensing element comprises: (i) an inducible activation motif that is activated in response to changes in tumor microenvironment conditions, and (ii) a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to changes in the tumor microenvironment conditions. [Item A14] The T cell of any one of the above claims, wherein the tumor microenvironment sensing element comprises: (i) an inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to T cell activation binds, (ii) a nuclear localization-binding inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to a decrease in oxygen concentration binds, and (iii) a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading the glucose transporter is inactivated in response to the decrease in oxygen concentration. [Item A15] The T cell of any one of the above items, wherein the nucleic acid sequence encoding the glucose transporter comprises a construct operably linked to an inducible activation motif that is activated in response to changes in tumor microenvironment conditions and / or a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to changes in the tumor microenvironment conditions. [Item A16] The T cell of any one of the above items, wherein the nucleic acid sequence encoding the glucose transporter comprises a construct operably linked to a nuclear translocation-binding inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to T cell activation binds. [Item A17] The T cell of any one of the above items, wherein the nuclear translocation factor is selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families. [Item A18] The T cell of any one of the above items, wherein the nuclear translocation-binding inducible activation motif is selected from the group consisting of an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ ID NO: 1), a STAT binding sequence (TTCNNNGAA), or a modified sequence thereof.[Item A19] The T cell according to any one of the above items, wherein the combination of the nuclear localization factor and the nuclear localization binding-inducible activation motif comprises: (1) a combination of NFAT and an NFAT-binding domain; (2) a combination of NF-κB and a κB motif (GGGACTTTCC (SEQ ID NO: 1)); or (3) a combination of a STAT transcription factor family and a STAT binding sequence (TTCNNNGAA). [Item A20] The T cell according to any one of the above items, wherein the nucleic acid sequence encoding the glucose transporter comprises a construct operably linked to a nuclear localization binding-inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to a decrease in oxygen concentration binds. [Item A21] The T cell according to any one of the above items, wherein the nuclear localization factor is selected from the group consisting of HIF-1. [Item A22] The T cell according to any one of the above items, wherein the nuclear localization binding-inducible activation motif is selected from the group consisting of HIF-RE. [Item A23] The T cell according to any one of the above items, wherein the combination of the nuclear localization factor and the nuclear localization binding-inducing activation motif is selected from the group consisting of: (1) a combination of HIF-1 and HIF-RE. [Item A24] The T cell according to any one of the above items, comprising a construct in which a nucleic acid sequence encoding the glucose transporter is operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter in response to a decrease in oxygen concentration is inactivated. [Item A25] The T cell according to any one of the above items, wherein the factor to be inactivated is selected from the group consisting of oxygen-dependent degradation factors (ODDs). [Item A26] The T cell according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item A27] The T cell according to any one of the above items, wherein the T cell has effector function. [Item A28] The T cell is an effector T cell (T. ｅｆｆThe T cell according to any one of the above items, wherein the effector T cell precursor is a progenitor cell of a T cell that has been activated. [Item A29] The T cell according to any one of the above items, wherein the memory function of the effector T cell precursor is extended. [Item A30] The T cell according to any one of the above items, wherein the memory T cell precursor maintains the phenotype of a memory T cell. [Item A31] The T cell according to any one of the above items, wherein the effector T cell is activated or activity is maintained, and the effector T cell precursor has extended memory function or the memory T cell phenotype is maintained. [Item A32] The T cell according to any one of the above items, wherein the expression and / or enhancement is achieved in a tumor environment-specific manner. [Item A33] The T cell according to any one of the above items, wherein the CAR is expressed in the T cell. [Item A34] The T cell according to any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item A35] The T cell of any one of the above items, wherein the glucose transporter is GLUT3. [Item A36] The T cell of any one of the above items, wherein the T cell is a human T cell. [Item A37] A cell population comprising T cells with enhanced glucose uptake ability on demand. [Item A38] A cell population comprising the T cell of any one of the above items. [Item A39] The cell population of any one of the above items, wherein the T cells have the property of having effector function when introduced into the body. [Item A40] The cell population of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item A41] The cell population of any one of the above items, wherein the T cells are modified such that expression of a glucose transporter is regulated in the T cell in response to changes in tumor microenvironment conditions, and / or comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. [Item A42] The cell population according to any one of the above items, wherein the T cells have effector function. [Item A43] The cell population is T ｅｆｆ[Item A44] The cell population according to any one of the above items, comprising: ｅｆｆ[Item A45] The cell population of any one of the above items, comprising progenitor cells of the T cell. [Item A46] The cell population of any one of the above items, wherein the glucose transporters comprise GLUT1, GLUT2, GLUT3, and GLUT4. [Item A47] The cell population of any one of the above items, wherein the glucose transporter is GLUT3. [Item A48] The cell population of any one of the above items, wherein the T cell is a human T cell. [Item A49] A pharmaceutical composition comprising the T cell of any one of the above items or the cell population of any one of the above items. [Item A50] The pharmaceutical composition of any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item A51] The pharmaceutical composition of any one of the above items, which is for the prevention or treatment of cancer. [Item A52] The pharmaceutical composition according to any one of the above items, which is used to cure cancer. [Item A53] The pharmaceutical composition according to any one of the above items, which is used to prevent cancer metastasis or recurrence. [Item A54] The pharmaceutical composition according to any one of the above items, which is used to prevent or treat cancer to prevent recurrence. [Item A55] A pharmaceutical composition for preventing or treating a disease in a subject, the pharmaceutical composition comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population comprising said T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population comprising said T cells to the subject in an appropriate dosage and administration. [Item A56] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item A57] The pharmaceutical composition according to any one of the preceding items, wherein the T cells comprise a chimeric antigen receptor (CAR).[Item A58] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or an infectious disease. [Item A59] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer. [Item A60] The T cells with enhanced glucose uptake ability have not been modified to regulate the expression of a glucose transporter in response to changes in the tumor microenvironment conditions, and the expression of the glucose transporter is not regulated in response to changes in the tumor microenvironment conditions. ｅｆｆ and a glucose transporter modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. [Item A60A] The T cells with enhanced glucose uptake ability are not modified to include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and are not T cells that do not include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. ｅｆｆand a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and / or a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. [Item A61] The composition of any one of the above items, wherein the glucose transporter is GLUT3. [Item A62] The T cell or cell population of any one of the above items for use as a pharmaceutical. [Item A63] The cell or cell population of any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item A64] The cell or cell population of any one of the above items, which is for the prevention or therapy of cancer. [Item A65] The cell or cell population of any one of the above items, which is for the cure of cancer. [Item A66] The cell or cell population of any one of the above items, which is for the prevention of cancer metastasis or recurrence. [Item A67] The cell or cell population according to any one of the above items, which is used to prevent or treat cancer so that it does not recur. [Item A68] A cell or cell population for preventing or treating a disease in a subject, the cell or cell population being characterized by: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population comprising said T cell, which has an appropriate expression characteristic according to the index; and (C) administering the T cell with enhanced glucose uptake ability or the cell population comprising said T cell to the subject in an appropriate dosage and administration. [Item A69] The cell or cell population according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item A70] The cell or cell population according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR).[Item A71] The cell or cell population according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item A72] The cell or cell population according to any one of the above items, wherein the disease comprises cancer. [Item A73] The T cells with enhanced glucose uptake ability have not been modified to regulate the expression of a glucose transporter in response to changes in the tumor microenvironment conditions, and the expression of the glucose transporter is not regulated in response to changes in the tumor microenvironment conditions. ｅｆｆ and the cell or cell population according to any one of the above items, wherein the glucose transporter has been modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. [Item A73A] The T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and the T cells or cell population according to any one of the above items, wherein the glucose transporter has been modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. [Item A73B] The T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and the T cells or cell population according to any one of the above items, wherein the glucose transporter has been modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. ｅｆｆand a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and / or the cell or cell population comprises a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. [Item A74] The cell or cell population of any one of the above items, wherein the glucose transporter is GLUT3. [Item A75] A method for treating or preventing a subject in need thereof, comprising administering to the subject an effective amount of the T cell or cell population of any one of the above items. [Item A76] The method of any one of the above items, wherein the treatment or prevention in the subject is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item A77] The method of any one of the above items, wherein the treatment or prevention in the subject includes the prevention or treatment of cancer. [Item A78] The method of any one of the above items, wherein the treatment or prevention in the subject includes curing cancer. [Item A79] The method of any one of the above items, wherein the treatment or prevention of the subject includes prevention of cancer metastasis or recurrence. [Item A80] The method of any one of the above items, wherein the treatment or prevention of the subject includes preventing or treating cancer so that it does not recur. [Item A81] A method for preventing or treating a disease in a subject, the method comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate dosage and administration. [Item A82] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item A83] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR).[Item A84] The method according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item A85] The method according to any one of the above items, wherein the disease comprises cancer. [Item A86] The T cells with enhanced glucose uptake ability have not been modified to regulate the expression of a glucose transporter in response to changes in the tumor microenvironmental conditions, and the expression of the glucose transporter has not been regulated in response to changes in the tumor microenvironmental conditions. ｅｆｆ and a glucose transporter modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. [Item A86A] The T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and the T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. ｅｆｆand a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and / or the nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. [Item A87] The method of any one of the above items, wherein the glucose transporter is GLUT3. [Item A88] Use of the T cell of any one of the above items or the cell population of any one of the above items for manufacturing a medicament comprising the cell or cell population. [Item A89] The use of any one of the above items, wherein the medicament is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item A90] The use of any one of the above items, wherein the medicament is for the prevention or therapy of cancer. [Item A91] The use of any one of the above items, wherein the medicament is for the cure of cancer. [Item A92] The use of any one of the above items, wherein the medicament is for the prevention of cancer metastasis or recurrence. [Item A93] The use according to any one of the above items, wherein the medicament is for preventing or treating cancer so that it does not recur. [Item A94] The use according to any one of the above items, wherein the medicament is for preventing or treating a disease in a subject, the use being characterized by: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate dosage and administration method. [Item A95] The use according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item A96] The use according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR).[Item A97] The use according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or an infectious disease. [Item A98] The use according to any one of the above items, wherein the disease comprises cancer. [Item A99] The T cells with enhanced glucose uptake ability have not been modified to regulate the expression of a glucose transporter in response to changes in the tumor microenvironment conditions, and the expression of the glucose transporter is not regulated in response to changes in the tumor microenvironment conditions. ｅｆｆ and the use according to any one of the above items, wherein the glucose transporter has been modified so that its expression is regulated in response to changes in tumor microenvironment conditions, and / or the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions. [Item A99A] The T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and the use according to any one of the above items, wherein the T cells with enhanced glucose uptake ability do not comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. ｅｆｆ and a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, and / or a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, wherein the tumor microenvironment sensing element is modified to include a nucleic acid sequence encoding a glucose transporter operably linked to the tumor microenvironment sensing element, and wherein the tumor microenvironment sensing element senses changes in tumor microenvironment conditions, and / or the tumor microenvironment sensing element is modified to include a nucleic acid sequence encoding a glucose transporter operably linked to the tumor microenvironment sensing element, and wherein the glucose transporter is GLUT3.

[0010] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated, and further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0011] Note that features and significant actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings.

[0012] The present disclosure provides a method for producing effector T cells (T ｅｆｆ ) can be provided, and by using such cells, chimeric antigen receptor (CAR) T cells that function without starvation or exhaustion even in a tumor environment can be provided.

[0013] The cells disclosed herein can be described as metabolically enhanced T cells, which utilize the mechanisms by which tumor cells survive in the tumor environment. The cells disclosed herein are capable of maintaining effective cytotoxic activity and cytokine production in low-glucose environments, such as tumor microenvironments, a feat not achievable with conventional immune checkpoint inhibitor therapy, and thus can exert a high antitumor effect in vivo. Furthermore, by using an on-demand glucose transporter expression system that expresses glucose transporters in response to increased glucose demand, it is possible to avoid hyperdifferentiation and exhaustion of T cells due to excessive activation, maintain long-term efficacy, and induce memory phenotype T cells that are significantly effective in preventing recurrence. Such on-demand expression can be achieved by regulating glucose transporter expression in the T cells in response to changes in tumor microenvironment conditions, or by including a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

