T cells having enhanced glucose uptake ability when necessary, cell populations, and pharmaceutical compositions comprising the same

Abstract

The present disclosure provides a cell