[0014] Figure 1 shows an example of the structure of a metabolically engineered anti-EGFRvIII CAR (3C10 CAR_GLUT3) lentiviral vector plasmid. A single-chain fragment (scFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII, which is expressed in glioblastoma (GBM), etc., was constructed and linked to CD8 hinge, CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BBICD, and CD3z to construct a CAR. A high-affinity glucose transporter (GLUT3) was encoded by the CAR via a P2A sequence. The figure shows an example of the same 3C10 CAR_GLUT3 incorporated into a third-generation lentiviral vector plasmid containing the EF-1a promoter. Figure 2 shows enhanced T cell surface GLUT3 expression and glucose uptake. The upper panel shows the structures of the conventional CAR (3C10 CAR) and the metabolically engineered CAR (GLUT3 CAR). The lower left shows images of CAR and GLUT3 expression on T cells for 3C10 CAR and GLUT3 CAR under a fluorescence microscope. The lower middle shows GLUT3 expression quantified by fluorescence intensity. The lower right shows the results of quantifying intracellular glucose uptake using the 2-NBDG method. The conventional CAR (3C10 CAR) and the metabolically engineered CAR (GLUT3 CAR) were transfected into T cells derived from healthy donors to obtain CAR-T cells. Fluorescence microscopy confirmed homogeneous GLUT3 expression on the GLUT3 CAR-T cell membrane. Furthermore, GLUT3 CAR-T cells exhibited enhanced glucose uptake compared to T cells and conventional CAR-T cells (3C10 CAR-T cells). Figure 3 shows results demonstrating T cell expansion efficiency equivalent to that of conventional CAR-T cells. The X axis represents the number of days after transduction, and the Y axis represents LOG2 (FC). Peripheral blood mononuclear cells (PBMCs) containing T cells were stimulated with anti-CD3 / 28 beads, followed by CAR gene transduction, and T cell proliferation efficiency was analyzed. Under both normal glucose conditions (10 mM) and low glucose conditions (0.5 mM), GLUT3 CAR-T cells had establishment efficiency equivalent to that of conventional CAR-T cells (3C10 CAR-T cells). Figure 4 shows results demonstrating enhanced T cell glycolytic capacity due to GLUT3 expression.The graph on the left shows the glycolytic capacity of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu conc. 10 mM) and low glucose conditions (Glu conc. 0.5 mM). The graph below shows the glycolytic capacity of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu conc. 10 mM). The left panel on the right shows glycolysis values ​​observed after the addition of glucose, and the right panel shows glycolytic capacity values ​​observed after the addition of oligomycine. The metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Fluxanalyzer. The GLUT3 CAR had enhanced glycolytic activity compared to the 3C10 CAR in both low-glucose and normal-glucose environments. Figure 5 shows results demonstrating that GLUT3 CAR-T cells have a competitive advantage under low glucose conditions. The left panel shows the experimental procedure. The middle panel shows the cell count ratios of 3C10 CAR-T cells and GLUT3 CAR-T cells on days 0 and 3. The right panel shows the ratio of 3C10 CAR-T cells to GLUT3 CAR-T cells on day 3. 3C10 CAR-T cells (unlabeled) and GLUT3 CAR-T cells (CSFE-labeled) were co-cultured under normal glucose and low glucose conditions. Both cells showed comparable survival under normal glucose conditions, but significant survival of GLUT3 CAR-T cells was observed under low glucose conditions. Figure 6 shows results demonstrating the enhanced cytokine production ability of GLUT3 CAR-T cells. The left panel shows intracellular IFNg and IL-2 analysis of CD8-positive cells at the top, and the amount of intracellular IFNg and IL-2 at the bottom of CD4-positive cells. The right panel shows the percentage of cells positive for IFNγ, IL-2, and TNF-α, respectively. After antigen stimulation of 3C10 CAR-T cells and GLUT3 CAR-T cells, the percentage of cytokine-producing cells was analyzed by FCM. Significant increases in the expression of cytokines (IFN-g, IL-2, TNF-α) that are important for T cell maintenance and activation and the exertion of antitumor activity were observed. Figure 7 shows results demonstrating that the function of GLUT3 CAR-T cells is maintained even under low glucose conditions.The panels, starting from the left, show the percentage of cells producing IFNγ, IL-2, and TNF-α under normal glucose conditions (10 mM) and low glucose conditions (0.5 mM), respectively. Cytokine production capacity under normal glucose and low glucose was compared. Conventional CAR-T cells (3C10 CAR-T cells) showed an extreme decrease in function under low glucose. On the other hand, GLUT3 CAR-T cells showed high cytokine production capacity under normal glucose, and even under low glucose, they exhibited function comparable to the cytokine production capacity of 3C10 CAR-T cells under normal glucose. Figure 8 shows the results demonstrating the enhanced cytotoxic activity of GLUT3 CAR-T cells. The left panel shows the results of the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against the U87d cell line at a glucose concentration of 10 mM, and the right panel shows the results of the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against the U87d cell line at a glucose concentration of 0.5 mM. Cytotoxic activity was analyzed using an EGFRvIII-expressing cell line as a target. Cytotoxic activity was analyzed under low glucose and normal glucose conditions, and under both conditions, GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells. Figure 9 shows the results showing the properties of GLUT3 CAR that confer T cell memory phenotype. The left panel shows the expression of CD45 and CD27 before (preco-culture) and after (co-culture) co-culture with the U87d tumor cell line, and the right panel shows, from left to right, the percentage of CD45RA-positive and CD27-negative fractions before and after co-culture. The memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. A stronger tendency for differentiation into effector T cells was observed in GLUT3 CAR-T cells compared with 3C10 CAR. Figure 10 shows the suppression of inhibitory molecule expression in GLUT3 CAR-T cells. From left to right, data for PD-1, LAG3, and TIM3 are shown. The expression of inhibitory molecules under stimulation with EGFRvIII antigen-positive cells was compared. In GLUT3 CAR-T cells, suppression of PD-1, LAG3, and TIM3 expression was observed. Figure 11 shows mRNA analysis (evaluation of metabolism, exhaustion, activation, and differentiation) of GLUT3 CAR-T cells.The upper left panel shows, from left to right, PDCD1 expression, LAG3 expression, and TIM3 expression. The upper right panel shows, from left to right, enrichment plots related to lactate metabolism and glucose metabolism. The lower panel shows factors with increased expression based on gene ontology. Detailed analysis of 3C10 CAR-T and GLUT3 CAR-T cells was performed using mRNA expression. Similar to FCM, GLUT3 CAR-T cells showed a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors. These factors are associated with enhanced T cell effector functions, such as activation and cytokine production. Figure 12 demonstrates the generality of this gene, demonstrating that similar effects can be achieved not only with the 3C10 CAR but also with the CD19 CAR. The left panel shows an experiment using the 3C10 CAR, and the right panel shows an experiment using the CD19 CAR. In each panel, the upper left shows intracellular IL-2, IFNg, and TNF-a analysis of mock-transfected T cells, 3C10 CAR-T cells, and GLUT3 CAR-T cells, the upper right shows the percentage of IFNg-, IL-2-, and TNFa-positive cells (from left) for 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (10 mM), and the lower left shows the percentage of IFNg-, IL-2-, and TNFa-positive cells (from left) for 3C10 CAR-T cells and GLUT3 CAR-T cells under low glucose conditions (0.5 mM). The lower right shows the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against (from left) U87d and U251MGd cell lines under low glucose conditions (0.5 mM). We confirmed that the same effect as the 3C10 CAR was obtained when GLUT3 was loaded onto the CD19 CAR. It was also shown to be effective against pancreatic cancer cells in which CD19 was expressed in a pancreatic cancer cell line. Figure 13 shows that rapid tumor eradication was achieved in an intracranial xenograft model. The upper left panel shows a simple scheme of the experiment. Briefly, 2.5 x 10 cells were intracranially injected into immunodeficient NSG mice. ４ U87d tumor cell line was transplanted, and 5 days later, 1x10 ６This is a model in which CAR-T cells are administered via the tail vein and tumor burden is quantified over time by bioluminescence imaging (BLI). The middle left panel shows tumor imaging images on days 0 and 11. The graph on the bottom left shows luminescence intensity (reflecting tumor burden) over time. The middle-middle panel shows Kaplan-Meier curves for each treatment group. Below the middle panel, tumor imaging images on days 34, 38, and 42 in tumor-rechallenged mice are shown. The right column shows peripheral blood CD3 ＋Figure 14 shows the number of T cells. Antitumor activity was observed in a U87Δ intracranial xenograft model. The GLUT3 CAR-T cell treatment group demonstrated more rapid tumor eradication, improved survival, and significant T cell expansion compared to the 3C10 CAR-T cell treatment group. Furthermore, tumor rejection was confirmed by tumor rechallenge in mice cured with the GLUT3 CAR-T cell group. Figure 14 shows an overview of on-demand GLUT3-expressing CAR-T cells. Under steady-state conditions, only CAR is expressed, but GLUT3 is not. A schematic diagram of this system is shown, in which GLUT3 is expressed upon increased glucose demand, such as during CAR-T cell activation or in the tumor environment. Figure 15 shows the design of an inducible GLUT3 expression system. P indicates constitutive expression, A indicates NFATi-GLUT3, B indicates HIF1i-GLUT3, and B' indicates a hypoxia-sensing alternative version of HIF1i-GLUT3. The symbols in the schematic diagram represent the EF-1 promoter (EF-1a), anti-EGFRvIIICAR (EGFRvIII CAR = 3C10 CAR), P2A sequence (P2A), NFAT response element (NFAT-RE), HIF1a response element (HIF-RE), high-affinity glucose transporter (GLUT3), and oxygen-dependent degradation domain (ODD). The asterisk (*) indicates the stop codon. CAR constructs were designed to express GLUT3 in a tumor-environment-specific or activation-specific manner. This enables the expression of GLUT3 upon sensing activation-induced NFAT elevation (A) and tumor hypoxia (B and B'). A CAR construct was designed that expresses GLUT3 in a tumor environment-specific or activation-specific manner. This enables the expression of GLUT3 upon sensing activation-induced NFAT elevation (A) and tumor hypoxia (B). In A, GLUT3 is encoded via a minimal promoter (MP) under an octa-repeat NFAT-RE, to which NFAT translocated into the nucleus upon T cell activation binds and induces transcription, independently of a constitutively expressed CAR (e.g., EGFRvIIICAR) driven by the EF-1a promoter.In B, GLUT3 is encoded via a minimal promoter under an octa-repeat HIF-RE, which similarly encodes GLUT3 independently of CAR and to which HIF-1a, which translocates into the nucleus under hypoxic conditions in T cells, binds and induces transcription. In B', a GLUT3-ODD element that degrades GLUT3 in the presence of oxygen is encoded via a P2A sequence directly downstream of CAR driven by the EF-1a promoter. Figure 16 shows the structure of the above-mentioned NFATi-GLUT3 CAR when incorporated into a lentiviral vector plasmid as an example. Details of the structure are described in the Examples section. This is an all-in-one lentiviral vector plasmid incorporating 3C10 CAR, which is constitutively driven by the EF-1a promoter, and GLUT3, which is driven in an NFAT nuclear import-dependent manner by eight repeats of the NFAT binding site and a minimum promoter, in a single plasmid. Figure 17 shows the operation and basic functions of the inducible GLUT3 CAR system. The structure of the NFAT-inducible GLUT3 CAR is shown in the upper left. The center left panel shows fluorescent microscopic images of CAR and GLUT3 expression in T cells under non-stimulation (pre-stimulation) and stimulation (post-stimulation). The lower left panel shows, from the right, IFN-γ, TNFα, and IL-2 after stimulation with the U87d cell line. The middle right panel shows a flow plot of the intracellular expression intensities of IFN-γ, IL-2, and TNFα in each CAR-T cell under stimulation with the U87d cell line. The bottom right panel shows a graph demonstrating that under glucose conditions (0.5 mM), the cytotoxic activity was stronger than that of 3C10 CAR-T cells and equivalent to that of GLUT3 CAR-T cells. A system that expresses GLUT3 in conjunction with NFAT nuclear translocation was constructed (NFATi GLUT3 CAR-T), and its operation was confirmed. The cytotoxic activity of NFATiGLUT3 CAR-T cells (NFATi GLUT3 CAR-T) was equivalent to that of constitutively expressing GLUT3 CAR-T cells (GLUT3 CAR-T), and cytokine production was intermediate between that of 3C10 CAR-T (without GLUT3) and constitutively expressing CAR-T cells (GLUT3 CAR-T).Figure 18 shows the evaluation of the antitumor effects of mock T cells, 3C10 CAR-T cells, GLUT3 CAR-T cells, and NFATiGLUT3 CAR-T cells in a mouse model similar to that shown in Figure 15. A schematic diagram of the experiment is shown in the upper left. A Kaplan-Meier survival curve is shown in the upper right. The bottom shows the time-dependent changes in tumor burden quantified by bioluminescence imaging (BLI). Four CRs were obtained with GLUT3 CAR-T and two with NFATiGLUT3 CAR-t. Figure 19 shows tumor challenge and tumor rechallenge experiments (sudden death of mice in the GLUT3 CAR-T cell group). The left panel shows tumor burden by bioluminescence imaging on day 1 (post-challenge), day 5 of rechallenge, and day 12 of rechallenge, demonstrating survival of 3C10 CAR-T, GLUT3 CAR-T, and NFAT CAR-T, respectively. Areas lacking BLI images indicate dead mice. Cured mice from the previous experiment were re-implanted with tumors (U87d) to assess rejection potential (a "surrogate for memory formation"). 1x10 ５Tumor cells were re-implanted, and tumor rejection was not achieved with 3C10 CAR-T, but was achieved with GLUT3 CAR-T and NFATiCAR-T. However, three out of four mice in the GLUT3 CAR-T group died (non-tumor amplification death). Figure 20 shows the suppression of apoptosis after stimulation with NFATiGLUT3. The left panel shows staining for cell death and apoptosis markers 7-AAD and Annexin V in Mock T cells, 3C10 CAR-T cells, GLUT3 CAR-T cells, and NFATi CAR-T cells in an unstimulated state (top row of the left panel), 7 days after the first stimulation with U87d cell line (middle row), and 7 days after the second stimulation with U87d cell line (bottom row). The graphs on the right panel show, from top to bottom, the percentage of late apoptotic cells, pre-apoptotic cells, and viable cells for each CAR-T cell type 7 days after the first stimulation and 7 days after the second stimulation. CAR-T cells were repeatedly stimulated with U87d, and CAR-T cell apoptosis was observed using Annexin V and 7-ADD. GLUT3 has an inhibitory effect on apoptosis (considered to be the sum of activation-induced cell death and cell death resulting from terminal differentiation). NFAT CAR-T is suggested to have a stronger inhibitory effect on apoptosis than GLUT3 CAR-T. Figure 21 shows enhanced long-term survival after stimulation. Each CAR-T cell type was observed over time under the above conditions. This figure shows the in vitro (CAR) T cell stimulation and culture test plan. Non-CAR-transfected T cells (UTD), 3C10 CAR-T cells, GLUT3 CAR-T cells, and NFATi CAR-T cells were evaluated, with an unstimulated group and a stimulated group (stimulated with U87d cell line or EGFRvIII magnetic beads). Furthermore, each group was cultured under glucose-free conditions (0 mM), low glucose conditions (0.5 mM), and normal glucose conditions (10 mM) (36 groups in total). The analyses shown in the schedule on the right were performed over time for each group. Under low glucose conditions, normal CAR-T (3C10) did not exhibit effective cytokine production, whereas GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibited effective cytokine production.However, excessive glucose uptake in GLUT3 CAR-T leads to terminal differentiation into CCR7-negative Tem or Temra over time (day 3 or day 7), loss of stemness, and apoptosis. On the other hand, apoptosis is suppressed in on-demand NFATi GLUT3 CAR-T cells, and CCR7-positive Tcm or Tscm (memory cells, cells with stemness) are maintained. When CAR-T cells (established with normal glucose) are suddenly placed under low glucose conditions of 0 mM or 0.5 mM (day 0), the highly glucose-dependent GLUT3 CAR-T are prone to apoptosis (⇔NFATi GLUT-3 CAR-T cells are resistant to low glucose). Figure 22 shows enhanced long-term survival after stimulation. The antitumor effect, survival, and adverse events were examined using the same mouse system as described above. This figure demonstrates enhanced long-term survival in vivo. The antitumor effect, survival, and adverse events were examined using the same mouse system as described above. U87d tumor cell lines were implanted intracranially into immunodeficient NSG mice. Five days later, mock-transduced T cells (UTD), 3C10 CAR-T cells, GLUT3 CAR-T cells, or NFATi CAR-T cells were administered intravenously via the tail. Antitumor effect quantification and survival were analyzed using bioluminescence imaging. In Experiment 1, GLUT3 CAR-T and NFATi GLUT3 CAR-T showed comparable antitumor activity. In Experiment 2, glucose exposure caused overactivation of GLUT3 CAR-T, leading to apoptosis and hyperdifferentiation, resulting in the loss of antitumor activity. On the other hand, excessive activation of NFATiCAR-T cells is suppressed, resulting in effective antitumor effects. Even in rechallenge, NFATiCAR-T cells reject tumors. In Experiment 4, superior CAR-T cell engraftment and tumor T cell infiltration were observed in NFATiGLUT3, in proportion to tumor efficacy. Experiment 3 was an exploratory experiment using a liver tumor model in which immunosuppression due to a low glucose, high lactate environment has been suggested, and is expected to produce results similar to those of Experiment 2. Figure 23 shows data (in vitro data) demonstrating enhanced long-term survival after stimulation.The three left panels show the results on day 1, the three middle panels show the results on day 3, and the three right panels show the results on day 7. Each panel shows 0 mM, 0.5 mM, and 10 mM from left to right, and from the top row shows the results for mock, 3C10 CAR-T, and GLUT3 CAR-T. At each time point, staining data for 7-AAD and Annexin V, which reflect cell death and apoptosis, are shown. Cell death in each CAR-T cell type was examined with Annexin V and 7-AAD. In the unstimulated state, differences in glucose concentration were observed, but no significant differences were observed among each CAR-T cell type. Figure 24 shows in vitro data demonstrating enhanced long-term survival after stimulation. The three left panels show results from day 1, the three middle panels show results from day 3, and the three right panels show results from day 7. Each panel shows 0 mM, 0.5 mM, and 10 mM from left to right, and from the top to bottom, the results for mock, 3C10 CAR-T, and GLUT3 CAR-T. At each time point, staining data for 7-AAD and Annexin V, which reflect cell death and apoptosis, are shown. The survival and cell death of each CAR-T cell after stimulation were evaluated in the same manner as in the previous figure. Compared to 3C10 CAR and GLUT3 CAR-T cells, NFATi GLUT3 CAR-T cells had enhanced survival (suppressed cell death). Figure 25 shows in vitro data on enhanced long-term survival after stimulation. The three left panels show the results from day 1, the three middle panels show the results from day 3, and the three right panels show the results from day 7. In each panel, glucose concentrations of 0 mM, 0.5 mM, and 10 mM are shown from left to right, and from the top to bottom, the results are for mock, 3C10 CAR-T, and GLUT3 CAR-T. The percentage of CAR-expressing cells at each time point is shown. The CAR-positive percentage of each CAR-T cell type after stimulation was analyzed. After stimulation, the percentage of CAR-positive cells decreased, particularly in constitutively expressing GLUT3 CARCAR-T, suggesting that this fraction underwent cell death. Figure 26 shows the enhanced long-term survival after stimulation. The number of T cells on day 7 is shown.As a model reflecting post-administration kinetics in the patient's body (the kinetics of infiltration from blood with sufficient glucose concentration into a low- to no-glucose tumor environment after administration), cells were established and cultured under normal glucose conditions (10 mM glucose), and then stimulated with EGFRvIII-conjugated beads. Subsequently, cells were cultured under glucose-free (0 mM), low-glucose (0.5 mM), or normal glucose (10 mM) conditions, and the viable cell count after 7 days was assessed. The graph shows the relative cell count, with the number of mock T cells defined as 1. Most GLUT3 CAR-T cells died at 0 mM. On the other hand, many NFATi CAR-T cells remained viable. These results suggest that cell death due to overactivation caused by constitutive GLUT3 expression and a highly glucose-dependent metabolism may have led to cell death. Figure 27 shows the expression of an inhibitory molecule (PD-1). The three left panels show the results of PD-1 expression on day 1 of culture in glucose-free (0 mM), low glucose (0.5 mM), or normal glucose (10 mM) conditions, and the three right panels show the results on day 3 of culture. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from the left, and from the top row, the results are for Mock, 3C10 CAR-T, and GLUT3 CAR-T. NFATi GLUT3 CAR-T cells had lower expression of the inhibitory molecule (PD-1) than GLUT3 CAR-T cells. Figure 28 shows the suppression of expression of the inhibitory molecule (TIM3). The three left panels show the results of TIM3 expression on day 1 of culture under glucose-free (0 mM), low glucose (0.5 mM), or normal glucose (10 mM) conditions, and the three right panels show the results on day 3 of culture. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from the left, and from the top row, the results are for Mock, 3C10 CAR-T, and GLUT3 CAR-T. NFATi GLUT3 CAR-T cells had lower expression of the inhibitory molecule (TIM3) than GLUT3 CAR-T cells. Figure 29 shows that low glucose conditions impair the function of CAR-T cells. (a-c) Interstitial fluid was collected by low-speed centrifugation from surgical or biopsy specimens of patients with glioblastoma (GBM), non-small cell lung cancer, and colon cancer (n=5 each), and glucose concentrations were measured. (a) Experimental scheme. (b) Glucose concentrations in interstitial fluid and paired serum samples from GBM specimens. Data are shown as mean ± SEM.Statistical analysis by Student's t-test: ***, P<0.001. (c) Glucose concentrations in interstitial fluid of GBM, non-small cell lung cancer (lung cancer), and colorectal cancer (colorectal cancer) specimens. Data are shown as mean ± SEM. Statistical analysis by one-way analysis of variance: *, P<0.05. (d, e) Mock-transduced T cells (Mock T cells) and conventional EGFRvIII CAR-T cells (conventional EGFRvIII CAR-T cells) were prepared from PBMCs of healthy subjects (n=5). (d) Cytokine production in mock-transformed T cells (Mock T) and conv EGFRvIII CAR-T cells (conv CAR-T) upon stimulation with EGFRvIII-expressing tumor cells (U-87mGΔ) under low glucose (0.5 mM) or high glucose (10 mM) conditions was examined by intracellular cytokine staining. Representative contour plots (left) and summaries of the frequencies of cytokine-producing cells (IFN-γ, IL-2, and TNFα) are shown (right). Data are presented as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01. (e) Cytokine (IFN-γ, IL-2, TNFα) production by conv EGFRvIII CAR-T cells cocultured with U-87mGΔ cells for 16 hours under low glucose (0.5 mM) or high glucose (10 mM) conditions was measured by ELISA. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001; ***, P<0.0001. (f) Gene expression in EGFRvIII CAR-T cells 24 hours after stimulation with EGFRvIII beads under low glucose (0.5 mM) or high glucose (10 mM) conditions. Pathways enriched under low glucose conditions compared to high glucose conditions are shown according to the Molecular Signatures Database. (g) Gene expression of selected genes is shown in a heatmap. Gene expression was normalized by row. All experiments were performed at least twice. Figure 30 shows that overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells and increases cytotoxicity and cytokine production under low glucose conditions.(a) Constructs of conventional EGFRvIII CAR (conv CAR, top) and EGFRvIII CAR stably expressing GLUT3 (GLUT3 CAR, bottom). (b) Immunofluorescence staining of conv EGFRvIII CAR-T cells (conv CAR-T) and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T). The white dotted circle indicates T cells not transduced with CAR. Katyusha (red), 4,6-diamino-2-phenylindole (DAPI: blue), GLUT3 (green). (c) Expression levels of GLUT1 (left) and GLUT3 (right) in CAR-T cells. A summary of mean fluorescence intensity (MFI) is shown. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ****, P<0.0001. (d) Glucose uptake by CAR-T cells measured by glucose analog 2-NBDG uptake assay. Representative histograms (left) and mean fluorescence intensity (MFI) summaries (right) are shown. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; **, P<0.01. (e, f) Glycolytic capacity (e) and metabolic phenotype (f) of CAR-T cells under low glucose (0.5 mM) conditions as determined by flux analyzer. Data are presented as mean ± SD. (g, h) CAR-T cells were prepared from PBMCs of healthy donors (n=5). (g) Cytokine production upon stimulation of EGFRvIII-expressing tumor cells (U-87mGΔ) under low glucose (0.5 mM) conditions was examined by intracellular cytokine staining. Representative contour plots (left) and a summary of the frequency of cytokine (IFN-γ, IL-2, TNFα)-producing cells (right) are shown. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; *, P<0.05; **, P<0.01; ****, P<0.0001. (h) Cytokine (IFN-γ, IL-2, TNFα) production by CAR-T cells cocultured with U-87mGΔ cells under low glucose (0.5 mM) conditions for 16 hours was measured by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ****, P<0.0001. (i) Cytotoxicity of CAR-T cells under low glucose (0.5 mM) conditions was measured by a luciferase-based assay.CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc) were cocultured for 24 hours at the indicated effector-to-target (E:T) ratios, and specific lysis was calculated based on luciferase activity. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; **, P<0.01. (j, k) Expression of exhaustion markers (PD-1 and Tim-3) by CAR-T cells. PD-1. ＋ CAR-T cells (j) and PD-1 ＋ Tim-3 ＋Figure 31 shows a summary of the frequency of the CAR-T cell (k) population. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ****, P<0.0001. Figure 31 shows experimental results in an intracranial human GBM cell xenograft model. In this model, stable GLUT3 expression in CAR-T cells did not improve survival due to severe toxicity. (a) Experimental schema. NSG mice (n=6) intracranially inoculated with U-87mGΔ-Luc cells were intravenously administered either mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), or GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) on day 5 after tumor inoculation. Mice in which U-87mGΔ-Luc tumors were eradicated by treatment with conv EGFRvIII CAR-T cells or GLUT3 EGFRvIII CAR-T cells were re-challenged with the same tumor on day 35. Tumor burden was monitored by BLI. (b) Tumor burden measured by BLI. Each line represents an individual mouse treated with the indicated CAR-T cells. Crosses indicate the cause of death. Numbers in the upper right panel indicate the number of mice that achieved complete response (CR) by day 35 and the number of mice that died. The horizontal dotted line indicates the background BLI signal level obtained from mice not inoculated with tumor. (c) Kaplan-Meier curves. Statistical analysis of the Kaplan-Meier curves using the log-rank test. (d) Body weight change of each mouse during CAR-T cell treatment. Crosses indicate death from any cause, and colored dotted lines indicate mice that experienced treatment-related mortality (TRM). (e) Stacked bar graph showing the results at the end of the observation period. TRM; treatment-related mortality. (f, g) Pathological analysis of brain and lung tissues of mice that showed severe weight loss on day 14. (f) Human CD3 in brain tissue. ＋ IHC staining of cells. (g) Hematoxylin and eosin (HE) staining in lung tissue (left: low magnification, middle: high magnification) and human CD3 　 Staining (right). The arrow indicates the hyaline membrane. (h) Phenotype of circulating human T cells (CAR-T cells) 14 days after CAR-T cell administration. Representative contour plot (left) and effector memory (CD45RA CCR7 －－) A summary of T cell frequencies (right) is shown. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05. (i) Comparison of gene expression between conv EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells 7 days after stimulation with EGFRvIII beads under low (0.5 mM) glucose conditions. Pathways enriched in GLUT3 EGFRvIII CAR-T cells compared to conv EGFRvIII CAR-T cells are shown according to the Molecular Signatures Database. Figure 32 shows that on-demand expression of GLUT3 confers superior effector activity to CAR-T cells. (a) A construct (on-demand GLUT3 CAR) in which GLUT3 expression is driven by NFAT binding to the NFAT-binding domain (NFAT-BD), enabling on-demand GLUT3 expression in CAR-T cells. BD: Binding domain. (b) Immunofluorescence imaging of on-demand GLUT3 EGFRvIII CAR-T cells for the CAR transduction markers Katyusha (red), 4,6-diamino-2-phenylindole (DAPI: blue), and GLUT3 (green). (c) Time course of GLUT3 expression in conv EGFRvIII CAR-T cells (conv CAR-T) and on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T). Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; t***, P<0.0001. MFI, mean fluorescence intensity. (d) Glucose uptake capacity measured by glucose analog 2-NBDG uptake assay. A representative histogram (left) and MFI summary (right) are shown. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001. (e, f) The glycolytic capacity (e) and metabolic phenotype (f) of CAR-T cells cultured under low (0.5 mM) glucose conditions were examined by a flux analyzer on days 3 and 7 after stimulation with EGFRvIII beads, as indicated. Data are presented as mean ± SD. (g, h) CAR-T cells were prepared from PBMCs of healthy donors (n=5).(g) Cytokine production following stimulation with EGFRvIII-expressing tumor cells (U-87mGΔ) under low glucose (0.5 mM) conditions, as determined by intracellular cytokine staining. A representative contour plot (left) and a summary of the frequency of cytokine (IFN-γ, IL-2, TNFα)-producing cells (right) are shown. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ***, P<0.001; ****, P<0.0001. (h) Cytokine (IFN-γ, IL-2, TNFα) production by CAR-T cells cocultured with U-87mGΔ cells under low glucose (0.5 mM) conditions for 16 hours was measured by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ****, P<0.0001. (i) The cytotoxicity of CAR-T cells against U-87mGΔ and U-251mGΔ cells under low (0.5 mM) glucose conditions was measured by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc or U-251mGΔ-Luc) were cocultured for 24 hours at the indicated effector-to-target (E:T) ratio under low (0.5 mM) glucose conditions, and specific lysis was calculated based on luciferase activity. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Figure 33 shows that on-demand expression of GLUT3 results in the reduction of differentiated T cells and improves T cell survival by avoiding overactivation. (a) Apoptotic status measured by Annexin V and 7-AAD staining of the indicated CAR-T cell populations [Mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), or on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T)] 72 hours after antigen stimulation under low glucose (0.5 mM) conditions. Representative contour plots (left) and live cells (Annexin V) are shown. － 7-AAD － ), Pre-apoptosis (Annexin V＋ 7-AAD － ), Apoptosis (Annexin V ＋ 7-AAD ＋ (b) A summary of the frequency of cell populations (right) is shown. (b) Heat map of gene expression related to apoptosis and T cell activation in CAR-T cells 72 hours after stimulation with EGFRvIII beads under low (0.5 mM) glucose conditions. Gene expression was normalized by row. (c) Comparison of gene expression between conv EGFRvIII CAR-T cells (conv CAR-T) and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) 72 hours after stimulation with EGFRvIII beads under low (0.5 mM) glucose conditions. Pathways enriched in GLUT3 EGFRvIII CAR-T cells are compared with conv EGFRvIII CAR-T cells, demonstrating their interrelationships. (d, e) CAR-T cells were prepared from PBMCs of healthy donors (n=5) and stimulated with EGFRvIII-expressing tumor cells (U-87mGΔ) under low (0.5 mM) glucose conditions. (d) T cell phenotype (CD62L) of the indicated CAR-T cell populations. ＋ CD45RA ＋ :naive / stem cellmemory;CD62L ＋ CD45RA － :centralmemory;CD62L － CD45RA － :effector memory; and CD62L － CD45RA ＋ : terminally differentiated effector memory). Representative contour plot (left) and naive / stem cell memory T cells (CD62L ＋ CD45RA ＋ ) A summary of the population frequencies (right) is shown. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.01; ***, P<0.0001. (e) Expression of exhaustion markers (PD-1 and Tim-3) by CAR-T cells. PD-1 ＋ Tim-3 ＋A summary of the frequencies of CAR-T cell populations is shown. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; ****, P<0.0001. (f) Comparison of gene expression between GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) and on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) after stimulation with EGFRvIII beads under low (0.5 mM) glucose conditions. Principal component analysis of the RNA-seq transcriptome profiles of GLUT3 EGFRvIII CAR-T cells and on-demand GLUT3 EGFRvIII CAR-T cells on days 3 and 7 post-stimulation. (g) Heatmap of metabolic scores of CAR-T cells analyzed by RNA-seq on day 7 after stimulation with METAFlux. Gene expression was normalized by row. FA; fatty acids. Figure 34 shows on-demand GLUT3 expression by CAR-T cells. On-demand GLUT3 expression by CAR-T cells induces durable anti-tumor effects with a significant survival benefit in an intracranial human GBM cell xenograft model. (a) Experimental schema. NSG mice (n=10, excluding the mock T cell group; n=5) intracranially inoculated with U-87mGΔ-Luc cells were intravenously administered either mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), or on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) on day 5 after tumor inoculation. Mice in which U-87mGΔ-Luc tumors were eradicated by treatment with conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, or on-demand GLUT3 EGFRvIII CAR-T cells were re-challenged with the same tumor on day 21. Tumor burden was monitored by BLI. (b) Tumor burden measured by BLI. Each line represents an individual mouse treated with the indicated CAR-T cells. Crosses indicate the cause of death. Numbers in the upper right panel indicate the number of mice that achieved a complete response (CR) by day 21 and the number of mice that died.The horizontal dotted line indicates the background BLI signal level obtained from mice not inoculated with tumors. (c) Body weight change of each mouse during CAR-T cell therapy. Crosses indicate death from any cause, and colored dotted lines indicate mice with treatment-related mortality (TRM). (d) Mouse body weight 42 days after CAR-T cell infusion. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05. (e) Kaplan-Meier curves of survival time of tumor-bearing U-87mGΔ mice treated with the indicated CAR-T cells (n=10, excluding the mock T cell group; n=5). Statistical analysis of Kaplan-Meier curves by log-rank test; ns, not significant; *, P<0.05. (f) Phenotype of circulating human T cells 14 days after CAR-T cell administration. Representative contour plots (left) and each T cell population [CD45RA CCR7. ＋＋ :naive / stem cell memory (SCM), CD45RA CCR7 -＋ : central memory (CM), CD45RA CCR7 -－ : effector memory (EM), and CD45RA CCR7 ＋－(g) Kaplan-Meier curves for the survival rate of mice bearing U-251mGΔ cells treated with the indicated CAR-T cells (n=10, excluding the mock T cell group; n=5). Statistical analysis of Kaplan-Meier curves by log-rank test; ns, not significant; *, P<0.05. (h) Cytokine (TNFα and GM-CSF) concentrations in serum on day 14 after CAR-T cell administration. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05. (i) The rate of decrease in serum TNFα and GM-CSF on day 14 relative to day 7 after CAR-T cell administration. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ns, not significant; *, P<0.05. The results in Figure 34 demonstrate that stably expressed GLUT3 enhances the antitumor effect (i.e., effector function) of EGFRvIII CAR-T cells. On-demand GLUT3 expression not only enhances the effector function of EGFRvIII CAR-T cells, but also extends memory function and enhances the maintenance of the memory T cell phenotype. Furthermore, it can be concluded that stably expressed GLUT3 prevented adverse events such as weight loss and treatment-related death, resulting in a significant improvement in survival rate, including cure. Figure 35 shows that low glucose conditions impair CAR-T cell function. (a) Volcano plot showing differentially expressed genes in conv EGFRvIII CAR-T cells cultured under low glucose (0.5 mM) conditions compared to high glucose (10 mM) conditions. X-axis is log ２ (fold change) (log ２ FC), and the Y axis is -log １０ (adjusted p value) [(-log １０ (padj)) ]. The dotted line represents log ２ (fold change) cutoff value, x = ±1.4 and -log １０The cutoff value for the adjusted p value, y = 0.1, is shown. The top 5 and bottom 5 genes are highlighted in green and purple, respectively. Figure 36 shows that GLUT3 overexpression enhances the metabolic fitness of CAR-T cells. (a) Expression of GLUT1 (left) and GLUT3 (right) in conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), U-87mGΔ cells, and U-251mGΔ cells. Representative histograms are shown. (b) Glycolytic parameters of CAR-T cells cultured under low glucose (0.5 mM) conditions were measured by a glycolytic stress test. Data are shown as mean ± SD. Statistical analysis was performed by Student's t-test; ns, not significant; ***, P<0.001. (c, d) Competition assay of CAR-T cells under low glucose (0.5 mM) or high glucose (10 mM) conditions (72-hour culture). (c) Experimental schema (n=3). (d) Representative contour plot (left) and summary of the proportion of each CAR-T cell population (right). Data are shown as mean ± SEM. Statistical analysis was performed by Student's t-test; ***, P<0.001. Figure 37 shows that stable expression of GLUT3 improves the function of CAR-T cells under low glucose conditions. (a-c) Cytokine production by mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) (n=5) upon stimulation with EGFRvIII-expressing tumor cells (U-87mGΔ) under high glucose (10 mM) conditions was examined by intracellular staining. (a) CD3 ＋ Summary of the frequency of cytokine (IFN-γ, IL-2, TNFα) producing CAR-T cells. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; ***, P<0.001. (b) Representative contour plot (left) and cytokine (IFN-γ, IL-2, TNFα) producing CD8 ＋Summary of CAR-T cell frequencies (right). Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ***, P<0.001. (c) Representative contour plots (left) and cytokine (IFN-γ, IL-2, TNFα)-producing CD4 ＋Summary of CAR-T cell frequency (right). Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; **, P<0.01. (d, e) The cytotoxicity of CAR-T cells against U-87mGΔ cells and U-251mGΔ cells under low glucose (0.5 mM) or high glucose (10 mM) conditions was determined by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc and U-251mGΔ-Luc) were cocultured at the indicated effector-to-target (E:T) ratio for 24 hours, and specific lysis was calculated based on luciferase activity. (d) Cytotoxicity of CAR-T cells against U-251mGΔ-Luc cells under low glucose (0.5 mM) conditions. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01. (e) Cytotoxicity of CAR-T cells against U-87mGΔ-Luc or U-251mGΔ-Luc cells under high glucose (10 mM) conditions. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. (F, G) Expression of exhaustion markers (PD-1, f; PD-1 and Tim-3, g) by the indicated CAR-T cell populations. Representative contour plots are shown. Figure 38 shows that stable expression of GLUT3 improves CAR-T cell function independently of the type of scFv. (a, b) Mock T cells (Mock T), conv CD19 CAR-T cells (conv CD19 CAR-T), and GLUT3 CD19 CAR-T cells (GLUT3 CD19 CAR-T) were prepared from PBMCs of healthy volunteers (n=5). (a) Cytokine production by the indicated CAR-T cells after stimulation with CD19-expressing tumor cells (NALM6) under low glucose (0.5 mM) conditions, as determined by intracellular cytokine staining. Representative contour plots (left) and a summary of the frequency of cytokine (IFN-γ, IL-2, TNFα)-producing cells (right) are shown. Data are presented as mean ± SEM.Statistical analysis by Student's t-test: *, P<0.05; ***, P<0.001; ***, P<0.0001. (b) Cytokine (IFN-γ, IL-2, TNFα) production by conv CD19 CAR-T cells and GLUT3 CD19 CAR-T cells cocultured with NALM6 cells for 16 hours under low glucose (0.5 mM) conditions was measured by ELISA. Data are shown as mean ± SEM. Statistical analysis by Student's t-test: *, P<0.05; ***, P<0.001; ***, P<0.0001. (c) The cytotoxicity of conv CD19 CAR-T cells and GLUT3 CD19 CAR-T cells against NALM6- and CD19-expressing AsPC-1 cells under low glucose (0.5 mM) conditions was determined by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (NALM6-Luc and CD19-expressing AsPC-1-Luc) were cocultured for 24 hours at the indicated effector-to-target (E:T) ratio, and specific lysis was assessed based on luciferase activity. Data are shown as mean ± SEM. Statistical analysis was performed by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01. Figure 39 shows that on-demand GLUT3 expression enhances metabolic capacity and improves cytokine production and CAR-T cell cytotoxicity. (a) Glycolysis parameters (measured by glycolysis stress test) of conv EGFRvIII CAR-T cells (conv CAR-T) and on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) cultured under low glucose (0.5 mM) conditions are shown. Data are shown as mean ± SD. Statistical analysis was performed by Student's t-test; **, P<0.01; ***, P<0.001; ****, P<0.0001. (b, c) Conv EGFRvIII CAR-T cells (conv CAR-T) and on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) were prepared from PBMCs of healthy volunteers (n=5). (b) Cytokine production by the indicated CAR-T cells was examined by intracellular cytokine staining when EGFRvIII-expressing tumor cells (U-87mGΔ) were stimulated under high glucose (10 mM) conditions.A summary of the frequencies of cytokine (IFN-γ, IL-2, TNFα)-producing cells is shown. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ***, P<0.001. (c) The cytotoxicity of CAR-T cells against U-87mGΔ cells and U-251mGΔ cells under high glucose (10 mM) conditions was determined using a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc or U-251mGΔ-Luc) were cocultured at the indicated effector-to-target (E:T) ratio under high glucose (10 mM) conditions for 24 hours, and specific lysis was calculated based on luciferase activity. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01; ****, <0.0001. Figure 40 shows that on-demand expression of GLUT3 improves the survival rate of CAR-T cells. (a, b) CD3 24 hours after stimulation with anti-EGFRvIII beads under low glucose (0.5 mM) conditions. ＋ T cell proliferation and CAR in cells ＋ T cell frequencies. Mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), and on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) were prepared from PBMCs of healthy individuals (n=3). (c) Phenotype of CAR-T cells 7 days after stimulation with U-87mGΔ cells. Representative contour plots (left) and central memory (CD45RA CCR7 －＋(b) Summary of T cell frequencies (right). Data are shown as mean ± SEM. Statistical analysis was by Student's t-test; ns, not significant; ****, P<0.0001. (d) Expression of excretion markers (PD-1 and Tim-3) by the indicated CAR-T cell populations 72 hours after stimulation with EGFRvIII-expressing tumor cells (U-87mGΔ). Representative contour plots are shown. Figure 41 shows on-demand GLUT3 expression by CAR-T cells. On-demand GLUT3 expression by CAR-T cells enhances survival while maintaining durable anti-tumor efficacy in an intracranial human GBM cell xenograft model. (a) BLI of U-87mGΔ-Luc tumor-bearing mice. (b, c) NSG mice (n=10, excluding the mock CAR-T cell group; NSG mice (n=5) intracranially inoculated with U-251mGΔ-Luc cells were intravenously administered either mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), or on-demand GLUT3 EGFRvIII CAR-T cells (on-demand GLUT3 CAR-T) on day 5 after tumor inoculation. U-87mGΔ-Luc tumors were intravenously administered with EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, or on-demand GLUT3 EGFRvIII CAR-T cells. Mice eradicated by CAR-T cells were rechallenged with the same tumor on day 21. Tumor burden was monitored by BLI. (b) Tumor burden measured by BLI. Each colored line represents an individual mouse. Crosses indicate the cause of death. The numbers in the upper right corner indicate the number of mice that achieved complete response (CR) by day 21 and the number of mice that died. The horizontal dotted line indicates the baseline BLI signal level obtained from tumor-uninoculated mice. (c) Body weight change in mice during CAR-T cell treatment. Crosses indicate all causes of death, and colored dotted lines indicate mice that experienced treatment-related death (TRM). (d) Serum cytokine and chemokine concentrations (IL-5, IP-10, MDC) were examined by ELISA 14 days after CAR-T cell administration. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01.

[0015] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0016] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0017] As used herein, "about" means ±10% of the numerical value that follows. For example, "about 20" includes "18 to 22." Numerical ranges include all values ​​between and at the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0018] As used herein, the term "T cells" is used in the broad sense used in the art and refers to lymphocytes produced in the bone marrow that have migrated to the thymus and matured. T cells may be CD45-positive and CD3-positive cells among normal fractions of peripheral blood and bone marrow-derived mononuclear cells. T cells used may be, but are not limited to, T cells isolated from a donor, particularly a human donor. Examples of T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions. It is known that there are multiple types of T cells based on their functions, including effector memory T cells (T ｅｆｆExamples of peripheral T cells include T cells (Tnv), stem cell memory T cells (Tscm), central memory T cells (Tcm), and terminally differentiated RA-positive T cells (Temra). As used herein, "peripheral T cells" refers to T cells present outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues. As used herein, the term "peripheral T cells" refers to T cells present outside the thymus, and it is sufficient that the cell population contains peripheral T cells; the T cells do not need to be isolated. In addition to T cells, cell fractions containing various lymphocytes, such as peripheral blood mononuclear cells (PBMC), may also be used.

[0019] As used herein, a "cell population" refers to a population containing two or more cells. For example, it may be a flat cluster of cells or a cell mass formed by three-dimensional adhesion of cells. Furthermore, a "cell population" may be formed by a single type of cell or may contain multiple types of cells. When referring to a cell population of T cells as described herein, it is sufficient that the population contains at least one cell capable of effector function in the location where it is to function (typically in the body), such as Tnv, Tscm, Tcm, or Tempra. As used herein, "flow cytometry" refers to a technique for measuring the number of cells, individuals, and other biological particles suspended in a liquid, as well as their individual physical, chemical, and biological properties. A device using this technique is referred to as a "flow cytometer." In the present disclosure, "positive" and "negative" for cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are determined by flow cytometry, as commonly used in the art. More specifically, in flow cytometry, cells are lined up and flowed, and the number of cells is counted using spectroscopic techniques. For example, cells labeled with fluorescent or luminescent enzymes are irradiated with laser light, and the fluorescent or luminescent signals emitted by the cells are detected by a detector such as a photodiode, thereby counting the number of target cells. The detection results from the detector can also be input into a computer, and a two-dimensional plot can be generated and displayed. This makes it easy to determine the presence and number of target cells.

[0020] As used herein, the term "effector function" refers to biological activities such as cytotoxic activity, cytokine production, and division / proliferation that are caused by signals mediated by TCR or CAR in T cells, and to biological activities caused by the Fc region of an antibody in other immune cells. Effector functions include, for example, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells. In the present disclosure, whether or not a T cell has an effector function can be determined by confirming that the T cell has cytotoxic activity, cytokine production, and division / proliferation using the techniques exemplified in the Examples and the like.

[0021] As used herein, the term "effector cell" or "effector T cell" refers to a T cell having an effector function and exerting cytotoxic effects on target cells, and is a T ｅｆｆ As used herein, whether a cell is an "effector" (cell) can be determined by methods such as cytotoxicity assays, surface antigen analysis by flow cytometry, and intracellular cytokine staining. Effector cells are immune cells that perform effector functions, such as mediating antibody-dependent cellular cytotoxicity (ADCC). Effector cells include, for example, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils, and can be isolated from natural tissues such as blood.

[0022] As used herein, "effector T cells (T ｅｆｆEffector T cell precursors (also called effector T cell precursors) are cells that are not effector cells but acquire effector function by migrating to a site of function (e.g., the body) or by other stimuli. These cells include lymphoid precursors, Tnv, Tscm, Tcm, and Tempra. Effector T cell precursors are activated early in the immune response and mature into effector T cells through proliferation and differentiation. However, some precursors have the potential to differentiate into memory T cells. These precursors have specific gene expression patterns and metabolic states that contribute to the prolongation of memory function. Several molecular and metabolic characteristics contribute to this prolongation of memory function. First, effector T cell precursors have specific metabolic programs. These cells rely predominantly on oxidative phosphorylation rather than glycolysis, which is characteristic of effector T cells. This metabolic pathway is highly energy efficient and allows for long-term cell survival. This metabolic characteristic is a key factor supporting the long-term maintenance of memory T cell function. Second, the role of transcription factors is also important. Effector T cell precursors express transcription factors such as T-bet and Eomes, which promote differentiation into memory T cells and enable their long-term survival and function. Furthermore, these precursor cells, expressing surface markers such as CD62L and CCR7, possess the ability to migrate to secondary lymphoid tissues. This homing function allows precursor cells to receive necessary survival signals in secondary lymphoid tissues, contributing to the maintenance of memory function. Furthermore, suppression of antigenic stimulation also contributes to the extension of memory function. Release from antigenic stimulation inhibits complete differentiation into effector T cells, instead retaining the properties of memory T cells. This mechanism suppresses excessive effector responses while preserving memory function. In addition, the cytokine environment surrounding the cells also plays an important role. In particular, cytokines such as IL-7 and IL-15 provide survival signals necessary for the maintenance of memory T cells, which contribute significantly to the extension of memory function.These factors allow effector T cell precursors to survive longer than normal effector T cells and retain memory functions that enable rapid and powerful secondary immune responses. This property is thought to play an important role not only in immune defense against infectious diseases but also in cancer immunotherapy and vaccine development. Thus, understanding the properties of effector T cell precursors is of great significance for both basic immunological research and clinical applications.

[0023] The maintenance of memory T cell phenotype in effector T cell precursors refers to the phenomenon in which some precursor cells retain memory T cell-specific phenotypes (e.g., surface markers, metabolic state, transcription factor expression, etc.) during the differentiation process into effector T cells. These precursor cells survive even after the end of an immune response and have the ability to provide long-term immunological memory. This phenomenon is thought to be an important mechanism for T cells to balance the process of exerting effector function with memory function, which allows long-term survival and rapid secondary immune responses. The characteristics of this phenomenon are explained below. First, effector T cell precursors maintain surface markers specific to memory T cells. These include homing molecules such as CD62L and CCR7, and the expression of these molecules allows precursor cells to maintain their ability to migrate to secondary lymphoid tissues. Second, these precursor cells also maintain a characteristic metabolic state. Specifically, oxidative phosphorylation predominates over glycolysis, maintaining an energy-efficient metabolic program that enables long-term survival. This metabolic characteristic is important for maintaining memory T cell function. Furthermore, progenitor cells maintain a characteristic balance in the expression of transcription factors. Appropriate expression of transcription factors such as T-bet and Eomes ensures both differentiation into effector T cells and maintenance of the memory T cell phenotype. Furthermore, progenitor cells are responsive to cytokine signals and can receive survival signals such as IL-7 and IL-15. This responsiveness allows progenitor cells to survive for long periods while retaining memory T cell characteristics. Finally, these progenitor cells are capable of antigen-independent maintenance. In other words, memory T cell progenitors retain their phenotype even in the absence of antigen and maintain a state of readiness for a rapid immune response. Due to these characteristics, the maintenance of the memory T cell phenotype by effector T cell progenitors plays a crucial role in the flexibility of the immune system and the establishment of long-term memory immunity. These characteristics are thought to be of great significance in infectious disease prevention, cancer immunotherapy, and vaccine design.

[0024] As used herein, the term "antigen receptor" refers to any molecule that specifically binds to a target antigen and transmits a signal into a cell that expresses the receptor. In the present disclosure, an antigen receptor is one that can be introduced into a T cell and may be a natural or artificial molecule, and includes, for example, a T cell receptor (TCR) / B cell receptor (BCR) as well as a chimeric antigen receptor (CAR).

[0025] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered receptor that can confer antigen specificity to cells (e.g., immune cells). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. A CAR is an engineered receptor that transfers antigen specificity to immune system cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, or a combination thereof, NK cells, macrophages, etc.). CARs may include, for example, an antigen-specific targeting region, an extracellular domain, a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. They may also include bispecific CARs that use multiple (usually two) types of antigen-specific targeting regions. Preferably, the CAR of the present disclosure includes at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0026] As used herein, "T cell receptor (TCR)" refers to a receptor present on T cells. TCRs are heterodimeric receptor molecules consisting of two TCR polypeptide chains. There are two types: αβ TCRs, which are expressed by normal T cells, and γδ TCRs, which have specialized functions. α and β chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transduce intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens proliferated within cells following viral infection and cancer antigens derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Furthermore, antigens derived from foreign microorganisms are taken up by antigen-presenting cells by endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules. Regarding the αβ TCR, which is one of the main objects of this specification, the gene products of α and β are said to express specificity through unique combinations.

[0027] As used herein, the term "glucose uptake ability" refers to the ability of a cell to take up glucose per unit time. The uptake of glucose into a cell can be evaluated by a technique such as the 2-NBDG uptake assay.

[0028] As used herein, "enhanced glucose uptake ability" refers to improving glucose uptake ability by any method. Improvement of glucose uptake ability can be achieved by modifying immune cells (e.g., T cells) to express one or more glucose transporters and / or by modifying them to enhance glucose transporter expression. Such genetically modified immune cells are predicted to exhibit high glucose uptake, for example, in a low-glucose environment (e.g., a tumor microenvironment). Therefore, immune cells co-expressing one or more glucose transporters and a chimeric receptor polypeptide may exhibit superior physiological activities (e.g., in a tumor microenvironment, such as low-glucose conditions, optionally in the presence of a therapeutic antibody), such as cell proliferation, activation (e.g., increased cytokine production, e.g., IL-2 or IFNγ production), cytotoxicity, and / or in vivo anti-tumor activity.

[0029] As used herein, the term "glucose transporter" (GLUT) refers to a transporter that transports glucose (grape sugar) by facilitated diffusion. Molecular species of GLUT, GLUT1 to GLUT14, have been reported, and among them, for example, GLUT1 is expressed in a wide range of tissues and is responsible for constant baseline glucose transport, GLUT2 is activated when the sugar concentration increases and enhances glucose uptake, GLUT3 plays a central role in the central nervous system and exerts transport function even at low glucose concentrations, and GLUT4 is activated in response to insulin stimulation.

[0030] As used herein, the term "tumor microenvironment" is used interchangeably with "tumor environment." The tumor microenvironment (TME) refers to the local biological environment surrounding tumor tissue and encompasses a variety of factors involved in tumor progression, growth, metastasis, drug resistance, and other processes. This environment includes not only tumor cells but also non-tumor cells such as immune cells (e.g., T cells and macrophages), fibroblasts, vascular endothelial cells, and adipocytes. Humoral factors such as extracellular matrix (ECM), cytokines, chemokines, and growth factors are also important components. Furthermore, the tumor microenvironment is influenced by physical factors such as oxygen concentration, pH, and nutritional status. Changes in intercellular interactions and physical factors in the tumor microenvironment are believed to play important roles in tumor malignancy and therapeutic responsiveness. In particular, immunosuppressive microenvironments have attracted attention as factors that reduce the efficacy of immunotherapy. In the present invention, the "tumor microenvironment" refers to the collection of biological and physical factors, including these elements and their interactions, and is an important target in tumor treatment and diagnosis.

[0031] As used herein, the term "tumor microenvironmental conditions" refers to the tumor microenvironment or equivalent conditions. Therefore, "tumor microenvironmental conditions" refers to a broad concept encompassing not only the conditions constituting the tumor microenvironment but also similar or equivalent environmental conditions to which tumors are exposed within the body. Specifically, it encompasses not only the local biological environment surrounding tumor tissue, but also endogenous and exogenous factors that affect tumors when they reside within the body. This environment includes interactions between tumor cells and non-neoplastic cells (e.g., immune cells, fibroblasts, vascular endothelial cells, etc.), humoral factors such as the extracellular matrix (ECM), cytokines, chemokines, and growth factors, and physical factors such as oxygen concentration, pH, nutritional status, and physical pressure. Furthermore, "tumor microenvironmental conditions" encompasses situations in which tumors interact with a broader physiological environment within the body, and is therefore not limited to the local environment surrounding the tumor. In the present invention, "tumor microenvironmental conditions" refers to a comprehensive range of environmental conditions that tumors encounter within the body, including factors that affect tumor progression and therapeutic response. Thus, tumor microenvironment conditions include the interior of the tumor, the transduction of TCR-associated signals, the matrix and periphery (external), and similar conditions.

[0032] As used herein, "modification" broadly refers to any structural, functional, or expression-related change made to a gene (including nucleic acids and their corresponding proteins), including naturally occurring or artificial modifications. Specifically, this term includes sequence changes due to nucleotide substitution, deletion, insertion, addition, or a combination thereof; changes to the amino acid sequence or properties of the encoded protein; and chemical modifications (e.g., glycosylation, phosphorylation, acetylation, etc.) aimed at adding or improving functionality. "Modification" also includes exogenous gene introduction (e.g., transfection or viral vector-mediated gene introduction), transformation, knockout or knock-in of specific genes using genome editing technology, and suppression of gene expression (e.g., suppression using RNA interference or CRISPR technology). Furthermore, epigenetic modifications (e.g., DNA methylation, histone modifications, etc.) and modifications of expression regulatory elements are also included. As used herein, "modification" refers comprehensively to any form of change that affects the structure, expression, function, or biological behavior of a gene, including not only endogenous changes but also changes introduced or manipulated by exogenous means.

[0033] As used herein, "on-demand" expression refers to the expression of a gene of interest when it is needed. Demand refers to, for example, when glucose is about to become depleted (e.g., when T cells infiltrate a low-glucose environment, such as a tumor or bone marrow, or when glucose consumption increases due to metabolic changes to support T cell activation, cytotoxic activity, cytokine production, proliferation, etc., or both). The duration of demand can be any, for example, hours, days, or weeks, but is not limited thereto. The definition of "on-demand" also includes cases where the demand period extends beyond a few days. "On-demand" may refer to a property in which the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions, or may include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. In some cases, the gene of interest is expressed intracellularly without being integrated into the host's genome. In other cases, the gene of interest is integrated into the host's genome and expressed intracellularly. Methods known to those skilled in the art can be used for on-demand expression, and transfection or transduction can be performed using kits provided by manufacturers. Selection may be performed to select cells capable of achieving on-demand expression, for example, by drug selection or magnetic bead selection, both of which are well known to those skilled in the art. If the constructed construct already possesses on-demand properties, it can be used as is. On-demand expression can be achieved, for example, by modifying the glucose transporter in the T cells so that its expression is regulated in response to changes in the tumor microenvironment conditions.

[0034] As used herein, "tumor microenvironment conditions" refers to any conditions associated with a tumor, including the interior, matrix and periphery (exterior) of the tumor, and similar conditions.

[0035] As used herein, "glucose transporter expression is regulated (in cells such as T cells) in response to changes in tumor microenvironment conditions" means that when tumor microenvironment conditions worsen (the cancer state worsens), the expression or level of the glucose transporter is reduced or its degradation is promoted, and when tumor microenvironment conditions improve (the healthy state improves), the expression or level of the glucose transporter is less enhanced and its degradation is reduced. Regulation of glucose transporter expression (in cells such as T cells) in response to changes in tumor microenvironment conditions can be achieved by modifying cells, for example by introducing such a gene construct.

[0036] Here, "deterioration of tumor microenvironment conditions" refers to a change in the tumor microenvironment (TME), which promotes tumor growth and progression, to a state more favorable for the tumor, or to conditions equivalent thereto. This tumor microenvironment is composed of not only tumor cells but also the surrounding blood vessels, immune cells, fibroblasts, extracellular matrix (ECM), signaling molecules, and the like. The "deterioration" of this environment encompasses the following specific changes: 1) Progression of hypoxia: As tumors grow, blood vessels become insufficient, resulting in a lack of oxygen supply, leading to hypoxia around the tumor. This condition promotes tumor cell proliferation and metastasis. Therefore, examining oxygen status can detect a deterioration of tumor microenvironment conditions. 2) Activation of abnormal angiogenesis: Tumor growth promotes angiogenesis (the formation of new blood vessels), but these newly formed blood vessels are often structurally abnormal, leading to an imbalance in the supply of oxygen and nutrients, which promotes tumor progression. Therefore, examining the state of angiogenesis can detect a deterioration in tumor microenvironmental conditions. 3) Formation of an immunosuppressive environment: Tumors suppress antitumor immunity by increasing immunosuppressive cells (e.g., regulatory T cells and myeloid-derived suppressor cells), reducing the immune system's ability to eliminate tumors. Therefore, examining the state of an immunosuppressive environment can detect a deterioration in tumor microenvironmental conditions. 4) Progression of fibrosis: Activation of fibroblasts and abnormal accumulation of extracellular matrix around the tumor increase tumor cell mobility and drug resistance. Therefore, examining the state of fibrosis can detect a deterioration in tumor microenvironmental conditions. 5) Activation of chronic inflammation: Persistent chronic inflammatory responses in the tumor microenvironment promote tumor cell proliferation, angiogenesis, and metastasis. Therefore, examining the state of (chronic) inflammation can detect a deterioration in tumor microenvironmental conditions. 6) Progression of metabolic abnormalities: Tumor cells consume excessive glucose and activate anaerobic metabolism (the Warburg effect). This leads to the accumulation of lactic acid, acidifying the microenvironment and impairing immune cell function. Therefore, by examining metabolic status (e.g., glucose consumption, lactate accumulation, etc.), it is possible to detect deterioration of tumor microenvironment conditions.

[0037] The effects of a deterioration in the tumor microenvironment are wide-ranging, and a deterioration in the tumor microenvironment affects tumor characteristics, including, but not limited to, the following: 1) promotion of tumor growth (tumor cells become activated, accelerating their division and proliferation); 2) promotion of metastasis (tumor cells become more likely to spread to other organs via blood and lymphatic vessels); 3) increased resistance to treatment (changes in the tumor microenvironment can make chemotherapy and immunotherapy less effective); etc.

[0038] As used herein, "modifying a cell (such as a T cell) so that its expression is regulated in response to changes in tumor microenvironment conditions" refers to introducing a new glucose transporter into a cell or modifying an existing glucose transporter so that the expression of the glucose transporter is regulated in response to changes in tumor microenvironment conditions in the cell. Such modification can be achieved by operably linking an element that regulates the expression or degradation of the glucose transporter in response to changes in tumor microenvironment conditions in the cell to a nucleic acid sequence encoding the glucose transporter. Alternatively, an existing glucose transporter or an element that directly or indirectly regulates it can be modified using genetic engineering techniques such as gene manipulation or genome editing to regulate the expression or degradation of the glucose transporter.

[0039] As used herein, the term "TCR-associated signal" refers to any signal associated with a T cell receptor (TCR), and includes not only signals directly associated with the TCR (for example, naturally expressed unmodified T cell receptors), but also chimeric antigen receptor (CAR)-associated signals, which are complexes of a part of the TCR and a costimulatory factor, expression of tumor antigens (referring to antigens recognized by the TCR or CAR) or costimulatory ligands, fluctuations in cytokine conditions, etc. For example, a TCR-associated signal can be mediated by any element involved in TCR signal transduction, and may include a TCR signal or a part thereof, a chimeric signal of a TCR signal and another antigen (also referred to as a CAR signal), etc.

[0040] As used herein, the term "TCR signal domain" refers to any domain involved in TCR signaling, and a representative example thereof is an immunoreceptor tyrosine-based activation motif (ITAM) present in the CD3 molecules (CD3ε, CD3δ, CD3γ) and ζ chain (CD247, zeta chain) that constitute the CR complex. ITAMs are characterized by the presence of tyrosine residues (ITAMs contain two tyrosine residues, which initiate signal transduction when phosphorylated), the recruitment of kinases (phosphorylation of tyrosine residues recruits signal transduction kinases (mainly Lck and ZAP-70), thereby activating the signal cascade), and the initiation of signal transduction cascades (following ITAM phosphorylation, calcium signals, the MAPK pathway, the NF-κB pathway, etc. are activated, inducing immune responses such as T cell proliferation, differentiation, and cytokine secretion). Examples of TCR signal domains include the CD3ζ chain (zeta chain; the CD3ζ chain is a major signal transduction molecule of the TCR complex and has three ITAMs, which enable amplification of the TCR signal), the CD3ε domain (epsilon domain; CD3ε has one ITAM and contributes to early signal transduction via Lck after antigen recognition), modified TCR signal domains (application in CAR-T cells), and chimeric antigen receptor (CAR) therapy, in which a TCR signal domain is artificially designed and combined with the ITAM of the CD3ζ chain and a costimulatory molecule domain such as CD28 or 4-1BB to achieve stronger signal transduction).

[0041] As used herein, the term "tumor antigen," also known as "cancer antigen," refers to an antigen expressed by tumor cells and a molecule recognized by the immune system. Tumor antigens are primarily classified into those specifically expressed in tumor cells and those that are also expressed in normal cells but are abnormally highly expressed in tumor cells. These antigens are important targets in tumor immunity and are used in cancer immunotherapy (e.g., vaccine therapy, T-cell therapy, CAR-T therapy). Tumor antigens are primarily classified into two categories: tumor-specific antigens (TSA) and tumor-associated antigens (TAA). TSAs are antigens that are specifically expressed in tumor cells and are rarely expressed in normal cells. These are usually caused by the following: genetic mutations (mutated peptides (neoantigens) resulting from genetic mutations in RAS, p53, etc.), and virus-derived antigens (tumors caused by viral infection (e.g., E6 / E7 antigens derived from human papillomavirus (HPV)). TAAs are antigens that are expressed in normal cells but are abnormally highly expressed in tumor cells. The main characteristics are as follows: embryonic antigens (antigens expressed during fetal development but hardly expressed in adult tissues (e.g., CEA, AFP)), and overexpressed antigens (antigens expressed at low levels in normal tissues but Antigens that are expressed at high levels in tumor cells (e.g., HER2, EGFR). Examples of tumor antigens include: neoantigens (antigens generated by mutation in tumor cells): KRAS mutant peptide; tumor virus-derived antigens (antigens expressed in virus-infected tumors): HPV-derived E6 / E7 (cervical cancer); embryonic antigens (expressed only in fetal stages in normal cells and silent in adults): carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP); and overexpressed antigens: HER2 (breast cancer), EGFR (lung cancer), etc.

[0042] As used herein, the term "co-stimulatory ligand" refers to a ligand that binds to a costimulatory molecule to complement antigen presentation (recognition by the T cell receptor (TCR) or B cell receptor (BCR)), which is a major signal in the activation of T cells and other immune cells. Costimulatory ligands are expressed on the surface of antigen-presenting cells (APCs), tumor cells, and other cells, and play a role in enhancing or modulating the immune response. The interaction between these ligands and costimulatory molecules is important for T cell activation, proliferation, cytokine secretion, and the formation of immunological memory, contributing to the appropriate control of the immune response. Costimulatory ligands are molecules that play an important role in the immune response and are involved in the activation and regulation of immune cells, including T cells. Their characteristics include, first, that they provide a second signal following antigen recognition (first signal) via the T cell receptor (TCR), enabling full activation of T cells. The expression and interaction of costimulatory ligands (co-ligands) regulates the strength and nature of immune responses, maintaining the proper function of the immune system, and is important for maintaining the balance between immune response and tolerance, since their absence can lead to T cell anergy and the induction of immune tolerance.

[0043] Specific examples of costimulatory ligands include those that promote activation and those that have inhibitory roles. Activation-promoting costimulatory ligands include B7-1 (CD80) and B7-2 (CD86), which bind to the CD28 receptor on T cells to promote initial T cell activation. Furthermore, OX40L (CD252) binds to OX40 (CD134) and supports T cell proliferation and survival, thereby promoting the formation of immunological memory. 4-1BBL (CD137L) binds to 4-1BB (CD137) to support the proliferation and sustained activation of activated T cells. ICOSL (CD275) binds to the ICOS receptor and is involved in the activation of helper T cells (Tfh cells) and B cell help. Inhibitory costimulatory ligands include PD-L1 (Programmed Death-Ligand 1) and PD-L2, which inhibit T cell function and induce immune tolerance by binding to the PD-1 receptor on T cells. In addition, B7-H3 (CD276) and B7-H4 are also thought to have inhibitory functions and are believed to be involved in immunosuppression and the induction of inhibitory T cells. The application of costimulatory ligands holds great potential in the fields of immunotherapy and disease treatment. In cancer immunotherapy, treatments that enhance T cell activation using 4-1BBL and OX40L have been developed, and immune checkpoint inhibitors targeting inhibitory ligands such as PD-L1 / PD-1 have become an important strategy in cancer treatment. Furthermore, in vaccine development, efforts are being made to enhance immune responses using adjuvants that express costimulatory ligands. Additionally, in the treatment of autoimmune diseases, research is progressing on the use of inhibitory costimulatory ligands to control excessive immune responses.

[0044] As used herein, the term "degree of TCR-associated signal transduction" refers to the strength and quality of signal transduction that occurs when a T cell receptor (TCR) recognizes a peptide-MHC complex on an antigen-presenting cell (APC). This degree significantly influences the outcome of immune responses, such as T cell activation, proliferation, cytokine production, differentiation, or apoptosis. The degree of TCR-associated signal transduction is an important factor in determining T cell activation and immune responses and is regulated by various factors. Specifically, the higher the binding strength (affinity) between the peptide-MHC complex and the TCR, the stronger the degree of signal transduction. Furthermore, when the density of peptide-MHC complexes present on the surface of antigen-presenting cells is high, TCR-mediated signal transduction is further enhanced. In addition, the presence of ligands for costimulatory molecules such as CD28 and ICOS (e.g., B7 family molecules) amplifies TCR signaling, whereas signaling is suppressed when immunosuppressive molecules such as PD-1 and CTLA-4 are involved. Furthermore, the phosphorylation state and expression level of signaling molecules downstream of the TCR (e.g., Lck, ZAP-70, LAT, SLP-76) are also important factors that affect the strength of the signal. In addition, metabolic conditions such as cytokines, oxygen concentration, and glucose concentration also contribute to signal enhancement or suppression. The degree of TCR-associated signal transduction is crucial in determining the quality of the immune response. For example, strong signals induce effector T cell activation and cytotoxicity, while weak signals are involved in immune tolerance and the induction of regulatory T cells (Tregs). Taking advantage of these characteristics, modulation of TCR-associated signal transduction has been applied to immunotherapy and disease treatment. In cancer immunotherapy, attempts are being made to induce T cell responses that effectively attack cancer cells by increasing TCR signal strength. Furthermore, in the treatment of autoimmune diseases, approaches are being investigated that control excessive immune responses by suppressing TCR signaling. Furthermore, in T cell engineering, when designing chimeric antigen receptors (CARs) or artificial TCRs, ensuring the appropriate degree of signal transduction is key to improving therapeutic efficacy and safety.

[0045] As used herein, the term "degree of TCR stimulation" refers to the strength and quality of stimulation that occurs when a T cell receptor (TCR) recognizes a peptide-MHC complex on an antigen-presenting cell (APC) or target cell. The degree of stimulation is an important factor that determines the outcome of T cell activation and immune responses, and is determined by the strength, duration, and frequency of the signal. As used herein, the term "TCR co-stimulation" refers to the additional signal transduction required to fully activate a T cell when the T cell recognizes a peptide-MHC complex on an antigen-presenting cell (APC) with its T cell receptor (TCR). Antigen recognition by the TCR alone (first signal) often results in incomplete activation of the T cell or in an unresponsive (anergy) state. Therefore, TCR co-stimulation is important for effectively inducing adaptive immune responses, including T cell proliferation, cytokine production, effector function, and the formation of immunological memory. TCR costimulation is provided by the interaction of costimulatory molecules on the surface of T cells with costimulatory ligands on antigen-presenting cells, amplifying TCR signals and modulating the immune response.

[0046] As used herein, "humoral factor(s)" refers to soluble molecules present in body fluids that are involved in intercellular communication and maintaining homeostasis in the body. These factors are involved in various physiological processes, such as immune responses, inflammatory responses, tissue repair, and metabolic regulation, and act primarily in body fluids such as blood, lymph, and tissue fluid. Humoral factors primarily play important roles in the immune system, and representative examples include molecules such as cytokines and complement. These molecules bind to cell surface receptors to initiate signal transduction and regulate the response of target cells.

[0047] As used herein, the term "transcription factor that translocates into the nucleus upon T cell activation" refers to a transcription factor that translocates into the nucleus when a T cell recognizes a peptide-MHC complex presented by an antigen-presenting cell (APC) with its T cell receptor (TCR). This triggers a series of signal transduction cascades, resulting in the activation and translocation of a transcription factor into the nucleus. This transcription factor plays an important role in the activation, proliferation, differentiation, and expression of effector functions of T cells. For example, major transcription factors that translocate into the nucleus upon T cell activation include NFAT, NF-κB, AP-1, and the STAT family. NFAT (Nuclear Factor of Activated T-cells) is activated by calcium signals, dephosphorylated by calcineurin, and then translocates into the nucleus, promoting the expression of interleukin-2 (IL-2) and other cytokine genes. On the other hand, NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) is activated through IκB degradation by TCR signaling and controls the transcription of genes related to inflammatory cytokines and cell proliferation. AP-1 (Activator Protein-1) is activated by MAPK signaling pathways such as ERK and JNK, contributing to the expression of IL-2 and interferon-γ (IFN-γ). Furthermore, the STAT family (Signal Transducer and Activator of Transcription) (which includes at least seven types: STAT1-4, STAT5A, STAT5B, and STAT56) is activated by signaling via cytokine receptors. For example, STAT3 is activated by IL-6 and IL-10 signals and regulates the expression of genes related to inflammatory responses and immunosuppression. STAT5 is activated by IL-2 and IL-7 signals and plays an important role in T cell proliferation and survival. Thus, NFAT, NF-κB, AP-1, and the STAT family translocate into the nucleus via distinct pathways and function cooperatively in regulating T cell responses.

[0048] As used herein, the term "degree of oxygen concentration" refers to an index of the amount of oxygen present in a particular environment or tissue, typically measured as oxygen partial pressure (unit: mmHg) or oxygen saturation (%). In vivo, oxygen concentration is an important factor that directly affects the metabolic activity of cells and tissues and is broadly classified into four major states, including normoxia, hypoxia, and hyperoxia. Each state is described below. Normoxia refers to a state in which the oxygen partial pressure is within the normal physiological range, typically an oxygen concentration of approximately 21% and an oxygen partial pressure of approximately 100 mMHg at atmospheric pressure. This state represents a typical oxygen concentration found in healthy tissues and organs; for example, the oxygen partial pressure in arterial blood is typically 80-100 mMHg. Within this range, cells efficiently produce energy and maintain normal physiological functions. Hypoxia, on the other hand, refers to a state in which the oxygen partial pressure is below the normal range. For example, the tumor microenvironment may experience a lack of oxygen supply, resulting in an oxygen partial pressure below 20 mMHg. This hypoxic state promotes tumor growth and metastasis and may even cause resistance to radiation therapy. Hypoxia also occurs at high altitudes (above 3,000 meters) and is known to cause acute mountain sickness. Hyperoxia (hyperoxia) is a state in which oxygen partial pressure exceeds the normal range. Hyperbaric oxygen therapy utilizes this hyperoxia to increase the oxygen partial pressure and thereby increase tissue oxygen supply. This technique is sometimes used in wound healing and infection treatment, and is expected to be effective in the treatment of diabetic foot ulcers. Finally, extreme hypoxia (anoxia) refers to a state in which there is little or no oxygen. For example, in cardiac arrest or severe ischemia (insufficient blood flow), oxygen supply is completely cut off, causing tissue oxygen deprivation and ultimately necrosis. Such extreme oxygen deprivation can have serious consequences for life. As described above, oxygen levels are deeply involved in both physiological and pathological processes, and understanding how each state affects bodily functions and disease progression is important for effective treatment and management.

[0049] In one embodiment, "tumor microenvironment condition-sensing elements" refer to elements that sense changes in tumor microenvironment conditions, and refer to molecules or structures that sense specific conditions or stimuli (e.g., hypoxia, acidification, nutrient deficiency, inflammatory cytokines, changes in metabolic products, etc.) within the tumor microenvironment (TME) and transmit that information to cells. These elements function as molecular mechanisms that allow tumor cells and surrounding immune cells, fibroblasts, vascular endothelial cells, etc., to adapt to the tumor microenvironment, contributing to tumor progression, metastasis, and even treatment resistance. Condition-sensing elements in the tumor microenvironment sense specific environmental conditions and trigger adaptive responses in tumor cells accordingly. Examples of major conditions and their corresponding sensing elements are shown below.

[0050] First, in hypoxic conditions (hypoxia), HIF (Hypoxia-Inducible Factor) functions as a sensing element. This element senses the hypoxic environment and induces tumor cell metabolism and the expression of angiogenic factors (VEGF). HIF-1α, in particular, plays a central role in tumor cell adaptation to hypoxic conditions. Next, in acidification (a decrease in pH), pH sensors such as proton-sensitive ion channels and carbonic anhydrase IX (CAIX) function as sensing elements. These sense the acidic environment within the tumor and adjust the intracellular and extracellular pH balance, thereby promoting tumor cell survival. Under conditions of nutrient deficiency (glucose or amino acid deficiency), the mTOR pathway and AMPK (AMP-activated protein kinase) function as sensing elements. These sense nutrient deficiency and regulate energy metabolism, thereby maintaining cell survival. For inflammatory cytokines (e.g., IL-6 and TNF-α), cytokine receptors (e.g., IL-6R and TNF receptors) and the NF-κB signaling pathway act as sensing elements. These sense the inflammatory environment, promote tumor cell proliferation, and induce the expression of immunosuppressive factors. Furthermore, for metabolic changes (e.g., lactate accumulation), monocarboxylate transporters (MCTs) act as sensing elements. This senses metabolic products such as lactate and promotes tumor cell survival and proliferation. Furthermore, for oxidative stress (increased ROS), NRF2 (nuclear factor erythroid 2-related factor 2) acts as a sensing element. NRF2 senses reactive oxygen species (ROS) and activates the antioxidant response to protect tumor cells. These condition-sensing elements provide important mechanisms for tumor cells to adapt to the microenvironment and are deeply involved in tumor progression and treatment resistance.

[0051] As described herein, a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment condition sensing element that senses changes in tumor microenvironment conditions, having such a configuration, plays a role in responding to changes in tumor microenvironment conditions and regulating glucose transport by containing the following key components: First, the sensing element sequence has the function of responding to specific conditions in the tumor microenvironment (e.g., hypoxia, acidification, nutrient deprivation). Specifically, it includes a portion encoding a HIF-binding domain that responds to hypoxia and a protein region that functions as a pH sensor that senses acidification. This sequence enables the detection of specific changes in the tumor microenvironment. Second, the glucose transporter sequence encodes a glucose transporter (e.g., GLUT1, GLUT3) that transports glucose across the cell membrane. This sequence ensures efficient glucose transport in and out of cells and meets the metabolic needs of tumor cells. Furthermore, the linking region sequence encodes a linker region that functionally connects the sensing element and the glucose transporter. This linker region provides flexibility and stability, allowing the sensing element and the glucose transporter to operate in concert. However, by targeting the function of the protein encoded by this nucleic acid sequence, it becomes possible to block the metabolic adaptation of tumor cells. Specifically, to inhibit tumor adaptation, the protein senses changes in the tumor microenvironment, such as hypoxia and acidification, and inhibits glucose transport based on these changes, thereby cutting off the energy supply to tumor cells. Furthermore, to block environmental responsiveness, the protein prevents tumor cells from detecting changes in the tumor microenvironment via the sensing element, preventing tumor cells from adapting to harsh environmental conditions. Such functional inhibition makes it possible to realize a therapeutic strategy that effectively suppresses the survival and growth of tumor cells.

[0052] Examples of the tumor microenvironment sensing element include, but are not limited to, (i) an inducible activation motif (e.g., HIH-RE, NFAT-RE) that is activated in response to changes (e.g., deterioration) in the tumor microenvironment conditions, and (ii) a nucleic acid sequence that encodes a degradation factor (e.g., ODD) of the glucose transporter that is inactivated in response to changes (e.g., deterioration) in the tumor microenvironment conditions.

[0053] As used herein, the term "inducible activation motif responsive to tumor microenvironmental changes" refers to a molecular motif that controls signal transduction and is selectively activated in response to changes in specific conditions in the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deficiency, inflammatory signals, increased oxidative stress, etc.). This motif is involved in regulating metabolism, growth, and immune responses in tumor cells and cells surrounding the tumor, affecting tumor progression, metastasis, and therapeutic resistance. The inducible activation motif functions in specific processes of intracellular signaling pathways and alters gene expression and metabolic pathways in response to worsening tumor microenvironmental conditions (e.g., progression of hypoxia or nutrient deficiency). This enhances the ability of tumor cells to adapt to harsh environmental conditions.

[0054] As used herein, the term "hypoxia-responsive nuclear translocation and activation motif, to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds," refers to a DNA sequence or protein motif to which a specific factor (e.g., HIF-1α) that is activated in response to a decrease in oxygen concentration (hypoxic state, hypoxia) and translocates into the nucleus binds. This motif functions as a molecular switch that regulates gene expression under hypoxic conditions and plays an important role in enabling tumor cells and normal cells to adapt to a hypoxic environment. This inducible activation motif controls the transcription of hypoxia-responsive genes (e.g., angiogenic factors, metabolic enzymes, and oxidative stress resistance factors) and enhances the adaptive capacity of cells, while also being deeply involved in cancer progression and treatment resistance.

[0055] As used herein, the term "inducible activation motif bound by nuclear translocation factors that translocate into the nucleus in response to T cell activation" refers to a DNA sequence or protein domain to which a nuclear translocation factor (e.g., NFAT, NF-κB, AP-1, etc.) activated in response to T cell receptor (TCR) stimulation or signal transduction of a costimulatory molecule specifically binds. This motif receives signal transduction in the nucleus upon T cell activation and plays a role in inducing gene expression and transcriptional regulation. The inducible activation motif functions as a central molecular mechanism that controls T cell proliferation, differentiation, cytokine production, and expression of effector functions.

[0056] As used herein, the term "degradation factors of the glucose transporter responsive to tumor microenvironmental changes" refers to molecules or motifs that play a role in inactivating or degrading glucose transporters (GLUTs) in response to changes in specific conditions in the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deprivation, oxidative stress, etc.). These degradation factors play a role in inhibiting tumor growth and progression by suppressing the metabolic activity of tumor cells, particularly by controlling excessive glucose transport and metabolic adaptation. Specific examples include oxygen-dependent degradation domains (ODDs), which regulate the stability of target proteins depending on environmental conditions. As used herein, the term "inducible activation motif bound by nuclear translocation factors in response to T cell activation" refers to a DNA sequence or protein motif to which specific transcription factors (e.g., NFAT, NF-κB, AP-1, etc.) that are activated in response to T cell receptor (TCR) stimulation or signal transduction by costimulatory molecules and translocated into the nucleus bind. This motif controls gene expression related to T cell activation and plays a role in regulating T cell proliferation, differentiation, cytokine production, and the expression of effector functions. This inducible activation motif is a central molecular mechanism that regulates immune responses and is an important transcriptional regulatory element in T cell responses. It mainly contains the following components. First, it is a nuclear localization factor binding site. Transcription factors (e.g., NFAT, NF-κB, AP-1) that translocate into the nucleus upon T cell activation specifically bind to this site, reflecting the signal in gene transcription. Second, it is an environmentally responsive region that is dynamically activated in response to TCR stimulation and signals from costimulatory molecules.This region is inactive in a quiescent state. Third, it functions as a transcriptional regulator. It promotes or suppresses the transcription of target genes through the binding of nuclear import factors. This motif has a wide range of functions. First, it induces gene expression. It controls the gene expression of cytokines and effector molecules such as IL-2, IFN-γ, and TNF-α. Second, it regulates the immune response, controlling T cell proliferation, differentiation, and effector function to determine the quality and strength of the adaptive immune response. It also promotes or suppresses inflammatory responses and is involved in the formation of immunological memory, regulating the function of the entire immune system. Specific examples include the NFAT-binding motif in the IL-2 promoter region, the NF-κB response element in the TNF-α promoter region, and the region that binds AP-1 activated via the MAPK pathway. These motifs each regulate the expression of important genes associated with T cell activation. This motif has a wide range of potential applications. The development of drugs targeting this induction-activation motif is anticipated for cancer immunotherapy and the treatment of autoimmune diseases. Furthermore, in gene therapy and synthetic biology, this motif can be used to design artificial systems that control gene expression under conditions specific to T cells. Furthermore, analyzing the activation state of the motif can be useful for monitoring immune responses and evaluating therapeutic effects. Examples of such motifs include, but are not limited to, the NFAT-binding domain, the κB motif (GGGACTTTCC (SEQ ID NO: 1)), and the STAT-binding sequence (TTCNNNGAA), or modified sequences thereof.

[0057] As used herein, the term "nuclear translocation factor" is a general term for molecules that are activated in response to extracellular stimuli or intracellular signals and translocate from the cytoplasm into the nucleus, thereby regulating the transcription of target genes. These factors are activated by specific signal transduction pathways, pass through the nuclear membrane via a nuclear localization signal (NLS), and bind to DNA sequences or other transcription factors to regulate gene expression. Examples of nuclear translocation factors that can be used herein include NFAT, NF-κB (nuclear factor-kappaB), and the STAT transcription factor family (seven types: STAT1-4, STAT5A, STAT5B, and STAT56).

[0058] As used herein, the term "combination of nuclear translocation factors and nuclear translocation-inducible activation motifs" refers to a combination of DNA sequences or protein motifs (nuclear translocation-inducible activation motifs) to which a transcription factor (nuclear translocation factor) that is activated in response to extracellular or intracellular stimuli (e.g., T cell activation, hypoxic conditions, inflammatory signals, etc.) and translocates into the nucleus specifically binds. This combination precisely regulates gene expression in response to specific stimuli and plays a role in controlling cellular function and adaptation.

[0059] In this specification, this combination is responsible for linking signal transduction pathways with gene expression control, and is important in biological processes such as immune response, inflammation, metabolic regulation, and cell differentiation. It is also known to play a pathological role in various conditions, including cancer and autoimmune diseases. Examples of major combinations are listed below. NFAT and NFAT-binding motif in the IL-2 promoter Nuclear translocation factor: NFAT (Nuclear Factor of Activated T-cells) Binding motif: NFAT response element in the IL-2 promoter Function: In response to T cell activation, NFAT translocates into the nucleus, induces IL-2 gene transcription, and promotes T cell proliferation and immune response. - NF-κB and NF-κB response motifs in the TNF-α promoter Nuclear translocation factor: NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) Binding motif: NF-κB response element in the TNF-α promoter (e.g., κB motif (GGGACTTTCC)) Function: In response to inflammatory signals (e.g., cytokine stimulation), NF-κB translocates into the nucleus and induces the expression of TNF-α, thereby regulating the inflammatory response. - HIF-1α and HRE (Hypoxia-Responsive Element) Nuclear translocation factor: HIF-1α (Hypoxia-Inducible Factor-1α) Binding motif: HRE (Hypoxia-Responsive Element) Function: In response to hypoxia, HIF-1α translocates into the nucleus and induces the transcription of angiogenic factors (e.g., VEGF).・STAT family and cytokine response elements Nuclear translocation factor: STAT3 (Signal Transducer and Activator of Transcription 3) Binding motif: STAT binding site in cytokine response genes (e.g., STAT binding sequence (TTCNNNGAA)) Function: STAT3 translocates into the nucleus in response to cytokine (e.g., IL-6) stimulation, and induces immune regulation and inflammatory responses.・AP-1 and growth factor response motifs Nuclear import factor: AP-1 (Activator Protein-1) Binding motif: AP-1 response element in the promoters of genes related to cell proliferation and differentiation Function: AP-1 activated via the MAPK pathway translocates into the nucleus and promotes cell proliferation and differentiation.

[0060] As used herein, "factors that inactivate the degradation activity of glucose transporters in response to a decrease in oxygen concentration (factors that inactivate the degradation activity of glucose transporters in response to hypoxia)" refers to factors that suppress the activity of a system that induces the degradation of glucose transporters (e.g., GLUT1, GLUT3) in response to a decrease in oxygen concentration (hypoxic condition, hypoxia), thereby increasing the stability of glucose transporters. This factor acts to suppress the degradation of glucose transporters under hypoxic conditions via a structure such as an oxygen-dependent degradation domain (ODD), thereby enabling cells to maintain their required energy supply. Furthermore, in the tumor microenvironment, it plays an important role in promoting metabolic adaptation of tumor cells and contributing to their survival and proliferation. The following describes the components and characteristics of factors whose glucose transporter degrading activity is inactivated in response to a decrease in oxygen concentration. First, there is the hypoxia-responsive sensor domain. This domain senses a decrease in oxygen concentration and emits a signal to inhibit degradation under hypoxic conditions. A representative example is HIF-1α (Hypoxia-Inducible Factor-1α), which stabilizes under hypoxic conditions and regulates the activity of degrading factors. Next, there is the degradation regulatory domain (ODD: Oxygen-Dependent Degradation domain). This domain has the property of regulating degradation depending on oxygen concentration. Normally, when oxygen concentration is high, ubiquitination is induced via this domain, and degradation progresses. On the other hand, under hypoxic conditions, degrading activity is inactivated, increasing the stability of glucose transporters. Degradation inhibitors are also important components. This factor inhibits ubiquitination and degradation by the proteasome system, helping to stabilize glucose transporters. A specific example is the regulation of pVHL (von Hippel-Lindau protein) activity. It also has transcriptional and translational regulatory functions.This factor regulates the transcription and translation of glucose transporters under hypoxic conditions, preventing their degradation and ensuring their stability in the cell membrane. These components work together to suppress degradative activity in response to changes in oxygen concentration, playing an important role in enabling cells to maintain their energy supply even in hypoxic environments. Thus, "on-demand expression and / or enhancement specifically achieved in the tumor microenvironment" refers to the phenomenon in which the expression and / or enhancement of specific genes and proteins is induced in response to characteristic conditions of the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deficiency, presence of inflammatory cytokines, etc.), and is controlled so that its expression is minimized or absent in normal tissues other than the tumor. This phenomenon is important for developing tumor-specific therapeutic targets and designing therapeutic strategies with reduced side effects. Tumor-environment-specific regulatory mechanisms can enhance therapeutic efficacy while minimizing impact on normal tissues. Below, we explain the key features of the phenomenon of on-demand expression and / or enhancement achieved specifically in the tumor environment. First, environmental regulation is a characteristic of this phenomenon. This refers to the mechanism by which gene and protein expression is induced or enhanced in response to specific conditions in the tumor microenvironment, such as hypoxia, acidic pH, nutrient deprivation, and inflammatory signals. A specific example is the induction of gene expression in a hypoxic environment by HIF-1 (hypoxia response factor). Second, tumor specificity is a characteristic of this phenomenon. This refers to the property that these genes and proteins are rarely expressed or expressed at very low levels in normal tissues other than the tumor, while their expression and function are specifically activated in the tumor microenvironment. Furthermore, on-demand is an important feature. Expression and enhancement are regulated as needed when specific conditions are met, such as changes in the tumor environment, such as a decrease in oxygen concentration or an increase in inflammatory cytokines. This property allows for a mechanism that allows it to function efficiently only in the tumor environment.Finally, this phenomenon lends itself to therapeutic targeting. Genes and proteins that are expressed or enhanced specifically in tumors can be used as targets for cancer therapy. By utilizing this property, it is possible to design therapeutic strategies that enhance therapeutic efficacy against tumors while minimizing side effects on normal tissues. Due to these characteristics, a mechanism for achieving on-demand expression and / or enhancement specifically in the tumor environment is a very promising element in new approaches to cancer treatment. Utilizing these properties is expected to lead to the development of more effective and safer therapies.

[0061] As used herein, "modification" in relation to a gene (nucleic acid, protein, etc.) refers to inserting a base sequence of a nucleic acid in a cell, changing a base sequence of a nucleic acid, deleting a portion of a base sequence of a nucleic acid in a cell, or a combination thereof.

[0062] As used herein, the term "disease" is broadly interpreted to refer to a state of mental or physical discomfort or inconvenience in humans or animals, and refers to any condition that cannot be described as a healthy state, such as illness, disability, or various symptoms, which are not specifically defined. Diseases that may be the subject of the present disclosure include, but are not limited to, diseases in which an immune response may be associated, such as cancer, autoimmune diseases, allergies, and infectious diseases.

[0063] As used herein, a subject having an "immune response" to a certain component or substance means that some kind of immune reaction occurs against the component or substance. The component or substance can be identified by observing changes in various immune cells or increases or decreases in immune-related substances (e.g., cytokines) in the subject or biological components (e.g., cells) derived from the subject. The response can be determined by objective indicators or by subjective judgment based on the experience of a physician or other professional.

[0064] As used herein, whether a subject "has immunological memory" of a certain component or substance can be evaluated by measuring whether the component or substance (i) enhances cytokine production in an antigen-dependent manner in memory CD4-positive T cells or has a proliferation-promoting effect in the subject or in a biological component (e.g., cell, etc.) derived from the subject, (ii) alters the expression of a surface antigen on memory regulatory T cells, (iii) changes the ratio of Treg to Th1, (iv) induces IFN-γ production from T-bet-positive Th1 cells, (v) alters the ability to produce IFN-γ, (vi) alters the ability to produce IL-2, and (vii) alters the ability to produce TNF-α, or (viii) has an antibody specific to the component or substance in the blood, and confirming that at least one of these results is positive.

[0065] As used herein, the term "infectious disease" refers to any infectious disease, and encompasses any type of infectious disease, such as viral infections (including any viral form, such as single-stranded or double-stranded DNA viruses and RNA viruses), bacterial infections, protozoan infections, and mycoplasma infections. Examples of such infectious diseases include tuberculosis, coronavirus, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonella infection, pathogenic E. coli, toxoplasmosis, Zika virus, herpesvirus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza, MERS, rabies, and diphtheria.

[0066] As used herein, the term "immune abnormality" refers to any disease, disorder, or condition that is caused or suspected to be caused at least in part by an abnormality in the immune system. It refers to a condition in which an abnormality in immunity occurs due to some cause, resulting in susceptibility to infectious diseases or allergic reactions. Immune abnormalities include, but are not limited to, allergies, autoimmune diseases, etc. When the immune response is directed against a self-antigen, it is generally referred to as an autoimmune disease, and when directed against an external antigen, it is referred to as an allergy. A strong immune response can be said to result in an autoimmune disease state against self-antigens and an allergic state against non-self antigens, while a weak immune response can result in a cancer state against self-antigens and an infectious disease state against non-self antigens.

[0067] As used herein, the term "autoimmune disease" refers to an excessive immune response to a specific self-antigen. Autoimmune diseases are diseases caused by a breakdown of immune tolerance, in which the immune system, which is responsible for recognizing and eliminating foreign substances, overreacts and attacks the body's own normal cells and tissues, resulting in symptoms.Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus erythrothematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leakage of leukocytes; central nervous system disorders CNS (Clinical System) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP), autoimmune uveitis, or autoimmune thrombocytopenia.

[0068] As used herein, "allergy" refers to an excessive immune response to a specific non-self antigen, and is a disease in which an immune response occurs to an "allergen." An "allergen" refers to an antigen that can react with the antibody of a subject with an allergic disease, and includes allergens derived from the pollen of trees (acacia, alder, velvet beech, beech, birch, maple, mountain cedar, red cedar, cottonwood, cypress, American elm, autumn elm, Douglas fir, rubber tree, eucalyptus tree, Chinese hackberry, hickory, American linden, sugar maple, mesquite, paper mulberry, and kona). Allergens derived from plant pollen (cotton, larkspur, longgrass, bromegrass, corn, broadleaf fescue, sorghum, oat, orchard grass, ricegrass, morning glory, ryegrass, rice, morning glory, timothy grass, water hyacinth, pigweed, etc.), and allergens derived from plant pollen (cotton, larkspur, longgrass, bromegrass, corn, broadleaf fescue, sorghum, oat, orchard grass, rice bran, morning glory, timothy grass, pigweed, corn, Allergens derived from insects (silkworms, mites, honeybees, wasps, ants, cockroaches, etc.), allergens derived from bacteria (alternaria, aspergillus, botulinum, candida, cephalosporin, etc.), allergens derived from insects (silkworms, mites, honeybees, hornets ... Examples of allergies include, but are not limited to, allergens derived from animal hair (dog, cat, bird, etc.), allergenic proteins derived from house dust, and allergens derived from food (OVA, etc.). Representative diseases of "allergy" include atopic dermatitis, allergic rhinitis (hay fever, etc.), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergies, drug allergies, and urticaria.

[0069] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0070] In one aspect, the present disclosure provides immune cells such as T cells with enhanced glucose uptake ability on demand, cell populations containing such cells, pharmaceutical compositions containing such cells or cell populations, and other related technologies. The immune cells such as T cells with enhanced glucose uptake ability on demand and cell populations containing such cells are typically T cells with enhanced glucose uptake ability, which have or have been modified to regulate the expression of a glucose transporter in the T cells in response to changes in tumor microenvironment conditions, or which contain a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

[0071] In another aspect, the present disclosure provides immune cells such as T cells with enhanced glucose uptake ability, in which the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions, a cell population containing such cells, a pharmaceutical composition containing such cells or cell population, and other related technologies.

[0072] In yet another aspect, the present disclosure provides a T cell comprising a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

[0073] In one aspect of the present disclosure, T cells with enhanced glucose uptake capacity (T ｅｆｆThe present invention also provides T cells that have been modified to express a glucose transporter on demand and / or have enhanced glucose transporter expression on demand. T cells that have been modified to express a glucose transporter on demand and / or have enhanced glucose transporter expression on demand, or that regulate glucose transporter expression in the T cells in response to changes in tumor microenvironment conditions, or that contain a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions, are not previously anticipated. T cells with constitutively enhanced glucose uptake capacity differentiate into effector memory T cells and become exhausted, and significant T cell death is induced when activated under low- or no-glucose conditions. However, cells with enhanced glucose transporter expression on demand are notable in that they maintain equivalent cytotoxic activity while avoiding T cell death under low- or no-glucose conditions, maintaining a memory phenotype that is expected to maintain activity for a longer period, and inducing resistance to exhaustion. The cells of the present disclosure advantageously have enhanced glucose uptake capacity. Such cells with enhanced glucose uptake capacity on demand are significantly effective against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc.

[0074] Furthermore, when used in CAR-T therapy and the like, the cells of the present disclosure demonstrated a significantly high recurrence prevention effect. Additionally, sudden death in mice due to toxicity caused by non-tumor factors, which was observed in T cells with constitutively enhanced glucose levels, was avoided by T cells in which a glucose transporter was expressed or enhanced on demand, or in which glucose transporter expression was regulated in the T cells in response to changes in tumor microenvironment conditions, or which contained a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. These findings were unexpected, and without being bound by theory, may be explained by avoidance of host toxicity caused by excessive T cell activation or excessive glucose uptake, a significant reduction in exhaustion, nutrient supply in line with nutritional needs, and suppression of cell death and induction of memory due to appropriate activation. Furthermore, the cells of the present disclosure preferably have effector function after being introduced into the location where they are to function, particularly into the body. Examples of such cells include T ｅｆｆ These include, but are not limited to, lymphoid progenitor cells, Tnv, Tscm, Tempra, and the like.

[0075] In one embodiment, in a cellular aspect of the present disclosure, the tumor microenvironment conditions include the degree of TCR-associated signal transduction. In this preferred embodiment, the TCR-associated signal may include a TCR signal or a portion thereof, a chimeric signal of a TCR signal and another antigen (also referred to as a CAR signal), etc.

[0076] In a preferred embodiment, in the cellular aspect of the present disclosure, the TCR-associated signal is mediated by a molecule comprising a TCR signal domain. Such a molecule may be a nucleic acid molecule.

[0077] In a preferred embodiment, the alteration of tumor microenvironment conditions comprises the presentation of tumor antigens and / or costimulatory ligands, and / or alterations in cytokine conditions.

[0078] In certain embodiments, the degree of TCR-associated signal transduction as used in the present disclosure includes at least one selected from the group consisting of the degree of TCR stimulation, TCR costimulation, and humoral factors. In certain embodiments, the degree of TCR-associated signal transduction includes the degree of TCR stimulation and at least one selected from the group consisting of TCR costimulation and humoral factors. In another embodiment, the degree of TCR-associated signal transduction includes the degree of TCR stimulation. In another embodiment, the degree of TCR-associated signal transduction includes the degree of TCR stimulation, TCR costimulation, and humoral factors. One or more of the strength of TCR stimulation, the influence of costimulatory molecules, and the action of humoral factors may be combined. TCR stimulation is initiated by the T cell receptor recognizing an antigen peptide bound to a major histocompatibility complex (MHC) on an antigen-presenting cell (APC). The strength and duration of this recognition have a significant impact on the degree of signal transduction. Strong TCR stimulation promotes T cell activation, proliferation, and even effector function, while weak TCR stimulation can result in partial activation or anergy. Furthermore, the involvement of costimulatory molecules is essential for TCR signaling. Costimulatory molecules provide a second signal necessary for T cell activation and amplify TCR signals. For example, CD28 is a stimulatory costimulatory molecule that enables full T cell activation, while inhibitory CTLA-4 acts to suppress the immune response. The presence of appropriate costimulatory signals enhances the immune response and enables the formation of memory T cells and sustained immune responses. On the other hand, the lack of costimulation leads to immune tolerance, suppressing excessive immune responses to self-antigens. Furthermore, humoral factors, particularly cytokines, play an important role in regulating the direction of T cell activation and differentiation. For example, IL-2 promotes T cell proliferation, while IL-10 and TGF-β suppress the immune response. Furthermore, specific cytokines secreted in inflammatory environments, such as IL-12 and IL-4, induce differentiation into Th1 and Th2 cells, respectively, and determine the nature of the immune response. Thus, humoral factors fine-tune the immune response, enabling an effective response adapted to the pathogen.The immune response is adaptively and flexibly regulated by TCR stimulation, costimulation, and humoral factors, either singly or through the interaction of multiple factors. This enables rapid and specific immune defense against pathogens, while at the same time maintaining the overall balance of the immune system, preventing autoimmune diseases and maintaining immune tolerance. This mechanism is essential for T cells to respond optimally to the environment and stimuli, and supports the diversity and functionality of the immune system.

[0079] In a particular embodiment, TCR stimulation may involve NFAT, NF-κB (nuclear factor-kappaB), the STAT transcription factor family (including, but not limited to, seven types: STAT1-4, STAT5A, STAT5B, and STAT56), and the like, which translocate into the nucleus upon activation of T cells.

[0080] In another embodiment, the tumor microenvironment conditions include the level of oxygen concentration. The level of oxygen concentration in the tumor microenvironment is an important factor that significantly influences tumor progression and therapeutic response. Due to abnormal vascular formation and structural heterogeneity, tumor tissue often experiences a shortage of oxygen compared to normal tissue, a condition known as "hypoxia." Hypoxia has a wide range of effects on tumor cell metabolism, proliferation, immune evasion, and therapeutic resistance. First, tumor cells adapt to hypoxic conditions by activating hypoxia-inducible factors (HIFs). HIFs promote the expression of angiogenic factors (e.g., VEGF) in tumor cells and induce the formation of new blood vessels. However, these new blood vessels have abnormal structures, contributing to further hypoxia. HIFs also alter the metabolic pathways of tumor cells, promoting the "Warburg effect," which prioritizes glycolysis, which does not require oxygen. This metabolic change not only sustains tumor cell proliferation but also creates an acidic environment and suppresses immune cell activity.

[0081] In one embodiment, glucose transporters operably linked to tumor microenvironment-sensing elements that detect changes in tumor microenvironment conditions are used. In this context, glucose transporters linked to tumor microenvironment-sensing elements that detect characteristics of the tumor microenvironment, such as oxygen concentration, pH, redox state, and inflammatory cytokines, are the subject of innovative research that exploits tumor-specific metabolic properties. Such transporters may be useful for therapeutic and diagnostic applications by responding to changes in the tumor microenvironment and specifically participating in tumor cell energy supply and metabolic pathways. For example, linking a hypoxia-inducible factor-1 (HIF-1) response element, which is specifically activated in hypoxic environments, to a glucose transporter can be engineered to increase glucose uptake in hypoxic environments. Such transporters can be used for the development of drug delivery and diagnostic probes targeting hypoxic conditions. Furthermore, because the tumor environment is characterized by a decreased pH, incorporating an acidic environment-sensing element into a glucose transporter can construct a transporter that is specifically activated in the acidic environment in which tumor cells reside. This mechanism contributes to the design of therapeutic approaches tailored to acidic environments. Furthermore, glucose transporters can be engineered with redox-sensing elements to respond to the redox state of the tumor microenvironment. Because tumor cells are often exposed to higher levels of oxidative stress than normal, such transporters would function specifically in these environments and be useful as tumor-cell-targeted therapeutic and diagnostic tools. Furthermore, by incorporating inflammatory cytokine (e.g., IL-6 or TNF-α) response elements, transporters can be engineered to sense the inflammatory state in the tumor microenvironment and adjust glucose transport accordingly. This could potentially improve the identification and therapeutic efficacy of inflammatory tumors. Thus, glucose transporters with tumor microenvironment-sensing elements could serve as a platform for utilizing the specific metabolic environment of tumors to deliver more effective and specific diagnostic and therapeutic strategies.

[0082] In one embodiment, the tumor microenvironment sensing element comprises (i) an inducible activation motif (e.g., HIH-RE, NFAT-RE) that is activated in response to a change (e.g., deterioration) in the tumor microenvironment conditions, and (ii) a nucleic acid sequence encoding a degradation factor of the glucose transporter (e.g., ODD) that is inactivated in response to a change (e.g., deterioration) in the tumor microenvironment conditions.

[0083] In one embodiment, the tumor microenvironment sensing element comprises: (i) an inducible activation motif (e.g., NFAT-RE) to which a nuclear localization factor that translocates into the nucleus in response to T cell activation binds; (ii) a nuclear localization-binding inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to a decrease in oxygen concentration binds; and (iii) a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading a glucose transporter is inactivated in response to the decrease in oxygen concentration.

[0084] In another embodiment, the construct comprises a nucleic acid sequence encoding a glucose transporter operably linked to an inducible activation motif that is activated in response to changes in tumor microenvironment conditions and / or a nucleic acid sequence encoding a degradation factor of said glucose transporter that is inactivated in response to changes in said tumor microenvironment conditions.

[0085] In another embodiment, the construct comprises a nucleic acid sequence encoding a glucose transporter operably linked to a nuclear import-inducing activation motif to which a nuclear import factor that translocates into the nucleus in response to T cell activation binds.

[0086] In yet another embodiment, nuclear import factors that can be used include NFAT, NF-κB (nuclear factor-kappaB), the STAT transcription factor family (STAT1-4, STAT5A, STAT5B, STAT56, etc.), and the like.

[0087] In certain embodiments, the nuclear translocation binding-inducing activation motif may be a NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ ID NO: 1), a STAT-binding sequence (TTCNNNGAA), or a modified sequence thereof, which may contain one, two, three, or more substitutions (replacement of ATGC with each other) compared to the original sequence.

[0088] In certain embodiments, the combination of a nuclear transport factor and the nuclear transport binding-inducing activation motif includes: (1) a combination of NFAT and an NFAT-binding domain; (2) a combination of NF-κB (nuclear factor-kappaB) and a κB motif (GGGACTTTCC (SEQ ID NO: 1)); or (3) a combination of a member of the STAT transcription factor family (seven types: STAT1-4, STAT5A, STAT5B, and STAT56) and a STAT binding sequence (TTCNNNGAA).

[0089] In certain embodiments, the construct comprises a nucleic acid sequence encoding a glucose transporter operably linked to a nuclear translocation binding-induced activation motif to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds. In this embodiment, the nuclear translocation factor may be HIF-1 or the like. Also in this embodiment, the nuclear translocation binding-induced activation motif may be HIF-RE or the like.

[0090] In certain embodiments, the combination of a nuclear import factor and a nuclear import binding-inducing activation motif may be, for example, (1) a combination of HIF-1 and HIF-RE.

[0091] In another embodiment, the construct comprises a nucleic acid sequence encoding a glucose transporter operably linked to a nucleic acid sequence encoding a factor whose activity in degrading the glucose transporter in response to a decrease in oxygen concentration is inactivated. In this embodiment, the factor to be inactivated may be an oxygen-dependent degradation factor (ODD), etc.

[0092] In certain embodiments, the T cells of the present disclosure comprise a chimeric antigen receptor (CAR).

[0093] In another embodiment, the T cells have effector function. Alternatively, the cells are effector T cells (T ｅｆｆ ) precursor cells.

[0094] In certain embodiments, the precursors of effector T cells of the present disclosure have extended memory function. The property of extending the memory function of effector T cell precursors is highly advantageous in terms of improving the durability and effectiveness of immune responses. This property is described below. While precursors of effector T cells have the ability to exert effector function in response to antigen stimulation, memory T cells (Memory T These cells have the potential to differentiate into T cells (cells). This allows them to maintain immune memory against antigens for a long period of time, even after the initial immune response has subsided. In particular, extending memory function is advantageous in that it increases the ability to induce a rapid and powerful immune response in the event of reinfection. First, extending memory function strengthens immune defense against tumors and other diseases. The presence of long-term memory cells enables a rapid and effective secondary immune response when the same antigen invades again. This increases the probability that tumors and other diseases will be eliminated before they spread within the body, preventing the disease from becoming severe. Furthermore, extending memory function is also a factor in improving the effectiveness of vaccines. The progenitor cells of effector T cells formed after vaccination retain memory function for a long period of time, extending the duration of immune memory and making it possible to reduce the frequency of additional vaccinations (booster vaccinations). This is important for improving the efficiency of vaccine strategies and reducing the burden on patients. The memory function of effector T cell precursors is important in the field of tumor immunology. Tumor cells often hide antigens or create an immunosuppressive environment. However, the presence of T cells with long-term memory function is expected to maintain immune surveillance of tumor cells and reduce the risk of recurrence. In particular, combining these cells with immune checkpoint inhibitors or CAR-T cell therapy may result in more durable and potent antitumor effects. Furthermore, even in conditions such as chronic infections and autoimmune diseases, effector T cell precursors with memory function contribute to maintaining appropriate immune responses and pathological control. This may enable long-term surveillance and control of specific pathogens and abnormal cells, while suppressing unnecessary inflammation and tissue destruction. Overall, the ability of effector T cell precursors to extend their memory function offers significant advantages in infectious disease and tumor treatment, as well as vaccine development.Utilizing this property will enable the induction of more sustained and effective immune responses, which is expected to contribute to the development of medicine and biotechnology.

[0095] In another embodiment, the effector T cell precursors maintain the memory T cell phenotype. Maintaining the memory T cell phenotype in effector T cell precursors offers numerous advantages. This characteristic is crucial for improving long-term immune memory and the efficiency of adaptive immune responses. First, maintaining the memory T cell phenotype in effector T cell precursors enables rapid and efficient immune responses upon reinfection. Memory T cells retain antigen-specific memory and are rapidly activated and exert effector function upon antigen reinvasion. This effectively eliminates tumors and other pathogens before they spread, preventing their progression. Maintaining the memory T cell phenotype improves the durability of immune responses. While effector T cells are short-lived, memory T cells persist in the body for long periods, thereby prolonging the retention of immune memory formed after vaccination or natural infection. This characteristic is particularly advantageous in terms of reducing the frequency of booster vaccinations and providing sustained immune protection. Maintaining the memory T cell phenotype is important in tumor immunity. Tumor cells often evade immune system surveillance by exploiting antigenic mutations or an immunosuppressive environment. However, T cells with long-term immunological memory continuously monitor tumor recurrence and metastasis. This potentially reduces the risk of recurrence after treatment and improves patient outcomes. Furthermore, they contribute to the control of chronic infectious diseases. For example, sustained immunological memory is important for suppressing the progression of pathogens such as tuberculosis and HIV, and for managing the disease. Maintaining the memory T cell phenotype enables long-term surveillance and control of pathogens. This could also be applied to the control of autoimmune diseases and immunotherapy. By maintaining the memory T cell phenotype and appropriately adjusting the immune response, it is possible to induce selective immune responses against pathogens and abnormal cells while suppressing excessive immune responses. Overall, maintaining the memory T cell phenotype in effector T cell precursors offers significant advantages in the treatment and prevention of infectious diseases, tumors, and chronic diseases. Understanding and utilizing this property may lead to the development of more effective and sustained immunotherapies and vaccine strategies.

[0096] In certain embodiments, effector T cells are activated or activity is maintained, and precursors of said effector T cells have extended memory function or maintained memory T cell phenotype. Both extended memory function and maintained memory T cell phenotype may be achieved, resulting in a synergistic effect.

[0097] The present disclosure is effective against all tumors, including solid tumors. Clinical trials of CAR-T cells against solid tumors have failed to demonstrate efficacy. However, the present inventors have demonstrated that dysfunction of CAR-T cells in solid tumors and other tumors is due to glucose depletion in the tumor microenvironment (TME) or its equivalent tumor microenvironment (TMEC), and that on-demand, rather than continuous, metabolic supplementation significantly improves the antitumor efficacy of CAR-T cells. Massive glucose consumption by cancer cells reduces glucose levels in the TME of solid tumors and other tumors, resulting in CAR-T cell impairment. We have found that stably expressing a high-affinity glucose transporter (GLUT, e.g., GLUT3) in CAR-T cells restores cytokine production and killing activity. However, while CAR-T cells stably expressing GLUT induced tumor regression in solid tumor models, their overactivation led to adverse events and mouse death. CAR-T cells into which GLUT was introduced on demand showed that, upon stimulation with a target antigen, nuclear factor of activated T cells (NFAT), used as an example, was translocated into the nucleus and bound to tumor microenvironment-sensing elements, inducing GLUT transcription or inhibiting GLUT degradation, thereby improving metabolic fitness and enhancing antitumor efficacy, preventing adverse events and resulting in long-term tumor control in an intracranial human GBM cell xenograft model. The present inventors have demonstrated that on-demand yet stable metabolic fitness, such as upon exposure to tumor antigens, is a novel concept for maximizing the antitumor effect of CAR-T cells against solid tumors.

[0098] In certain embodiments, the present disclosure demonstrates that dysfunction of CAR-T cells in GBM is due to glucose deprivation in the tumor microenvironment (TME) and that on-demand, rather than continuous, metabolic supplementation significantly improves the antitumor efficacy of CAR-T cells. Massive glucose consumption by cancer cells reduces glucose levels in the TME of GBM, resulting in CAR-T cell impairment. Stable expression of the high-affinity glucose transporter GLUT3 in CAR-T cells restored cytokine production and killing activity. However, while CAR-T cells stably expressing GLUT3 induced tumor regression in a GBM model, their overactivation led to adverse events and mouse death. On-demand GLUT3 CAR-T cells, which induce GLUT3 transcription through nuclear translocation of nuclear factor of activated T cells (NFAT) upon target antigen stimulation, exhibit improved metabolic fitness and enhanced antitumor efficacy, preventing adverse events and resulting in long-term tumor control in an intracranial human GBM cell xenograft model. We propose that on-demand yet stable metabolic fitness, such as upon exposure to tumor antigens, represents a novel concept for maximizing the antitumor efficacy of CAR-T cells against solid tumors.

[0099] In one aspect, glioblastoma (GBM), targeted by the present disclosure, is a devastating malignant brain tumor with an annual incidence rate of 3.19 per 100,000 people. The advent of a combination therapy consisting of temozolomide and radiation in 2005 led to the development of oncolytic field therapy (Novo TTF) for primary GBM, improving the prognosis of GBM patients in clinical settings. However, the 5-year overall survival rate for GBM remains below 20% [References 1, 2]. Given the clinical success of chimeric antigen receptor T cell (CAR-T cell) therapy in hematological malignancies, several preclinical and clinical trials of CAR-T cell therapy for GBM are being actively investigated [References 3-10]. While some patients treated in clinical trials have shown promising results, such as CAR-T cell infiltration into the tumor microenvironment (TME) and reduction in tumor burden, most patients do not respond to CAR-T cell therapy [Reference 3].

[0100] GBM possesses a metabolically challenging TME, impairing the function of effector T cells, including CD8+ T cells, and adoptively transferring CAR-T cells into the TME. In the brain, neural cells, such as neurons and glial cells, require glucose as an energy source, and the brain consumes 25% of the total body's glucose-derived energy

[11] . Furthermore, aggressive GBM cells consume three times more glucose than normal neurons due to aerobic glycolysis (known as the Warburg effect), a characteristic metabolic pathway of cancer cells

[12] . This leads to glucose depletion in the GBM TME [13-16]. On the other hand, naive T cells shift their energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to meet their increased energy demands. Therefore, intense metabolic competition via glucose occurs between activated T cells and cancer cells in the GBM TME. GLUT3, encoded by SLC2A3 and physiologically expressed in neurons and glial cells, has a five-fold higher affinity for glucose than GLUT1, the major glucose transporter expressed in T cells [16-18]. As a result, GLUT3-expressing GBM cells can survive and proliferate continuously within the TME with sufficient glucose uptake, whereas activated T cells lose their function due to metabolic competition failure [19, 20].

[0101] Given the significant glucose uptake by GBM cells via high-affinity GLUT3, equal or greater expression of GLUT3 by CAR-T cells may enable them to fully compete with GBM cells. In this study, we developed a construct linking SLC2A3 to CAR to induce GLUT3 expression in CAR-T cells. However, stable GLUT3 expression induced excessive activation of CAR-T cells, indicating that effector T cells require delicate energy replenishment. Therefore, we altered GLUT3 expression to be driven by glucose demand (on-demand expression) upon activation of CAR-T cells following target antigen exposure in the TME. CAR-T cells with on-demand GLUT3 expression restored metabolic fitness through nuclear factor of activated T cells (NFAT) nuclear translocation upon target antigen stimulation, thereby restoring metabolic fitness and resulting in enhanced and sustained antitumor efficacy and prolonged survival in the harsh TME of a low-glucose GBM model. In summary, the concept of restoring T cell function in the TME by increasing metabolic fitness needs to be explored in an on-demand manner rather than through steady replenishment.

[0102] (Cell Population) In one aspect of the present disclosure, a cell population is provided that includes immune cells, such as T cells, with enhanced glucose uptake capacity on demand. Alternatively, a cell population is provided that includes T cells with enhanced glucose uptake capacity, wherein the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions. Alternatively, a cell population is provided that includes T cells with enhanced glucose uptake capacity that have been modified so that the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions. Alternatively, a cell population is provided that includes T cells that include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. It has been unexpectedly shown that at least a portion of the T cells included in such a cell population have been modified and / or enhanced to express a glucose transporter, and that when applied to CAR-T cells, etc., they have a remarkable effect on diseases such as cancer ( FIG. 19 ). Advantageously, the cell population of the present disclosure has enhanced glucose uptake capacity. Furthermore, the cell population of the present disclosure preferably contains cells that have effector functions after being introduced into the place where they are to function, particularly into the body. ｅｆｆ These include, but are not limited to, lymphoid progenitor cells, Tnv, Tscm, Tempra, and the like.

[0103] While cell populations containing T cells with constitutively enhanced glucose uptake capacity have been observed to differentiate into effector memory T cells and become exhausted, cell populations containing cells with enhanced glucose transporter expression on demand are notable in that they maintain equivalent cytotoxic activity while also maintaining a memory phenotype that is expected to maintain its effect for a longer period of time and inducing exhaustion resistance. Advantageously, the cell population of the present disclosure is a cell population containing cells with enhanced glucose uptake capacity. When applied to CAR-T cells, TCR-T cells, etc., such cell populations exhibit remarkable effects against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases.

[0104] Furthermore, when used with CAR-T cells and the like, the cell population of the present disclosure demonstrated a significantly high recurrence prevention effect. Additionally, sudden death in mice due to toxicity caused by non-tumor factors, which was observed in T cells with constitutively enhanced glucose levels, was avoided in T cells in which a glucose transporter was expressed or enhanced on demand, or in which glucose transporter expression was regulated in the T cells in response to changes in tumor microenvironment conditions, and the T cells contained a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions. These findings were unexpected, and without being bound by theory, possible explanations for them include avoidance of host toxicity caused by excessive T cell activation or excessive glucose uptake, a significant reduction in exhaustion, nutrient supply in line with nutritional needs, and suppression of cell death and induction of memory due to appropriate activation. Furthermore, cell populations containing the cells of the present disclosure preferably have effector function after being introduced into the location where they are to function, particularly into the body. Examples of such cells include T ｅｆｆ Examples include, but are not limited to, T ｅｆｆ The progenitor cells may be lymphoid progenitor cells, such as lymphoid progenitor cells, Tnv, Tcm, and Temra.

[0105] In one embodiment of the present disclosure, a cell population comprising immune cells such as T cells as described above is advantageous in that the cell population comprises about 10% or more T cells having the characteristics of the present disclosure (e.g., on-demand enhancement or expression of GLUT, effector function, or both). In one embodiment, the cell population of the present disclosure can have a percentage of T cells having the characteristics of the present disclosure of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0106] In one embodiment, the cell population of the present disclosure comprises T cells that have the property of having effector function when introduced into the body.

[0107] In one embodiment, the cell population of the present disclosure, the T cells comprise a chimeric antigen receptor (CAR).

[0108] In one embodiment, in the cell population of the present disclosure, the T cells are engineered to express a glucose transporter on demand and / or have enhanced expression of a glucose transporter on demand. Alternatively, the cells in the cell population of the present disclosure can be T cells with enhanced glucose uptake capacity, wherein expression of the glucose transporter is regulated in the T cells in response to changes in tumor microenvironment conditions, or T cells with enhanced glucose uptake capacity, wherein expression of the glucose transporter is regulated in the T cells in response to changes in tumor microenvironment conditions, and wherein the T cells comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

[0109] In one embodiment, in the cell population of the present disclosure, the T cells have effector function. Alternatively, in the T cells in the cell population of the present disclosure, the tumor microenvironment conditions include the degree of TCR-associated signal transduction, and in certain embodiments, the TCR-associated signal is mediated by a molecule selected from the group consisting of a TCR signal or a portion thereof, and a chimeric signal of a TCR signal and another antigen (also referred to as a CAR signal), and preferably, the TCR-associated signal is mediated by a molecule comprising a TCR signal domain.

[0110] Alternatively, the change in tumor microenvironment conditions comprises the presentation of tumor antigens and / or costimulatory ligands and / or changes in cytokine conditions, and preferably, the degree of TCR-associated signal transduction comprises the degree of TCR stimulation. Alternatively, in another embodiment, the degree of TCR-associated signal transduction comprises at least one selected from the group consisting of the degree of TCR stimulation, TCR costimulation, and humoral factors.

[0111] In another embodiment, in cells in the cell population of the present disclosure, the degree of TCR-associated signal transduction comprises the degree of TCR stimulation and at least one selected from the group consisting of TCR costimulation and humoral factors. In one embodiment, the degree of TCR-associated signal transduction comprises the degree of TCR stimulation, TCR costimulation, and humoral factors. Preferably, the TCR stimulation is selected from the group consisting of NFAT, NF-κB, and the STAT transcription factor family, which translocate into the nucleus upon T cell activation. Alternatively, the tumor microenvironment conditions include the degree of oxygen concentration.

[0112] In another embodiment, in a cell population comprising T cells comprising a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element of the present disclosure that senses changes in tumor microenvironment conditions, the tumor microenvironment sensing element for each T cell comprises (i) an inducible activation motif that is activated in response to changes in tumor microenvironment conditions, and (ii) a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to changes in the tumor microenvironment conditions. Here, the tumor microenvironment sensing element preferably comprises (i) an inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to T cell activation binds, (ii) a nuclear translocation-binding inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds, and (iii) a nucleic acid sequence encoding a glucose transporter degradation enzyme whose activity of degrading the glucose transporter is inactivated in response to the decrease in oxygen concentration.

[0113] In another embodiment, with respect to a T cell in the cell population of the present disclosure, the nucleic acid sequence encoding a glucose transporter comprises a construct operably linked to an inducible activation motif that is activated in response to changes in tumor microenvironment conditions and / or a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to changes in the tumor microenvironment conditions. In one embodiment, the nucleic acid sequence encoding the glucose transporter comprises a construct operably linked to a nuclear translocation-binding inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to T cell activation binds. In a preferred embodiment, the nuclear translocation factor is selected from the group consisting of NFAT, NF-κB, and the STAT transcription factor family (e.g., TAT1-4, STAT5A, STAT5B, and STAT5, etc.), and the nuclear translocation-binding inducible activation motif is selected from the group consisting of an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ ID NO: 1), and a STAT binding sequence (TTCNNNGAA), or a modified sequence thereof. In a preferred embodiment, the combination of a nuclear transport factor and the nuclear transport binding-inducing activation motif comprises (1) a combination of NFAT and an NFAT-binding domain, (2) a combination of NF-κB and a κB motif (GGGACTTTCC (SEQ ID NO: 1)), or (3) a combination of a STAT transcription factor family and a STAT binding sequence (TTCNNNGAA).

[0114] In another embodiment, with respect to a T cell in the cell population of the present disclosure, the T cell comprises a construct in which a nucleic acid sequence encoding a glucose transporter is operably linked to a nuclear translocation-binding-inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds. Preferably, the nuclear translocation factor is selected from the group consisting of HIF-1, and the nuclear translocation-binding-inducible activation motif is selected from the group consisting of HIF-RE, or the combination of the nuclear translocation factor and the nuclear translocation-binding-inducible activation motif is selected from the group consisting of (1) a combination of HIF-1 and HIF-RE.

[0115] In another embodiment, with respect to a T cell in the cell population of the present disclosure, the T cell comprises a construct in which a nucleic acid sequence encoding a glucose transporter is operably linked to a nucleic acid sequence encoding a factor whose activity in degrading the glucose transporter in response to a decrease in oxygen concentration is inactivated, and preferably the factor to be inactivated is selected from the group consisting of oxygen-dependent degradation factors (ODDs).

[0116] In another embodiment, with respect to the T cells in the cell population of the present disclosure, the T cells have effector function and the T cells are effector T cells (T ｅｆｆ ) precursor cells, wherein the precursor cells of effector T cells have extended memory function and / or maintain memory T cell phenotype. Alternatively, effector T cells are activated or activity is maintained and the precursor cells of said effector T cells have extended memory function or maintain memory T cell phenotype.

[0117] In another embodiment, with respect to T cells in the cell populations of the present disclosure, expression and / or enhancement is achieved specifically in the tumor environment.

[0118] In one embodiment, the cell population of the present disclosure is T ｅｆｆ , and / or T ｅｆｆ Preferably, the cell population (or T ｅｆｆ The progenitor cells of the lymphoid lineage include at least one of lymphoid progenitor cells, Tnv, Tscm, Tcm, and Tempr.

[0119] In one embodiment, in the cell population of the present disclosure, the CAR is expressed by the T cells.

[0120] In one embodiment, in the cell population of the present disclosure, the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, preferably GLUT3.

[0121] In one embodiment, in the cell population of the present disclosure, the T cells are human T cells.

[0122] In one embodiment, the cell population of the present disclosure is provided as a medicament or pharmaceutical composition. Its use may be for the prevention or treatment of cancer, for the cure of cancer, or for the prevention of metastasis or recurrence of cancer. In a specific embodiment, the cell population of the present disclosure may be used to prevent or treat cancer so that it does not recur.

[0123] <Glucose transporter> Cancer cells reprogram their metabolic system to favor their own cell proliferation, actively utilizing the glycolytic pathway, which is less efficient in producing ATP even in the presence of oxygen, to increase glucose uptake and lactate production (Warburg effect). Therefore, cancer cells consume and deplete large amounts of glucose, and T cells, which require glucose as an energy source, are depleted. ｅｆｆ When tumor-specific T cells of this type infiltrate tumors, they receive signals from TCR but do not respond to the Ca ２＋ The concentration drops, leading to starvation and exhaustion.

[0124] Thus, in one embodiment of the present disclosure, T cells and cell populations are provided that are genetically engineered to express glucose transporters to promote aerobic glycolysis and increase the competitiveness and / or fitness of immune cells in a glucose-depleted tumor microenvironment (TME).

[0125] In one embodiment, glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, and among them, the glucose transporter with the highest affinity for glucose is GLUT3. Thus, in a preferred embodiment, the glucose transporter used in the cells of the present disclosure is GLUT3.

[0126] GLUT3 was first identified in the mouse brain and was initially defined as a neuronal glucose transporter. GLUT3 was subsequently shown to be expressed in other glucose-requiring cells, such as mouse sperm, which provide the energy needed for movement, and blastocysts, which are important for post-implantation development. Furthermore, GLUT3 is expressed in immune cells, such as lymphocytes, monocytes, macrophages, and platelets, where it is normally stored in intracellular vesicles and translocates to the cell surface upon activation to maintain metabolic switches. More recently, it has been shown to be expressed in T cells, particularly CD8 ＋ Since GLUT3 is highly expressed during differentiation and activation, it has been suggested that T cells depend not only on GLUT1 but also on GLUT3 for glucose uptake.

[0127] In one embodiment, T cells and T cells included in the cell populations of the present disclosure can be modified to express a glucose transporter on demand and / or have glucose transporter expression enhanced on demand. T cells with constitutively enhanced glucose uptake ability differentiate into effector memory T cells and become exhausted, and significant T cell death is induced when activated under low to no glucose conditions. However, T cells with enhanced glucose transporter expression on demand are notable in that they maintain equivalent cytotoxic activity while avoiding T cell death under low to no glucose conditions, maintaining a memory phenotype that is expected to maintain activity for a longer period of time, and inducing resistance to exhaustion.

[0128] Activation of glucose transporters such as GLUT3 in T cells enhances effector functions, such as cytotoxic activity and cytokine production, through glucose uptake. However, prolonged activation significantly induces differentiation into short-acting effector memory types and T cell exhaustion. Therefore, in one embodiment of the present disclosure, a NFAT-induced glucose-inducible expression system or a hypoxia-induced glucose-inducible expression system (e.g., Figure 12 ) that utilizes the mechanism of HIF1a, which induces glucose derivatives upon activation, which increases T cell glucose demand, or upon entry into a hypoxic tumor environment, can be used. This allows for transient expression specific to the tumor environment, tailored to increased demand. Therefore, the cells of the present disclosure can maintain memory phenotype T cells and avoid exhaustion while maintaining cytotoxic activity.

[0129] In one embodiment of the present disclosure, the T cells or T cells included in the cell population of the present disclosure may include effector cells.

[0130] In one embodiment of the present disclosure, the T cells and T cells included in the cell populations of the present disclosure may advantageously be human T cells.

[0131] <Chimeric Antigen Receptor (CAR)> In another aspect of the present disclosure, there is provided a T cell or a cell population comprising such a cell with enhanced glucose uptake ability, wherein the T cell comprises a chimeric antigen receptor (CAR), and the T cell has been modified to express a glucose transporter on demand and / or the expression of the glucose transporter has been enhanced on demand. The CAR contained in the T cell or T cells contained in the cell population of the present disclosure may be contained as a protein, or may comprise a nucleic acid molecule expressing the CAR, as long as it can function as a CAR. In one embodiment, the CAR of the present disclosure may be expressed in the T cell.

[0132] The CARs disclosed herein comprise at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0133] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing the antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. CARs feature MHC-independent antigen binding properties of monoclonal antibodies to redirect T cell specificity and reactivity toward selected targets. MHC-independent antigen recognition can confer the ability of CAR-expressing T cells or T cells in a cell population to recognize antigens independently of antigen processing, allowing tumor immune escape.

[0134] The intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen.

[0135] <T Cell Receptor (TCR)> In another aspect of the present disclosure, there is provided a T cell or cell population comprising such a cell with enhanced glucose uptake ability, wherein the T cell comprises a T cell receptor (TCR), and the T cell has been modified to express a glucose transporter on demand and / or the expression of the glucose transporter on demand is enhanced. The TCR contained in the T cells or T cells included in the cell population of the present disclosure may be contained as a protein, or may comprise a nucleic acid molecule expressing the TCR, as long as it can function as a TCR. In a representative embodiment, the TCR of the present disclosure may be expressed in the T cell.

[0136] As used herein, TCR refers to a heterodimeric receptor molecule consisting of two TCR polypeptide chains. There are αβ-type TCRs expressed by normal T cells and γδ-type TCRs with specialized functions. The α- and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transduce intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens proliferated within cells following viral infection and cancer antigens derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Furthermore, antigens derived from foreign microorganisms are taken up by antigen-presenting cells via endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules. In the case of αβ-type TCRs, the gene products of α and β can be combined to express specificity. TCRs can be modified as appropriate and may contain a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain includes the intracellular domain of a T cell receptor, for example, the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen.

[0137] (Extracellular Domain) In one embodiment, the CAR used in the T cells or T cells contained in the cell population disclosed herein comprises an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof can be selected appropriately depending on the type and number of ligands on the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a specific disease state. Thus, examples of cell surface markers that can act as a ligand for the antigen-binding domain in the CAR of the present disclosure include tissue-specific markers, tumor-specific markers, markers associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0138] The extracellular binding domain of the CAR can be composed of a single-chain variable fragment (scFv) obtained by fusing the variable heavy and light regions of a mouse or humanized monoclonal antibody. Alternatively, scFv derived from a Fab (not from an antibody, e.g., obtained from a Fab library) can be used. The scFv can be fused to a transmembrane domain and then to an intracellular signaling domain.

[0139] In one embodiment, the antigen-binding domain portion of the CAR of the present disclosure binds to: (1) alloantigens including MHC class I and MHC class II; (2) extracellular self-antigens including TSHR (thyroid stimulating hormone receptor), DSG3 (desmoglein 3), and Cytokeratin 8; (3) foreign antigens including Gliadin and Ara h2; and (4) CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin), and MadCam1 (mucosal vascular addressing cell adhesion molecule). 1), and the like, but the antigens that can be targeted by the antigen-binding domain portion of the CAR of the present disclosure are not limited thereto.

[0140] In one embodiment, depending on the desired antigen to be targeted, the CAR of the present disclosure can be modified to include an antigen-binding domain specific for the desired antigen target. For example, if CD19 is the target antigen, an antibody against a cancer antigen such as CD19 can be used as the antigen-binding domain in the CAR. Non-limiting examples of cancer antigens include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folate receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGE-4, and MAGE-5. A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, midkin BAGE, CASP-8, P-catenin, CA-125, CDK-1, ESO-1, gp75, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orfl l2, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2, AR T1, ART4, B cycling, Grill, Cav-1, Cathepsin B, CD74, E-Cadherin, EphA2 / Eck, Fra-1 / Fosl 1, GAGE-1, ganglioside / GD2, GnT-V, pl, 6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOXIO, SOX11, Survivin, phCG, WT1, mesothelin, Melan-A, NY-BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the effector cells disclosed herein are engineered to express one or more CARs to recognize one or more antigens.

[0141] Transmembrane Domains The CARs used in the T cells or T cells included in the cell populations disclosed herein can comprise one or more transmembrane domains fused to an extracellular domain.

[0142] In one embodiment, a linker domain derived from the extracellular domain may be connected to the transmembrane domain. The transmembrane domain may be natural or synthetic, and natural transmembrane domains may be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present disclosure may be derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0143] In one embodiment, the CAR used in the effector T cells disclosed herein may have a spacer domain disposed between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain may preferably have a sequence that promotes binding of the CAR to an antigen and enhances signal transduction into the cell.

[0144] (Intracellular Domain) The cytoplasmic signaling domain (or intracellular signaling domain) of a CAR is involved in activating at least one of the normal effector functions of an immune cell in which the CAR is expressed. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to carry out a specialized function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0145] In one embodiment, examples of intracellular signaling domains used in CARs include cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that initiate signal transduction following antigen receptor binding.

[0146] (Alloantigens, allergens and haptens associated with rejection reactions) CARs used in the T cells or T cells included in the cell populations disclosed herein can include those associated with alloantigens, allergens and haptens associated with rejection reactions.

[0147] <Medicinal Uses of T Cells or T Cells Contained in a Cell Population> In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population comprising such T cells, wherein the T cells have been modified to express a glucose transporter on demand and / or the expression of the glucose transporter has been enhanced on demand. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population comprising such T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR), etc., and have been modified to express a glucose transporter on demand and / or the expression of the glucose transporter has been enhanced on demand. In one embodiment of the present disclosure, the T cells or T cells contained in the cell population of the present disclosure can have one or more characteristics of the effector T cells described above.

[0148] The cells, cell populations, etc. disclosed herein can be used in immunotherapy. Immunotherapy is considered to be effective against diseases that cause antigenic lesions (e.g., cancer, autoimmune diseases, allergies, infectious diseases, etc.) and diseases in which an abnormal immune response to a specific antigen is involved in the onset or progression of the pathology. For example, the cells, etc. disclosed herein can be used to treat, cure, or prevent autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD), rejection, or graft failure during transplantation. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), Sjögren's syndrome, systemic lupus erythematosus (SLE), antiphospholipid syndrome, polymyositis / dermatomyositis, systemic sclerosis, mixed connective tissue disease, vasculitis syndrome, type I diabetes, Graves' disease, Hashimoto's disease, idiopathic Addison's disease, autoimmune hepatitis, Goodpasture's syndrome, glomerulonephritis, autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenic purpura, autoimmune neutropenia, myasthenia gravis, pemphigus, vitiligo, and idiopathic azoospermia. Examples of allergic diseases include, but are not limited to, hay fever, allergic rhinitis, bronchial asthma, and atopic dermatitis. The cells, cell populations, and the like disclosed herein can also be used to treat or prevent diseases in which an abnormal immune response to a specific antigen is involved in the onset or progression of the disease.

[0149] In certain embodiments, the present disclosure provides such immunotherapy, which includes TCR-T therapy or CAR-T therapy. As used herein, "TCR-T therapy" refers to a cell therapy that utilizes modification of T cell receptors (TCRs), and is used, for example, in cancer treatment. As used herein, "CAR-T therapy" refers to a gene and cell therapy method in which a chimeric antigen receptor (CAR) (e.g., a CAR that has been genetically engineered to overcome tumor immune evasion mechanisms) is introduced into a patient's T cells, and the T cells are expanded and cultured ex vivo and then infused back into the patient.

[0150] The cells, cell populations, etc. of the present disclosure may be appropriately combined with other cancer treatments and used as a combination therapy. Typically, they may be administered in combination with one or more additional drugs. Alternatively, the combination therapy may be combined with radiation therapy. The one or more additional drugs may be any chemotherapeutic drug or may include an immune checkpoint inhibitor. Alternatively, other cancer treatments used in the combination therapy include, but are not limited to, other cancer immunotherapies (e.g., immune checkpoint inhibitors), hyperthermia, surgical procedures, etc.

[0151] In another aspect, there is provided a therapeutic agent comprising the T cells or cell population of the present disclosure, which performs a diagnosis of a disease in a subject and selects an appropriate CAR, TCR, etc. contained in the T cells or the cell population based on the diagnosis.

[0152] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the immune cells described herein (e.g., T cells, such as effector T cells) or cell populations comprising such cells and a pharmaceutically acceptable carrier. When the immune cells express a polypeptide such as a CAR or TCR, the pharmaceutical composition may further comprise an Fc-containing therapeutic agent, such as a therapeutic antibody or an Fc-fusion protein. The Fc-containing therapeutic agent can bind to a target antigen, such as an immune cell specific for a tumor antigen, a pathogen antigen, or an autoantigen. The pathogen antigen may be a bacterial antigen, a viral antigen, or a fungal antigen.

[0153] In one embodiment, the Fc-containing therapeutic agent is selected from the group consisting of adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, brentuximab, canakinumab, cetuximab, certolizumab, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumab, The therapeutic antibody may be, but is not limited to, mab, infliximab, ipilimumab, labetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tositumomab, ustekinumab, mogamulizumab, and vedolizumab.

[0154] Additionally, the present disclosure provides a kit comprising: (i) a first pharmaceutical composition comprising any T cell described herein or a cell population comprising such a T cell and a pharmaceutically acceptable carrier; and (ii) an Fc-containing therapeutic agent described herein and a pharmaceutically acceptable carrier.

[0155] In another aspect of the present disclosure, there is provided a method for inhibiting cells expressing a target antigen in a subject (e.g., reducing the number of such cells, inhibiting cell proliferation, and / or suppressing the activity of cells), comprising administering to the subject a cell, cell population, and / or pharmaceutical composition of the present disclosure. In one embodiment, at least a portion of the cells expressing the target antigen may be in a low-glucose environment.

[0156] In one embodiment, the subject treated by the method of the present disclosure may be a human patient suffering from cancer, such as carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Exemplary target cancers include, but are not limited to, B-cell derived cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B-cell derived cancers include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0157] In addition to treating a target disease or disorder, such as cancer or an infectious disorder, use of the T cells or cell populations of the present disclosure for the manufacture of a medicament for a medical treatment of interest is also within the scope of the present disclosure.

[0158] In one embodiment, a pharmaceutical composition comprising T cells comprising the chimeric antigen receptor (CAR) or T cell receptor (TCR) of the present disclosure, or a cell population comprising such T cells, can be used for cell therapy. In cell therapy, T cells comprising the chimeric antigen receptor (CAR) or T cell receptor (TCR) of the present disclosure, can be infused into a subject in need thereof as a pharmaceutical composition or as a formulation of a therapeutically effective cell population expressing the CAR of the present disclosure. The infused T cells or a cell population comprising such T cells in the subject can treat a target disease or disorder, such as cancer or an infectious disorder, in the subject. The subject may be the same subject from whom the cells were obtained (autologous cell therapy), or the cells may be derived from another subject of the same species (allogeneic cell therapy).

[0159] In one embodiment, T cells comprising the CAR or TCR, etc., of the present disclosure, or a cell population comprising such T cells, can be formulated for administration to a subject using techniques known to those skilled in the art. In one embodiment, a formulation comprising therapeutically effective T cells comprising the CAR or TCR, etc., of the present disclosure, or a cell population comprising such T cells, can contain a pharmaceutically acceptable excipient (carrier or diluent). The excipients included in the formulation have different purposes depending on, for example, the properties of the antigen-binding domain of the CAR of the present disclosure. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants.

[0160] In this specification, when a gene name and its product are written in all capital letters, contrary to the usual usage, it may refer to both the gene and the protein. For example, the FOXP3 gene and the FOXP3 protein may be used interchangeably, and the term FoxP3 refers to both the concept and entity (whole) of the gene or protein.

[0161] A preparation containing therapeutically effective T cells containing the CAR or TCR of the present disclosure or a cell population containing such T cells can be administered to a subject using methods and techniques known to those skilled in the art. Exemplary methods include, but are not limited to, intravenous injection. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarticular, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluid) administration. In one embodiment, the present disclosure is provided as a pharmaceutical composition for preventing or treating cancer in a subject, the pharmaceutical composition comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population comprising the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population comprising the T cells to the subject in an appropriate manner and dose, wherein the T cells comprise a chimeric antigen receptor (CAR).

[0162] In one embodiment, the T cells with enhanced glucose uptake are T cells that have not been modified to express a glucose transporter on demand and do not have enhanced glucose transporter expression on demand. ｅｆｆ and modified to express a glucose transporter on demand and / or to enhance expression of a glucose transporter on demand.

[0163] In one embodiment, the glucose transporter is GLUT3.

[0164] (General Techniques) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press;Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). These are described in PCR Applications: Protocols for Functional Genomics, Academic Press, a special edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and other publications, the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0165] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) and the like can be used. Other examples include Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.; M. et al. (1996). These are described in "Nucleic Acids in Chemistry and Biology," Oxford University Press; "Hermanson, G. T. (1996) Bioconjugate Techniques," Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0166] As used herein, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." When "within the range of" two values ​​is specified herein, the range includes the two values ​​themselves. References cited herein, such as scientific literature, patents, patent applications, etc., are incorporated herein by reference in their entirety to the same extent as if each were specifically set forth.

[0167] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0168] In this example, various functions of T cells with enhanced glucose uptake were investigated. The reagents used were specifically those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0169] (Example 1; GLUT3-Expressing T Cells) (Methods and Materials) Construction of GLUT3 3C10-CAR Construct and Viral Vector Construction of GLUT3 3C10-CAR Construct and Viral Vector Construction of GLUT3 3C10-CAR Construct and Viral Vector Construction of GLUT3 3C10-CAR Construct A CAR-GLUT3-Katushka2S lentiviral vector was constructed (Figure 1). 293T cells were transfected with the lentiviral vector together with the packaging vectors psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259), and lentivirus was isolated from the supernatant. PBMCs containing T cells were transfected with this vector to obtain T cells expressing CAR and GLUT3.

[0170] Construction of an On-Demand GLUT3 Expression 3CAR Construct: NFAT-Inducible GLUT3-CAR An NFAT-inducible GLUT3 construct consisting of eight repeats of the NFAT binding site, a minimal promoter (minP), and an SLC2A3 (GLUT3) sequence was inserted into the GLUT3 3C10-CAR plasmid to construct an NFAT-inducible GLUT3 CAR plasmid (NFATiGLUT3 CAR: see Figure 15 , same lentiviral plasmid: see Figure 16 ). In this system, CAR is constitutively expressed driven by the EF1a promoter, and GLUT3 is expressed in response to the nuclear translocation of NFAT that occurs following activation of CAR-T cells.

[0171] Hypoxia-inducible GLUT3-CAR A HIF inducible GLUT3 construct consisting of xx repeats of the HIF1a binding site, minP, and SLC2A3 (GLUT3) sequence is inserted into the above-mentioned GLUT3 3C10-CAR plasmid to construct a hypoxia-inducible GLUT3 CAR plasmid ( FIG. 15 ). Alternatively, a 2A sequence is placed under 3C10-CAR, and a conjugate of GLUT3 and the HIF-1α oxygen-dependent degradation domain (ODD) is inserted. In both cases, CAR is driven by the EF1a promoter and is constitutively expressed; in the former, GLUT3 senses the nuclear translocation of NFAT that accompanies the activation of CAR-T cells; in the latter, GLUT3 continues to be degraded under normal oxygen conditions, but under hypoxia, the degradation of GLUT3 by ODD is suppressed, resulting in the induction of expression.

[0172] Gene transfer: Peripheral blood mononuclear cells from healthy donors were separated from heparinized whole blood by the Ficoll method (Ficoll-Paque PLUS, GE Healthcare), then stimulated with anti-CD3 / 28 beads (Dynabeads T-Activator CD3 / CD28, Veritas), and on day 1 after stimulation, CAR gene transfer was performed at an MOI of 4. The cells were cultured in the presence of 30 U / ml of IL-2 and either cryopreserved from days 8 to 12 or directly subjected to various evaluations.

[0173] Immunostaining 1×10 ６ GLUT3 3C10 CAR-T cells were fixed with 4% PFA and permeabilized with methanol. Blocking was performed with 3% BSA for 1 hour, followed by incubation with anti-GLUT3 antibody (ab15311) for 2 hours. After washing with 0.05% PBST, the cells were incubated with anti-rabbit IgG antibody (Alexa Flour 488) for 1 hour. After washing with 0.05% PBST, nuclei were stained with DAPI. After staining, the cells were attached to a slide glass using Cytospin (Thermo Fisher Scientific), mounted in VECTASHIELD (VECTOR LABORATORIES), and observed using a Keyence XZ-800 microscope.

[0174] Phenotype Analysis (FCM) The antibodies used in FCM analysis are shown in Table 1.

[0175]

[0176] The cells were washed with 4% FBS-PBS, and then stained for dead cells and surface staining. After washing, data were acquired using a FACSSymphony A3 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0177] In vitro CAR-T cell survival and function analysis (Figure 21) evaluated the long-term survival, memory formation, and functional maintenance of each CAR-T cell. CAR-T cells were stimulated at a 1:1 ratio with stimulatory factors (medium as a negative control, EGFRvIII stimulatory beads, U87d cell line). After 24 hours, the stimulatory factors were removed using magnetic beads or by sorting, and CAR-T cells were divided into 0 mM, 0.5 mM, and 10 mM groups and continued to be cultured. Cell counts were performed before stimulation and on days 1, 3, and 7 after stimulation. Cell survival and cell death were evaluated by analyzing memory phenotypes such as Annexin V / 7AAD, CD27, CCR4, and CD45RA, inhibitory factors such as PD-1, TIM3, and LAG3 (see Figures 23-28), and other factors (transcription factors, activation / senescence markers). In addition, RNA is extracted from each sample and detailed analysis such as RNA sequencing is performed. After this series of analyses, a portion of the day 7 cells are stimulated again in the same group, and cell survival and function analysis after repeated stimulation is similarly performed.

[0178] Cytokine analysis (FCM) CAR-T cells 1 x 10 ５ cells and EGFRvIII-expressing U87 cell line (U87Δ) 5 × 10 ５ The cells were co-cultured in 1 ml of medium for 12 hours, and then cultured for 6 hours in the presence of 5 μg / ml Monensin (BD 554724, BD Bioscience) to stimulate the CAR-T cells. The medium used was glucose-free RPMI 1640 (Wako) supplemented with 10% dialyzed FBS (Cytiva), with the glucose concentration adjusted to 10 mM and 0.5 mM with a glucose solution (Gibco). After fixation with Fixation / Permeabilization Diluent (Invitrogen), intracellular cytokines were stained with the antibodies shown in Table 1. Data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0179] Cytotoxicity analysis: 2 × 10 cells / well in a 96-well plate ５Target cells (luciferase-expressing U87Δ cells) were seeded and co-cultured with CAR-T cells at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1. After 16 hours, live cells were luminescently stimulated with VivoGlo Luciferin (Promega), and luciferase activity was measured using Cytation (Promega), and cytotoxic activity was calculated.

[0180] Metabolic analysis The metabolic activity of the cells was measured using an XFe24 Cell Flux Analyzer (Bioscience) and an XF Glycolysis Stress Kit (Agilent Technologies) according to the manufacturer's protocol. On the day before the analysis, the 24-well flat-bottom plate for analysis was coated with poly-D-lysine (0.1 mg / ml) and the sensor cartridge was hydrated with CO. ２ On the day of analysis, the cells for analysis were washed with analysis medium (Seahorse XF RPMI medium, L-glutamine 2 mM), and then plated at 2 × 10 cells on a poly-D-lysine-coated analysis plate. ５ The cells were seeded at the appropriate number and ２ The sample was left to stand at 37°C for 60 minutes. The sensor cartridge port was filled with glucose at a final concentration of 10 mM or 0.5 mM, oligomycin at a final concentration of 1 μM, and FCCP at a final concentration of 10 μM, and 2-DG at a final concentration of 50 mM. Analysis was then performed using a flux analyzer, and metabolic function was evaluated from OCR and ECAR.

[0181] Glucose uptake capacity evaluation 2 x 10 ４ GLUT3 CAR-T cells were seeded onto a 96-well plate and cultured for 5 hours in 200 μl of glucose-free RPMI 1640 (Wako). Five minutes before analysis, 1 μl of 2-NDBG (abcam) was added to each well. After washing, data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0182] Cranial tumor xenograft model: 2.5 x 10 luciferase-expressing U87Δ tumor cell line (U87Δ-luc) was implanted into the cranial cavity (right basal region) of NSG mice. ４ After engraftment, Mock, 3C10 CAR-T cells, GLUT3 CAR-T cells 2 x 10 ６ The cells were administered via the tail vein. The tumor burden was monitored over time by bioimaging, and survival was observed. In a tumor rechallenge test to verify the long-term survival, memory formation, and recurrence prevention effects of CAR-T cells, mice that achieved CR after the above treatment were administered 2.5 x 10 tumor cells (U87Δ-luc). ４ cells (Figure 13) or 1.0 x 10 ５ Cells (Figure 19) were again administered intracranially and BLI and survival were monitored.

[0183] (Method) Figure 1 shows the structure of metabolically engineered CAR (EGFRvIII_CAR_GLUT3). A single-chain antibody (asFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) was constructed, and the CAR was constructed by binding CD8 hinge, CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BB ICD, and CD3z. A high-affinity glucose transporter (GLUT3) was encoded under the CAR via the P2A sequence.

[0184] (Results) The results are shown in Figure 2 and subsequent figures. As shown in Figure 2, enhanced GLUT3 expression on the T cell surface and glucose uptake are demonstrated. CAR-T cells were obtained by gene transfer of a conventional CAR (3C10 CAR) and a metabolically engineered CAR (GLUT3 CAR) into T cells derived from a healthy donor. Fluorescence microscopy confirmed homogeneous GLUT3 expression on the membrane of GLUT3 CAR-T cells. Furthermore, glucose uptake was enhanced in GLUT3 CAR-T cells compared to T cells and conventional CAR-T cells (3C10 CAR-T cells).

[0185] Next, Figure 3 shows the T cell expansion efficiency, which is equivalent to that of conventional CAR-T cells. As shown in Figure 3, after stimulation with anti-CD3 / 28 beads, CAR was transfected and the T cell proliferation efficiency was analyzed. The GLUT3 CAR-T cells had an establishment efficiency comparable to that of conventional CAR-T cells (3C10 CAR-T cells).

[0186] Next, enhancement of T cell glycolytic capacity by GLUT3 expression is shown in Figure 4. As shown, the metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Flux analyzer. GLUT3 CAR enhanced glycolytic capacity in both low-glucose and normal-glucose environments.

[0187] Next, it was shown that GLUT3 CAR-T cells have a competitive advantage under low glucose conditions (Figure 5). As shown in Figure 5, 3C10 CAR-T cells and GLUT3 CAR-T cells were co-cultured under normal glucose and low glucose conditions. Under normal glucose conditions, both cells survived to a similar extent, but under low glucose conditions, GLUT3 CAR-T cells survived significantly longer, suggesting that they have an advantage in glucose uptake.

[0188] Next, Figure 6 shows that the cytokine production ability of GLUT3 CAR-T cells was enhanced. As shown in Figure 6, 3C10 CAR-T cells and GLUT3 CAR-T cells were stimulated with an antigen, and the percentage of cytokine-producing cells was analyzed by FCM. Significant increases in the expression of cytokines (IFN-g, IL-2, TNF-a) that are important for the maintenance and activation of T cells and the exertion of antitumor activity were observed.

[0189] Next, the function of GLUT3 CAR-T cells, which is maintained even under low glucose conditions, is shown in Figure 7. As shown, cytokine production capacity under normal glucose and low glucose conditions was compared. Conventional CAR-T cells (3C10 CAR-T cells) showed an extreme decrease in function under low glucose conditions. On the other hand, GLUT3 CAR-T cells showed high cytokine production capacity under normal glucose conditions, and even under low glucose conditions, they exhibited a function comparable to the cytokine production capacity of 3C10 CAR-T cells under normal glucose conditions.

[0190] Next, the enhanced cytotoxic activity of GLUT3 CAR-T cells is shown in Figure 8. As shown, cytotoxic activity was analyzed by targeting an EGFRvIII-expressing cell line. Cytotoxic activity was analyzed under low glucose and high glucose conditions, and under both conditions, GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells.

[0191] Next, the characteristics of GLUT3 CAR on memory phenotype are shown in Figure 9. As shown, the memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. A tendency for differentiation into effector T cells was observed in GLUT3 CAR-T cells.

[0192] Next, the suppression of inhibitory molecule expression in GLUT3 CAR-T cells is shown in Figure 10. The expression of inhibitory molecules was compared between unstimulated and stimulated with EGFRvIII antigen-positive cells. Suppression of PD-1, LAG3, and Tim3 expression was observed in GLUT3 CAR-T cells.

[0193] Next, mRNA analysis of GLUT3 CAR-T cells (evaluation of metabolism, exhaustion, activation, and differentiation) is shown in Figure 11. 3C10 CAR-T and GLUT3 CAR-T cells were analyzed in detail using mRNA expression. As with FCM, GLUT3 CAR-T cells showed a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors. Overall, the data indicate an increase in T cell effector functions, such as activation and cytokine production.

[0194] Next, Figure 12 shows that similar effects can be obtained not only with the 3C10 CAR but also with the CD19 CAR, demonstrating universality. We confirmed that GLUT3 loading on the CD19 CAR produced effects similar to those of the 3C10 CAR. Furthermore, CD19 expression in pancreatic cancer cell lines demonstrated efficacy against pancreatic cancer. As shown in Figure 13, rapid tumor eradication was achieved in an intracranial xenograft model. Antitumor activity was observed in a U87Δ intracranial xenograft model. GLUT3 CAR-T cells demonstrated more rapid tumor eradication than 3C10 CAR-T cells. Furthermore, tumor rejection was confirmed by tumor challenge.

[0195] (Example 2: On-demand expression) An on-demand GLUT3-expressing CAR was tested. An overview of this is shown in Figure 14. Figure 14 shows an overview of the on-demand GLUT3-expressing CAR. This is a system in which only the CAR is expressed, but not GLUT3, in the steady state, and GLUT3 is expressed when glucose demand increases, such as due to CAR-T cell activation or the tumor environment. To achieve on-demand expression of GLUT3, a CAR construct was designed that expresses GLUT3 in a tumor environment-specific or activation-specific manner (Figure 15). This enables the expression of GLUT3 in response to activation-induced NFAT elevation (A) or tumor hypoxia (B).

[0196] The function and antitumor effect of the CAR-introduced cells shown in Figure 15 will be analyzed.

[0197] (Example 3: Enhancement of glucose transporter expression) To achieve enhanced expression of glucose transporters, a CAR construct that constitutively expresses GLUT3 was designed (same as stbl-GLUT3=GLUT3 CAR in Figure 15).

[0198] The function and antitumor effect of the CAR-introduced cells shown in Figure 15 will be analyzed.

[0199] Figure 16 shows the structure of the NFATi-GLUT3 CAR plasmid. The details of the structure are as follows: In Figure 16, CAR and inducible GLUT3 are incorporated into a lentiviral vector. A CAR consisting of, in order, a CD8 hinge, a CD28 transmembrane domain, a CD28 intracellular domain (CD28I), 4-1BB, and CD3z is encoded by a single-chain antibody (3C10VH-linker-3C10VL) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) and the like. Katushka, a gene transfer marker, is encoded via a P2A sequence under the CAR. Furthermore, human GLUT3 (hGLUT3) is encoded under eight repeats of the NFAT binding site and minimum promoter.

[0200] Figure 17 shows the operation and basic function of the On Demand (Inducible) GLUT3 CAR system of the present disclosure. A system was constructed (NFATi GLUT3 CAR-T) that expresses GLUT3 in conjunction with NFAT nuclear translocation, and its operation was confirmed. The cytotoxic activity of NFATiGLUT3 CAR-T cells (NFATi GLUT3 CAR-T) was equivalent to that of constitutively expressing GLUT3 CAR-T cells (GLUT3 CAR-T), and cytokine production was intermediate between that of 3C10 CAR-T (without GLUT3) and constitutively expressing CAR-T cells (GLUT3 CAR-T).

[0201] The antitumor effect of NFAT1GLUT3 CAR-T cells was evaluated in a similar mouse model, and the results are shown in Figure 18. CR was obtained in four mice with GLUT3 CAR-T and two mice with NFATi GLUT3 CAR-T.

[0202] Figure 19 shows the results of tumor challenge and tumor rechallenge experiments. Specifically, it shows the sudden death of mice in the GLUT3 CAR-T cell group. Tumors (U87d) were re-implanted into cured mice from the previous experiment to evaluate their rejection ability (a "surrogate for memory formation"). ５ Tumor cells (four times the amount used in the previous experiment) were re-implanted. Tumor rejection was not achieved with 3C10 CAR-T, but was achieved with GLUT3 CAR-T and NFATiCAR-T. However, in the GLUT3 CAR-T group, three out of four mice died (non-tumor amplification deaths).

[0203] 20 shows that apoptosis was suppressed after stimulation with NFATiGLUT3. CAR-T cells were repeatedly stimulated with U87d, and CAR-T cell apoptosis was observed with Annexin V and 7-ADD.

[0204] Figure 21 shows that long-term survival after various stimuli is enhanced. Each CAR-T cell was observed over time under the above conditions. As a result, under low glucose conditions, normal CAR-T (3C10) did not exhibit effective cytokine production, while GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibited effective cytokine production. However, due to excessive glucose uptake, GLUT3 CAR-T differentiated over time (day 3 or day 7) into CCR7-negative Tem or Temra (terminal differentiation, loss of stemness, and apoptosis). On the other hand, apoptosis was suppressed in on-demand NFATi GLUT3 CAR-T cells, and CCR7-positive Tcm or Tscm (memory cells, cells with stemness) were maintained.

[0205] Furthermore, when CAR-T cells (established with normal glucose) are suddenly (on day 0) placed under low glucose conditions of 0 mM or 0.5 mM, the highly glucose-dependent GLUT3 CAR-T cells are prone to apoptosis (conversely, NFATi GLUT-3 CAR-T cells are resistant to low glucose).

[0206] Figure 22 shows that long-term survival after stimulation is enhanced. The antitumor effects, survival, and adverse events were examined using the same mouse system as described above. In Experiment 1, GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibited comparable antitumor activity. In Experiment 2, glucose exposure caused overactivation of GLUT3 CAR-T, leading to apoptosis and hyperdifferentiation, resulting in the loss of antitumor activity. On the other hand, NFATi CAR-T cells exhibited effective antitumor effects because excessive activation was suppressed. Even after rechallenge, NFATi CAR-T cells rejected tumors. In Experiment 4, NFATi GLUT3 cells exhibited superior CAR-T cell engraftment and tumor T cell infiltration, which were proportional to the tumor effect. Furthermore, Experiment 3 was conducted using a liver tumor model in which immunosuppression due to a low-glucose, high-lactic acid environment has been suggested, and it is expected that similar results to Experiment 2 will be obtained.

[0207] Figure 23 shows data (in vitro data) demonstrating enhanced long-term survival after stimulation. At each time point on the left, staining data for 7-AAD and Annexin V, which reflect cell death and apoptosis, are shown. Cell death in each CAR-T cell type was examined using Annexin V and 7-AAD. In the unstimulated state, differences in glucose concentration were observed, but no significant differences were observed among the CAR-T cells.

[0208] Figure 24 presents in vitro data demonstrating enhanced long-term survival after stimulation. As shown, survival and cell death of each CAR-T cell after stimulation were evaluated in the same manner as in the previous figure. Compared to 3C10 CAR and GLUT3 CAR-T cells, survival was enhanced (cell death was suppressed) in NFATi GLUT3 CAR-T cells.

[0209] Figure 25 shows in vitro data demonstrating enhanced long-term survival after stimulation. The CAR-positive rate for each CAR-T cell type after stimulation was analyzed. After stimulation, the rate of CAR-positive cells decreased, particularly in constitutively expressing GLUT3 CARCAR-T, suggesting that this fraction had undergone cell death.

[0210] Figure 26 shows in vitro data demonstrating enhanced long-term survival after stimulation. The number of T cells on day 7 is shown. As a model reflecting post-administration dynamics in patients (the dynamics of infiltration from blood with sufficient glucose concentration into a low- to no-glucose tumor environment after administration), cells were established and cultured under normal glucose conditions (10 mM glucose), and then stimulated with EGFRvIII-conjugated beads. Subsequently, cells were cultured under glucose-free (0 mM), low-glucose (0.5 mM), or normal glucose (10 mM) conditions, and the number of viable cells after 7 days was assessed. The graph shows the relative cell count, with the number of mock T cells set to 1. Most GLUT3 CAR-T cells died at 0 mM. On the other hand, many NFATi CAR-T cells remained viable. These results suggest that cell death due to overactivation caused by constitutive GLUT3 expression and a highly glucose-dependent metabolism may have led to cell death.

[0211] Expression of an inhibitory molecule (PD-1) is shown in Figure 27. Expression of an inhibitory molecule (PD-1) was lower in NFATi GLUT3 CAR-T cells than in GLUT3 CAR-T cells.

[0212] Figure 28 shows the suppression of expression of an inhibitory molecule (TIM3). In NFATi GLUT3 CAR-T cells, the expression of an inhibitory molecule (TIM3) was lower than in GLUT3 CAR-T cells.

[0213] (Example 4: Examples using precursor cells of other effector T cells) Similar experiments are performed using other T cells. For example, unpurified peripheral T cells include Tnv, Tcm, Tem, Temra, etc., and any fraction can be CAR-transduced (CAR, constitutively expressed GLUT3 CAR, or on-demand GLUT3 CAR).

[0214] In this example, a CAR (CAR, constitutively expressed GLUT3 CAR, on-demand GLUT3 CAR) is introduced into a purified and enriched T cell memory fraction. In a separate experiment, CAR-T cells enriched in a T cell memory fraction are induced under culture conditions.

[0215] Experimental procedure: ・Select T cells from peripheral blood, bone marrow, umbilical cord blood, etc., which are expected to survive longer and maintain stemness. ｅｆｆ Tnv, Tscm, and Tcm are precursor cells of T cells, or T cells that have already differentiated and mainly exert effector functions. ｅｆｆ and Temra fractions were analyzed using the CD45RA and CD45RA T cell memory-associated surface antigen expression patterns, respectively. ＋ CCR7 ＋ CD95 － , CD45RA ＋ CCR7 ＋ CD95 ＋ , CD45RA － CCR7 ＋ , CD45RA － CCR7 and CD45RA ＋ CCR7 －Based on this, the cells are purified and concentrated using FACS sorting or magnetic beads. Each fraction is stimulated with CD3 / 28 beads, etc., and CAR is introduced using a lentiviral vector. Alternatively, after gene transfer to bulk T cells, the type and amount of cytokines added can be adjusted (using IL-7 or IL-15, or using platelet lysate) to induce T cell proliferation. ｅｆｆ This induces the suppression of differentiation and the predominant proliferation of Tnv, Tscm, and Tcm, resulting in progenitor-predominant CAR-T cells. In vitro evaluation: These cells are co-cultured with U87d tumor cell lines or EGFRvIII beads, and after 1, 3, and 7 days, CFSE staining and cell counting are used to assess proliferation, and flow cytometry is used to assess activation and exhaustion. In vivo evaluation: These cells are administered to the aforementioned U87d intracranially injected mice, and antitumor activity and survival are assessed by BLI. Furthermore, mice in which cure is observed are rechallenged with tumors to assess tumor rejection.

[0216] Results: When introduced into bulk T cells or differentiated T ｅｆｆ Compared to the case of introducing Tmra, the precursor cells Tnv, Tscm, Tcm, etc. ｅｆｆ CAR-T cells introduced into progenitor cells or with predominantly amplified Tnv, Tscm, and Tcm are expected to have high proliferation and proliferative potential, and to demonstrate long-term proliferation. Furthermore, they are expected to have low expression of PD1, TIM3, and LAG3, and to be resistant to exhaustion. Furthermore, these CAR-T cells have demonstrated superior antitumor activity and improved survival in mouse models. Furthermore, efficient tumor rejection is expected during tumor rechallenge (a surrogate for long-term efficacy and prevention of recurrence).

[0217] Example 5: Demonstration This example provides a larger scale demonstration.

[0218] Materials and Methods

[0219] U-87 MG and U-251 MG GBM cell lines were purchased from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank, respectively, and cultured in D-MEM (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. EGFRvIII-packaged lentiviral vectors were introduced into U-87 MG and U-251 MG cells, and the transduced cell lines were designated U-87 MGΔ and U-251 MGΔ, respectively. SUP-T1, NALM6, and AsPC-1 cells were purchased from ATCC and cultured in RPMI 1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% FBS and 1% penicillin / streptomycin. A CD19 truncated vector was transfected into NALM6 cells to obtain the CD19-expressing AsPC1 cell line.

[0220] Flow cytometry assays were performed as previously described [40-43]. Briefly, cells were washed twice with FACS buffer and Fc-blocked using Fc receptor blocking solution (BioLegend, San Diego, CA) for 10 minutes at 4°C in the dark. Cells were washed twice with FACS buffer, and antibodies targeting cell surface molecules were added and incubated for 20 minutes at 4°C in the dark. Cells were then washed twice with FACS buffer. For intracellular antigen staining, cells were incubated in fixation / permeabilization solution (BD Biosciences, San Jose, CA) for 1 hour at room temperature and then washed twice with wash buffer (BD Biosciences) according to the manufacturer's instructions. Antibodies targeting intracellular antigens were added and incubated for 20 minutes at 4°C in the dark, followed by two washes. For the apoptosis assay, cells were washed once with FACS buffer and once with Annexin-binding buffer. Then, cells were stained with Annexin V and 7-AAD for 15 minutes at room temperature. After washing, flow cytometry (FCM) analysis was performed using an LSRFortessa X-20 cytometer (BD Biosciences) and FlowJo ver. 10 software (BD Biosciences). The staining solution was prepared according to the manufacturer's instructions.

[0221] Cytokine Staining Intracellular cytokine staining was performed as previously described [40-43]. Monensin was added to the culture medium for the final 5 hours of 6-hour T cell stimulation to retain cytokines intracellularly. After staining for cell surface markers, Cytofix / Cytoperm reagent (BD Biosciences) was added and incubated for 20 minutes at 4°C in the dark. The cells were then washed twice with washing buffer, and antibodies targeting cytokines were added. The cells were incubated for 20 minutes at 4°C in the dark. After washing, FCM analysis was performed using an LSRFortessa X-20 cytometer (BD Biosciences) and analyzed with FlowJo ver. 10 software (BD Biosciences). The antibodies used for cell staining are listed in the table. Antibodies for staining were prepared according to the manufacturer's instructions.

[0222]

[0223] Killing assay total 1x10 ５ Luciferase-expressing cells (U-87 MGΔ, U-251 MGΔ, AsPC-1, and NALM6) were co-cultured with CAR-T cells at the indicated ratios in 200 μL of RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with low glucose (0.5 mM) or high glucose (10 mM) in a flat-bottom 96-well white plate. After 24 hours of culture, Bio-Glo (Promega, Madison, WI) was added to each well, and luminescence was measured using a multiplate reader, Cytation 5 (Agilent Technologies, Santa Clara, CA). The % specific lysis was calculated using the following formula: % specific lysis = % specific lysis = [(experimental lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis)] x 100.

[0224] Immunofluorescence staining After washing, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with cool methanol at -20°C for 10 minutes. Cells were blocked with 3% BSA / PBS for 60 minutes at room temperature. After blocking, cells were incubated with primary antibodies for 2 hours at 4°C and secondary antibodies for 1 hour at 4°C. DAPI was then added and incubated at room temperature for 5 minutes. After washing, stained cells were fixed to slides using a Cytospin and analyzed using a BZ-X710 (KEYENCE, Osaka, Japan).

[0225] Immunohistochemical (IHC) Staining. IHC staining was performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded specimens. Antigen removal was performed using a steamer with citrate buffer (pH 6.0), followed by deparaffinization and rehydration. Slides were incubated with primary antibodies for 16 hours and HRP-conjugated secondary antibodies for 1 hour, and then developed with diaminobenzidine substrate. Primary staining was performed using anti-CD3ε antibody (clone: ​​SP7, catalog number: ab16669) (Abcam, Waltham, MA). Stained slides were counterstained with hematoxylin. Hematoxylin-eosin (HE) staining was performed according to standard protocols. Deparaffinized and hydrated slides were added to hematoxylin solution and incubated for 4 minutes. After washing, the slides were incubated with eosin solution for 2 minutes. The stained slides were scanned at 40x and 400x magnifications using a BZ-X710 (Keyence). Two pathologists independently evaluated the stained slides.

[0226] ELISA method Total 2.0 x 10 ５ U-87 MGΔ cells and 2.0 × 10 ５ CAR-T cells were co-cultured in 24-well plates. After 24 hours of culture, supernatants were collected and subjected to ELISA to measure cytokine concentrations. IFN-γ, IL-2, and TNFα ELISA kits (R&D Systems, Minneapolis, MN) were used according to the manufacturer's instructions. Cytokines in mouse serum were analyzed by a highly sensitive LUMINEX assay (Merck Millipore, Burlington, MA) according to the manufacturer's instructions.

[0227] CAR-T Cell Production Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL). T cells were isolated from PBMCs by negative selection using MojoSort (BioLegend). Isolated T cells were stimulated with anti-CD3 / 28 beads (Thermo Fisher Scientific, Waltham, MA) at a T cell:bead ratio of 1:1 (day 0). 24 hours after stimulation (day 1), T cells were transduced with CAR lentivirus at an MOI of 3 and cultured in 30 U / ml IL-2. Half of the medium was replaced 24 hours later. Five days later, the CD3 / 28 beads were removed. During the culture, half of the medium was replaced on days 4 and 10, and the T cell concentration was maintained at 0.7 × 10 ６ The developed CAR-T cells were collected on day 10 and subjected to subsequent analysis.

[0228] Animal Model: Female NSG mice (6 weeks old) were purchased from Jackson Laboratory. Before painful procedures, an anesthetic was injected intraperitoneally to prevent pain. Tumor cells were inoculated using a stereotaxic frame at a location 2 mm to the right of bregma, 3 mm behind, and 3 mm deep from the brain surface. A total of 2.0 × 10 cells were inoculated in 5 mL of PBS. ４ After injecting 1.0 × 10 cells over 1 minute, the 1 mm needle was removed and left for another 1 minute before being removed. Four days after tumor inoculation, tumor growth was monitored by bioluminescence imaging (BLI) and randomization to each treatment group was performed. On day 5, 1.0 × 10 cells were injected. ６ CAR-T cells were injected via the tail vein. Tumor growth after CAR-T cell injection was monitored twice weekly by BLI. Animal care and experiments were approved by the National Cancer Center Animal Experiment Ethics Committee and conducted in accordance with the guidelines of the National Cancer Center Animal Committee.

[0229] Tissue samples (5 × 5 mm) were washed to remove any blood. The samples were minced and centrifuged at 4°C for 10 minutes to collect interstitial fluid. Glucose concentrations in the interstitial fluid were measured using a Multiskan GO (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0230] Metabolic Analysis OCR (unit: pmol / min) and ECAR (unit: mpH / min) were assessed by a Seahorse XF-24 metabolic extracellular flux analyzer (Agilent Technologies). CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and on-demand GLUT3 EGFRvIII CAR-T cells) stimulated with EGFRvIII beads (ACRO Biosystems, Beijing, China) for 24 hours were resuspended in glucose-free unbuffered RPMI-1640 medium (Agilent Technologies) and plated onto poly-L-lysine (BD Bioscience)-coated Seahorse cell plates (2.0 × 10 cells per well). ５ Cells were plated onto a 1000-well plate (Figure 1). Perturbation profiling of metabolic pathway utilization by CAR-T cells was achieved by adding glucose (10 mM or 0.5 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (50 mM) (all from Agilent Technologies). Experiments using the Seahorse system were performed under the following assay conditions: 3 minutes of mixing, 2 minutes of waiting, and 3 minutes of measurement. Metabolic parameter values ​​were then calculated.

[0231] RNA sequencing and subsequent analysis. BD FACSymphony S6 (BD Biosciences) sorted CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and on-demand GLUT3 EGFRvIII CAR-T cells) were stimulated with EGFRvIII beads at a 1:1 ratio under low glucose (0.5 mM) or high glucose (10 mM) conditions, supplemented with 30 U / ml IL-2. CAR-T cells were harvested on days 3 and 7, and RNA was extracted using an RNeasy kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from the isolated RNA using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA). RNA-seq libraries were subjected to next-generation sequencing (paired-end reads) of 150 bp from both ends using Novaseq X plus (Illumina, San Diego, CA). For expression profiling of RNA-seq data, paired-end reads were aligned to the hg38 human genome assembly using STAR

[44] . RNAseqChef is a web-based platform for systematic transcriptome analysis, gene expression analysis, principal component analysis, and pathway analysis

[45] .

[0232] Statistical analysis: GraphPad Prism 9 (GraphPad Software, San Diego, CA) was used for statistical analysis. Relationships between groups were compared using t-tests or one-way analysis of variance. Survival rates were analyzed using the Kaplan-Meier method and statistically compared using the log-rank test. A P value of <0.05 was considered statistically significant.

[0233] Results: GBM exhibits low glucose concentrations in the TME, impairing CAR-T cell function. Accumulating evidence suggests that low glucose levels in the TME are a potential barrier to CAR-T cell therapy in solid tumors. However, the actual glucose concentration in the TME and how this low glucose state impacts CAR-T cell function remain unclear

[21] . We examined glucose concentrations in the interstitial fluid of surgical specimens and serum (Figure 1a). The glucose concentration in the interstitial fluid of GBM specimens was approximately 10-fold lower than that in serum [<0.5 mM (average 0.217 mM)] (Figure 1b). While low glucose concentrations are common in various cancer types, such as non-small cell lung cancer and colorectal cancer, GBM exhibits the lowest glucose concentrations in the TME. This indicates that low glucose concentrations in the TME are a hallmark of GBM (Figure 1c).

[0234] Next, we investigated the effects of a low-glucose environment on CAR-T cell functions, including cytokine production. CAR-T cells targeting the GBM-associated antigen EGFRvIII (conv EGFRvIII CAR-T cells) were cocultured with a human GBM cell line (U-87 MGΔ) expressing EGFRvIII under low-glucose conditions (0.5 mM), which mimic the GBM TME. Upon stimulation with U-87 MGΔ, cytokine production (IFN-γ, IL-2, and TNFα) was significantly reduced in the low-glucose environment, even after a short-term (16-hour) exposure ( Figures 29d and 29e ). To further investigate the functional changes of CAR-T cells under low-glucose conditions, CAR-T cells were stimulated under low-glucose (0.5 mM) or high-glucose (10 mM) conditions for 16 hours, and gene expression profiles were analyzed. The gene expression profile was significantly altered by exposure to low glucose conditions (Fig. 35a). Gene sets related to cell cycle, differentiation, and cytokine production were significantly decreased, whereas gene sets related to the cellular response to glucose starvation and intrinsic apoptosis were increased under low glucose conditions (Fig. 29f, g). Thus, the effector function of CAR-T cells is immediately impaired when exposed to low glucose conditions, such as the TME of GBM.

[0235] Overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells. Among the SLC2 family of glucose transporters (GLUTs), GLUT3, encoded by SLC2A3, has the highest affinity and is predominantly expressed in neurons and glial cells, which have a high glucose demand

[16] . Therefore, we investigated whether expression of GLUT3 by CAR-T cells could enhance CAR-T cell activity by promoting glucose uptake in the low-glucose TME of GBM. SLC2A3 was fused to a conventional anti-EGFRvIII CAR construct via a self-cleaving P2A sequence, enabling stable co-expression of CAR and GLUT3 (GLUT3 EGFRvIII CAR) (Figure 30a). GLUT3 was expressed on the membrane of T cells transfected with GLUT3 EGFRvIII CAR (GLUT3 EGFRvIII CAR-T cells) ( Figure 30b ). The expression level was approximately twice that of the parent anti-EGFRvIII CAR-T cells (conv EGFRvIII CAR-T cells) and was similar to that of the GBM cell line U-87 MGΔ, whereas no change was observed in the expression level of GLUT1 ( Figures 30c and 36a ). The amount of glucose uptake measured by a glucose uptake assay using the glucose analog 2-NBDG was greater in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells ( Figure 30d ). Accordingly, glycolysis and glycolytic capacity of GLUT3 EGFRvIII CAR-T cells were significantly improved (Fig. 30e and Fig. 36b). Metabolic analysis further revealed that GLUT3 EGFRvIII CAR-T cells had a significantly lower oxygen consumption rate (OCR) than conv EGFRvIII CAR-T cells. ｈｉｇｈ , extracellular acidification rate (ECAR) ｈｉｇｈThis confirmed a more energetic shift in glucose uptake in the low-glucose environment (Figure 30f). In a glucose competition assay in which carboxyfluorescein succinimidyl ester (CFSE)-labeled GLUT3 EGFRvIII CAR-T cells were cocultured with conv EGFRvIII CAR-T cells at a 1:1 ratio, the number of GLUT3 EGFRvIII CAR-T cells relative to conventional CAR-T cells increased at low glucose levels (0.5 mM) but remained similar under high glucose (10 mM) conditions, suggesting that GLUT3 EGFRvIII CAR-T cells are substantially competitive for glucose uptake, particularly in low-glucose environments (Figures 36c and 36d). Therefore, stable GLUT3 expression by CAR-T cells promotes glucose uptake, making CAR-T cells competitive for glucose uptake and enhancing the metabolic fitness of CAR-T cells in limited glucose environments.

[0236] GLUT3 expression improves the metabolic fitness of CAR-T cells, prompting us to investigate their effector function and safety profile. Therefore, we investigated the effector function of GLUT3 EGFRvIII CAR-T cells using both in vitro and in vivo assays. Compared with combo EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells significantly increased cytokine production (IFN-γ, IL-2, TNFα) at ​​both low (0.5 mM) and high (10 mM) glucose levels (Figures 30g, H and 37a). Cytokine production was significantly enhanced by CD8 ＋ T cell subsets and CD4 ＋GLUT3 EGFRvIII CAR-T cells also showed enhanced cytotoxicity against U-87 MGΔ cells and U-251 MGΔ cells, another EGFRvIII-expressing GBM cell line, under both low- and high-glucose conditions (Fig. 30i, Fig. 37d, e). Expression of efflux-related molecules, including PD-1 and Tim-3, after antigen stimulation was higher in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells (Fig. 30j, k and Fig. 37f, g). Furthermore, enhanced effector function due to stable GLUT3 expression was also observed in CAR-T cells targeting a different antigen, CD19 (GLUT3 CD19 CAR-T cells). This indicates that the enhanced metabolic fitness due to GLUT3 expression is universal for all CARs, regardless of the single-chain variable fragment (scFv) used or the antigen targeted (Figures 38a-c). Thus, stable GLUT3 expression allows CAR-T cells to be activated in the TME with low glucose levels, despite the presence of an exhausted phenotype.

[0237] Stable GLUT3 expression overactivates CAR-T cells and causes severe side effects. The impact of stable GLUT3 expression in CAR-T cells on antitumor efficacy and safety profile was further investigated using an intracranial U-87 MGΔ xenograft model (Figure 31a). GLUT3 EGFRvIII CAR-T cells were intravenously administered 5 days after intracranial tumor inoculation. As expected, GLUT3 EGFRvIII CAR-T cell therapy demonstrated enhanced antitumor efficacy (CR: 4 / 6) compared with conv EGFRvIII CAR-T cell therapy (CR: 2 / 6) (Figure 31b). Nevertheless, GLUT3 EGFRvIII CAR-T cell therapy failed to improve survival due to a high incidence of treatment-related mortality (TRM) associated with deterioration of general condition and severe weight loss (Figures 31c-e). Consistent with these findings, pathological analysis revealed hyperactivation of T cells in the brain tissue of mice treated with GLUT3 EGFRvIII CAR-T cells, with diffuse infiltration of the brain parenchyma, particularly the ventricular wall (Fig. 31f). Furthermore, diffuse alveolar damage was detected in the lung tissue, as indicated by thickening of the alveolar wall accompanied by T cell infiltration (Fig. 31g). Indeed, effector memory T cells (CD45RA CCR7 －－ The frequency of GLUT3 EGFRvIII CAR-T cells was significantly higher in mice administered with GLUT3 EGFRvIII CAR-T cells than in mice administered with conv EGFRvIII CAR-T cells (Figure 31h). Furthermore, gene expression assays revealed that gene sets associated with cytokine production, T cell differentiation, and T cell activation were significantly upregulated in GLUT3 EGFRvIII CAR-T cells even at day 7 post-stimulation (Figure 31i). Thus, stable expression of GLUT3 by CAR-T cells impairs the activation state of T cells induced by excessive glucose intake, highlighting the need for delicate control of metabolic capacity.

[0238] On-demand expression of GLUT3 upon antigen stimulation was achieved using the CAR-NFAT-GLUT3 construct. Considering the severe toxicity caused by overactivation of GLUT3 EGFRvIII CAR-T cells, we investigated whether expressing GLUT3 in response to glucose demand, i.e., upon T cell stimulation upon target antigen recognition in the TME, could maximize the benefits of GLUT3 expression and minimize toxicity. We developed a construct in which SLC2A3 was placed downstream of the NFAT-binding domain, enabling on-demand GLUT3 expression (on-demand GLUT3 CAR) (Figure 32a). T cell activation by antigen recognition induced nuclear translocation of NFAT, promoting GLUT3 expression. Target antigen recognition by T cells carrying the on-demand GLUT3 CAR construct (on-demand GLUT3 EGFRvIII CAR-T cells) induced GLUT3 expression. GLUT3 expression returned to its initial level 7 days after the last antigen stimulation (Fig. 32b, c). Indeed, glucose uptake and glycolysis were higher in on-demand GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells (Fig. 32d, e and Fig. 39a). On day 3, on-demand GLUT3 EGFRvIII CAR-T cells were in a metabolically more energetic state (OCR). ｈｉｇｈ ECAR ｈｉｇｈ) and recovered to a level comparable to that of conv EGFRvIII CAR-T cells by day 7 after the last antigen stimulation ( Figure 33f ). Cytokine production was significantly higher in on-demand GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells under both low (0.5 mM) and high (10 mM) glucose levels ( Figures 33g and 33h and 39b ). On-demand GLUT3 EGFRvIII CAR-T cells also showed increased cytotoxicity against U-87 MGΔ and U-251 MGΔ cells under both low and high glucose conditions ( Figures 33i and 39c ). Taken together, the on-demand GLUT3 CAR construct successfully improved metabolic fitness in an on-demand manner, and on-demand GLUT3 EGFRvIII CAR-T cells enhanced cytokine production and cytotoxicity under both low- and high-glucose conditions.

[0239] On-demand GLUT3 expression enables sustained effector function of CAR-T cells. Having successfully developed on-demand GLUT3 EGFRvIII CAR-T cells with enhanced cytokine production and cytotoxicity, we investigated their comprehensive immunological profile, focusing on their persistence, function, and molecular expression. We first examined cell proliferation after antigen stimulation. On-demand GLUT3 EGFRvIII CAR-T cells continued to expand after antigen stimulation, whereas GLUT3 EGFRvIII CAR-T cells did not (Figure 40a, b). This failure of GLUT3 EGFRvIII CAR-T cells to expand was primarily due to the induction of apoptosis, a process known as activation-induced cell death (AICD), which is an overactivation-induced phenomenon critical for T cell homeostasis

[22] (Figure 34a). Gene expression analysis further confirmed that under low glucose conditions, GLUT3 EGFRvIII CAR-T cells showed increased expression of apoptosis-related genes compared to on-demand GLUT3 EGFRvIII CAR-T cells and conv EGFRvIII CAR-T cells ( Figure 34b ). Furthermore, the expression of apoptosis-related genes was closely correlated with the expression of TNFα signaling genes, suggesting a role for TNFα in the apoptosis of GLUT3 EGFRvIII CAR-T cells ( Figure 34c ). Meanwhile, the expression of T cell activation-related genes was elevated in on-demand GLUT3 EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells compared to conv EGFRvIII CAR-T cells ( Figure 34b ). In accordance with this, on-demand GLUT3 EGFRvIII CAR-T cells are central memory (CD62L CCR7 ＋＋ CD45RA － ) and naive phenotype (CD62L CCR7 ＋＋ CD45RA ＋ ), whereas GLUT3 EGFRvIII CAR-T cells maintained a terminally differentiated phenotype (CD62L CCR7 －－ CD45RA ＋) (Figure 34d, Figure 40c). Furthermore, the expression of exhaustion-associated molecules, including PD-1 and Tim-3, was lower in on-demand GLUT3 EGFRvIII CAR-T cells than in GLUT3 EGFRvIII CAR-T cells and conv EGFRvIII CAR-T cells (Figure 34e and Figure 40d). Furthermore, the global gene expression profiles of GLUT3 EGFRvIII CAR-T cells and on-demand GLUT3 EGFRvIII CAR-T cells were comparable on day 3 post-stimulation but significantly different on day 7 post-stimulation (Figure 34f). RNA-seq analysis of each CAR-T cell population on day 7 post-stimulation further confirmed that GLUT3 EGFRvIII CAR-T cells were dependent on glycolysis, while on-demand GLUT3 EGFRvIII CAR-T cells maintained oxidative phosphorylation and fatty acid oxidation, reflecting an activated state, i.e., naive / stem cell memory and central memory phenotype (Figure 34g). These findings indicate that on-demand GLUT3 expression prevents CAR-T cell overactivation and maintains functional CAR-T cells for a long period of time. From these results, particularly those in Figure 34, it can be concluded that stably expressed GLUT3 enhances the antitumor effect (i.e., effector function) of EGFRvIII CAR-T cells. On-demand GLUT3 expression not only enhanced the effector activity of EGFRvIII CAR-T cells, but also prolonged memory function and enhanced maintenance of the memory T cell phenotype. Furthermore, it can be concluded that adverse events such as weight loss and treatment-related death induced by constitutively expressed GLUT3 were prevented, resulting in a significant improvement in survival rate, including cure.

[0240] On-demand GLUT3 EGFRvIII CAR-T cells demonstrated enhanced antitumor efficacy without severe toxicity in an intracranial human GBM cell xenograft model. Based on the favorable phenotype of on-demand GLUT3 EGFRvIII CAR-T cells, we investigated the antitumor efficacy and safety profile in an intracranial human GBM cell xenograft model (Figure 35a). Compared with conv EGFRvIII CAR-T cells, on-demand GLUT3 EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells demonstrated superior antitumor efficacy, with the majority of mice achieving CR (5 / 10 CR for conv EGFRvIII CAR-T cells, 9 / 10 CR for GLUT3 EGFRvIII cells, and 9 / 10 CR for on-demand GLUT3 EGFRvIII CAR-T cells) ( Figures 35b and 41a ). However, as observed in previous experiments ( Figures 32a–e ), GLUT3 EGFRvIII CAR-T cells induced severe toxicity accompanied by significant weight loss and failed to improve survival. In contrast, on-demand GLUT3 EGFRvIII CAR-T cells significantly improved survival without causing severe toxicity or weight loss ( Figure 35c-e ). Accordingly, tumor rechallenge was also suppressed only in mice treated with on-demand GLUT3 EGFRvIII CAR-T cells ( Figure 35b ). Accordingly, 14 days after CAR-T cell injection, high frequencies of naive / stem cell memory and central memory subsets were detected in the peripheral blood of mice treated with on-demand GLUT3 EGFRvIII CAR-T cells, whereas effector memory subsets were more abundant in mice treated with GLUT3 EGFRvIII CAR-T cells ( Figure 35f ). This increased anti-tumor effect induced by on-demand GLUT3 EGFRvIII CAR-T cells was similarly observed in another xenograft model (U-251 MGΔ); compared with mice in other treatment groups, mice treated with on-demand GLUT3 EGFRvIII CAR-T cells showed superior survival without severe toxicity ( FIG. 35 g and FIG. 41 b, c).Cytokine levels (TNFα and GM-CSF) remained elevated in the serum of mice treated with GLUT3 EGFRvIII CAR-T cells even on day 14, when tumors had already been rejected. The rate of cytokine reduction was significantly lower in mice treated with GLUT3 EGFRvIII CAR-T cells than in mice treated with conv EGFRvIII CAR-T cells or on-demand EGFRvIII CAR-T cells ( Figure 35h, i ). Furthermore, the levels of cytokines reported to be involved in the development of cytokine release syndrome (CRS), such as IL-5, IP-10, and MDC, were higher in the serum of mice treated with GLUT3 EGFRvIII CAR-T cells than in the serum of mice treated with on-demand GLUT3 EGFRvIII CAR-T cells ( Figure 41d ). Overall, on-demand GLUT3 expression by CAR-T cells is a promising strategy to achieve durable antitumor effects without severe toxicity in GBM.

[0241] Discussion: Enhancing the antitumor effect by improving the metabolic fitness of CAR-T cells in the TME is a novel concept for accelerating the clinical application of CAR-T cell therapy for refractory solid tumors. Although some clinical trials of CAR-T cells in GBM have failed to demonstrate clinical benefit, immunological monitoring of these trials revealed abundant CAR-T cells in the TME [Refs. 3-5]. Considering the presence of CAR-T cells in the TME, antigen loss and impaired migration are excluded as causes of failure, and it is possible that specific conditions in the GBM TME may be involved in inducing CAR-T cell dysfunction. Glucose is an essential nutrient for effector T cells, as they undergo glycolysis for survival and activation [Ref. 19]. Therefore, given the significantly low glucose environment detected in the GBM TME, low glucose levels in the GBM TME must be a major metabolic stressor for CAR-T cells. Indeed, conv EGFRvIII CAR-T cells are dysfunctional (low cytokine production and high expression of exhaustion markers). Furthermore, PD-1 signaling disrupts PI3K / Akt / mTOR signaling, further impairing the glycolytic pathway, potentially causing effector T cells to enter a negative metabolic cycle

[20] .

[0242] Considering that neural cells such as neurons and glial cells, as well as GBMs, especially those expressing EGFRvIII, meet their high glucose demands using the high-affinity glucose transporter GLUT3 [17, 18, 23], overexpression of GLUT3 in CAR-T cells is likely to overwhelm nutrient competition. Indeed, our GLUT3-EGFRvIII CAR construct enhanced glucose uptake and effector functions, such as cytokine production and cytotoxicity, in GLUT3-EGFRvIII CAR-T cells. However, unexpectedly, GLUT3-EGFRvIII CAR-T cells underwent apoptosis approximately 24 hours after antigen stimulation, and mice administered GLUT3-EGFRvIII CAR-T cells died of translocation-related leukemia (TRM). Global gene expression assays revealed that GLUT3 EGFRvIII CAR-T cells exhibited a distinct gene expression profile, with upregulated genes in the TNFα signaling pathway. This is consistent with AICD being caused by overactivation of GLUT3 EGFRvIII CAR-T cells due to continuous glucose influx via GLUT3, as TNFα, along with the FAS-FASL pathway, is a key mediator of AICD [24, 25]. TRM, characterized by severe inflammation in lung and brain tissues and weight loss, is also associated with increased levels of inflammatory cytokines, including TNFα, mimicking CRS observed in cancer patients treated with CAR-T cells. CRS has been reported to be induced by IFN-γ, TNFα, and GM-CSF produced by activated CAR-T cells

[26] . Accordingly, mice administered GLUT3 EGFRvIII CAR-T cells also showed increased cytokine production (IL-5, IP-10, MDC) associated with CRS severity. The present inventors used an in vivo animal model, NOD.Cg-Prkdc, which lacks innate and adaptive immune cells, including macrophages. ｓｃｉｄ Il2rg ｔｍ１ｌＷｊｌ Because / SzJ (NSG) mice were used, it was not possible to evaluate inflammatory cytokines produced by macrophages.

[0243] To overcome the complexity of stable GLUT3 expression in CAR-T cells, we developed an on-demand GLUT3 expression system that allows CAR-T cells to express GLUT3 on demand and take up glucose in response to glucose demand. This inducible GLUT3 expression was achieved by nuclear translocation of NFAT, a downstream signal of antigen stimulation. NFAT nuclear translocation occurs immediately after TCR / CAR signaling and peaks at 30 minutes

[29] . NFAT binds to a conserved DNA-binding domain (NFAT-binding domain) and promotes a panel of genes required for T cell activation, including genes that induce an energy shift from oxidative phosphorylation to aerobic glycolysis to meet increasing energy demands [30, 31]. Thus, GLUT3 expression is induced upon antigen encounter in the TME, and the kinetics of GLUT3 expression parallels those of NFAT. Additionally, stable overexpression of GLUT3 in anti-CD19 CAR-T cells is not beneficial in hematological malignancy models

[32] . Because CAR-T cells pass through various glucose conditions, including the high glucose environment of peripheral blood, before reaching the metabolically challenging, low glucose TME of GBM, on-demand expression of GLUT3 upon antigen exposure is desirable and necessary.

[0244] Furthermore, on-demand GLUT3 expression by CAR-T cells favors the formation of memory cells through improved metabolic fitness, resulting in sustained antitumor efficacy with a significant survival benefit in preclinical models. Furthermore, on-demand GLUT3 expression significantly reduced TRM by regulating the sustained production of inflammatory cytokines such as TNFα through stable GLUT3 expression. In a clinical trial in which bicistronic CAR-T cells targeting EGFR and interleukin-13 receptor subunit α2 (IL-13Rα2) were intrathecally administered to GBM, all patients (n=6) experienced TRM. All patients (n=6) experienced early onset of immune effector cell-associated neurotoxicity syndromes (ICANs), requiring the administration of high-dose dexamethasone and anakinra (an anti-IL-1R antibody). This indicates that CAR-T cell toxicities, such as CRS and ICANs, are a major concern. Additionally, high doses of steroids may reduce the antitumor efficacy of CAR-T cells [33, 34]. Therefore, on-demand GLUT3 expression may be an ideal strategy to achieve both sustained antitumor efficacy and a safe profile. These findings highlight the importance of the timing and persistence of immune cell infiltration.

[0245] Using an on-demand system that acts upon antigen stimulation, the inventors have successfully achieved enhanced and sustained antitumor activity of CAR-T cell therapy without systemic adverse events. Given that target molecules are ideally induced upon antigen recognition in the TME, it is conceivable that this on-demand system could be extended to other molecules, such as other metabolic molecules (including glutamine transporters and fatty acid metabolic enzymes), cytokines, and chemokines. For example, IL-12 has been introduced as a promising antitumor cytokine based on animal experiments in clinical studies [References 35-37]. However, severe side effects have hindered its clinical application [References 38, 39]. On-demand delivery of IL-12 to the TME may overcome these side effects and maximize antitumor efficacy.

[0246] In summary, on-demand GLUT3 expression was induced by antigen stimulation, and a novel CAR-T cell construct was developed. The on-demand GLUT3 CAR-T cells demonstrated durable antitumor efficacy with ideal metabolic competence without severe toxicity. Because delicate control of metabolic supply is required, the on-demand approach is a promising strategy. Overall, this study has profound implications for utilizing metabolic competence in immunotherapy in GBM and other solid tumors.

[0247] (Example 6: Example of TCR) A similar experiment is carried out in which a TCR is introduced instead of a CAR. ｅｆｆ TCR can also be introduced into fractions such as Tnv, Tcm, Tem, and Temra.

[0248] In this example, a TCR (CAR, constitutively expressed GLUT3 CAR, or on-demand GLUT3 CAR) is introduced into any purified and enriched T cell memory fraction. In a separate experiment, TCR- T cells enriched in any T cell memory fraction are induced under culture conditions.

[0249] Various in vitro and in vivo evaluations shown in Examples 1 to 5 are carried out.

[0250] Example 7: Prevention, prevention of reinfection, and treatment of bacterial infection This example examines the prevention, prevention of reinfection, and / or treatment of bacterial infection.

[0251] (Method) For prevention of bacterial infections (such as bacterial conjunctivitis), T cells of the present disclosure modified with an infectious agent of the bacterial infection are administered to a subject who does not have the bacterial infection.

[0252] In preventing recurrence and / or treating bacterial infections (such as bacterial conjunctivitis), T cells of the present disclosure are administered to a subject who has had and / or has a bacterial infection.

[0253] After administration, the subject is followed up for bacterial infection by techniques routinely used in the art.

[0254] Example 8: Prevention, reinfection prevention, and treatment of parasitic infections This example examines the prevention, reinfection prevention, and / or treatment of parasitic infections.

[0255] (Method) For prevention of a parasitic infection (such as Acanthamoeba keratitis), T cells of the present disclosure modified with an infectious antigen of the parasitic infection are administered to a subject who does not have the parasitic infection.

[0256] In preventing recurrence and / or treating a parasitic infection (such as, for example, Acanthamoeba keratitis), T cells of the present disclosure are administered to a subject who has had and / or has a parasitic infection.

[0257] After administration, the subject is followed up for parasitic infection by techniques routinely used in the art.

[0258] Example 9: Prevention of infection, onset, reinfection, and treatment of viral infections In this example, the prevention of infection, onset, reinfection, and / or treatment of viral infections is verified.

[0259] (Method) For the prevention of viral infections (e.g., which may include, but are not limited to, tuberculosis, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonella infection, pathogenic E. coli, toxoplasmosis, Zika virus, herpesvirus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza (virus), MARS, rabies, diphtheria, etc.), T cells of the present disclosure modified with an infectious antigen of the viral infection are administered to a subject who does not have the viral infection.

[0260] In preventing recurrence and / or treating the viral infection, the T cells of the present disclosure are administered to a subject who has had and / or is currently suffering from a viral infection.

[0261] After administration, the subject is monitored for prognosis of viral infection by techniques commonly used in the art.

[0262] Example 10: Prevention, relapse prevention, and treatment of allergies This example examines the prevention, relapse prevention, and / or treatment of allergies.

[0263] (Methods) In the prevention of allergies (eg, allergic conjunctivitis), T cells of the present disclosure modified with an allergic infection-causing antigen are administered to a subject who does not have the allergy-causing antigen.

[0264] In preventing recurrence and / or treating allergies (e.g., allergic conjunctivitis), T cells of the present disclosure are administered to a subject who has had and / or has the causative antigen of the allergy.

[0265] After administration, the subject is followed up for allergy by techniques routinely used in the art.

[0266] Example 11: Prevention, relapse prevention, and treatment of autoimmune disease This example examines the prevention, relapse prevention, and / or treatment of autoimmune disease.

[0267] (Method) In the prevention of autoimmune diseases (e.g., autoimmune uveitis), T cells of the present disclosure modified with an autoimmune disease infectious cause antigen are administered to a subject who does not have the allergy-causing antigen.

[0268] In preventing recurrence and / or treating an autoimmune disease (e.g., autoimmune uveitis), the T cells of the present disclosure are administered to a subject who has had and / or has the causative antigen of the autoimmune disease.

[0269] After administration, the subject is monitored for prognosis of the autoimmune disease by techniques commonly used in the art.

[0270] References 1. Stupp R, Taillibert S, Kanner A, et al (2017) Effect of Tumor-Treating Fields Plus Maintenance Temozolomide vs...

Claims

1. T cells with enhanced glucose uptake capacity, wherein the expression of glucose transporters in said T cells is regulated in response to changes in tumor microenvironment conditions.

2. The T cell of claim 1, wherein the tumor microenvironment conditions include the degree of TCR-associated signal transduction.

3. The T cell of claim 2, wherein the TCR-associated signal is mediated by a molecule selected from the group consisting of a TCR signal or a portion thereof, and a chimeric signal of a TCR signal and another antigen (also referred to as a CAR signal).

4. The T cell of claim 2 or 3, wherein the TCR-associated signal is mediated by a molecule containing a TCR signal domain.

5. The T cell of any one of claims 1 to 4, wherein the altered tumor microenvironment conditions include the presentation of tumor antigens and / or costimulatory ligands, and / or altered cytokine conditions.

6. A T cell according to any one of claims 2 to 5, wherein the degree of transmission of the TCR-associated signal comprises the degree of TCR stimulation.

7. A T cell according to any one of claims 2 to 6, wherein the degree of TCR-associated signal transmission includes at least one selected from the group consisting of the degree of TCR stimulation, TCR costimulation, and humoral factors.

8. A T cell according to any one of claims 2 to 7, wherein the degree of transmission of the TCR-associated signal comprises the degree of TCR stimulation and at least one selected from the group consisting of TCR costimulation and humoral factors.

9. A T cell according to any one of claims 2 to 8, wherein the degree of TCR-associated signal transduction includes the degree of TCR stimulation, TCR costimulation, and humoral factors.

10. A T cell according to any one of claims 6 to 9, wherein the TCR stimulation is selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families, which translocate into the nucleus upon activation of the T cell.

11. The T cell of any one of claims 1 to 10, wherein the tumor microenvironment conditions include a degree of oxygen concentration.

12. A T cell comprising a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

13. The T cell of claim 12, wherein the tumor microenvironment sensing element comprises: (i) an inducible activation motif that is activated in response to a change in the tumor microenvironment condition; and (ii) a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to a change in the tumor microenvironment condition.

14. The T cell described in claim 12 or 13, wherein the tumor microenvironment sensing element comprises: (i) an inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to T cell activation binds; (ii) a nuclear localization-binding inducible activation motif to which a nuclear localization factor that translocates into the nucleus in response to a decrease in oxygen concentration binds; and (iii) a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading a glucose transporter is inactivated in response to the decrease in oxygen concentration.

15. The T cell of any one of claims 1 to 14, comprising a construct in which a nucleic acid sequence encoding the glucose transporter is operably linked to an inducible activation motif that is activated in response to changes in tumor microenvironment conditions and / or a nucleic acid sequence encoding a degradation factor of the glucose transporter that is inactivated in response to changes in the tumor microenvironment conditions.

16. The T cell of any one of claims 1 to 15, comprising a construct in which a nucleic acid sequence encoding the glucose transporter is operably linked to a nuclear import-binding-inducing activation motif to which a nuclear import factor that translocates into the nucleus in response to T cell activation binds.

17. The T cell of any one of claims 1 to 16, wherein the nuclear import factor is selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families.

18. The T cell of claim 16 or 17, wherein the nuclear translocation binding-inducing activation motif is selected from the group consisting of an NFAT binding domain, a κB motif (GGGACTTTCC) (SEQ ID NO: 1), and a STAT binding sequence (TTCNNNGAA), or a modified sequence thereof.

19. The T cell according to any one of claims 16 to 18, wherein the combination of the nuclear transport factor and the nuclear transport binding-inducing activation motif comprises: (1) a combination of NFAT and an NFAT-binding domain; (2) a combination of NF-κB and a κB motif (GGGACTTTCC) (SEQ ID NO: 1); or (3) a combination of a STAT transcription factor family and a STAT binding sequence (TTCNNNGAA).

20. A T cell according to any one of claims 1 to 19, comprising a construct in which a nucleic acid sequence encoding the glucose transporter is operably linked to a nuclear translocation-binding-inducing activation motif to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds.

21. The T cell of any one of claims 16 to 20, wherein the nuclear import factor is selected from the group consisting of HIF-1.

22. A T cell according to any one of claims 13 to 21, wherein the nuclear translocation binding-inducing activation motif is selected from the group consisting of HIF-RE.

23. A T cell according to any one of claims 13 to 22, wherein the combination of the nuclear transport factor and the nuclear transport binding-inducing activation motif is selected from the group consisting of: (1) a combination of HIF-1 and HIF-RE.

24. A T cell according to any one of claims 1 to 23, comprising a construct in which a nucleic acid sequence encoding the glucose transporter is operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter in response to a decrease in oxygen concentration is inactivated.

25. The T cell of any one of claims 15 to 24, wherein the factor to be inactivated is selected from the group consisting of oxygen-dependent degradation factors (ODDs).

26. The T cell of any one of claims 1 to 25, wherein the T cell comprises a chimeric antigen receptor (CAR).

27. A T cell according to any one of claims 1 to 26, wherein the T cell has an effector function.

28. The T cells are effector T cells (T ｅｆｆ The T cell according to any one of claims 1 to 26, which is a precursor cell of a T cell of a T cell type.

29. The T cell of claim 28, wherein the precursor to the effector T cell has extended memory function.

30. The T cell of claim 28 or 29, wherein the precursor of the effector T cell maintains the phenotype of a memory T cell.

31. The T cells of any one of claims 28 to 30, wherein the effector T cells are activated or activity is maintained, and the precursors of the effector T cells have extended memory function or maintain memory T cell phenotype.

32. The T cell of any one of claims 1 or 26 to 31, wherein the expression and / or enhancement is achieved specifically in the tumor environment.

33. The T cell of any one of claims 26 to 32, wherein the CAR is expressed in the T cell.

34. The T cell of any one of claims 1 to 33, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4.

35. A T cell according to any one of claims 1 to 34, wherein the glucose transporter is GLUT3.

36. A T cell according to any one of claims 1 to 35, which is a human T cell.

37. A cell population containing T cells with enhanced glucose uptake capacity on demand.

38. A cell population comprising a T cell according to any one of claims 1 to 36.

39. The cell population described in claim 37 or 38, wherein the T cells include cells that have the property of having effector function when introduced into the body.

40. The cell population of any one of claims 37 to 39, wherein the T cells comprise a chimeric antigen receptor (CAR).

41. The cell population of any one of claims 37-40, wherein the T cells are modified such that expression of a glucose transporter is regulated in the T cells in response to changes in tumor microenvironment conditions, and / or comprise a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that senses changes in tumor microenvironment conditions.

42. The cell population of any one of claims 37 to 41, wherein the T cells have effector function.

43. The cell population is T ｅｆｆ The cell population according to any one of claims 37 to 42, comprising:

44. The cell population is T ｅｆｆ The cell population according to any one of claims 37 to 43, comprising progenitor cells of the above group.

45. A cell population described in any one of claims 38 to 44, wherein the CAR is expressed in the T cells.

46. ​​The cell population of any one of claims 37 to 45, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4.

47. The cell population of any one of claims 37 to 46, wherein the glucose transporter is GLUT3.

48. The cell population of any one of claims 37-47, wherein the T cells are human T cells.

49. A pharmaceutical composition comprising a T cell according to any one of claims 1 to 36 or a cell population according to any one of claims 37 to 48.

50. The pharmaceutical composition of claim 49, for the prevention or treatment of cancer.

51. The pharmaceutical composition of claim 49 or 50, for the treatment of cancer.

52. A pharmaceutical composition according to any one of claims 49 to 51, for preventing metastasis or recurrence of cancer.

53. A pharmaceutical composition according to any one of claims 49 to 52, for preventing or treating cancer so that it does not recur.

54. A T cell according to any one of claims 1 to 36 or a cell population according to any one of claims 37 to 48 for use as a medicament.

55. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a T cell described in any one of claims 1 to 36 or a cell population described in any one of claims 37 to 48.

56. Use of a T cell according to any one of claims 1 to 36 or a cell population according to any one of claims 37 to 48 for the manufacture of a medicament.

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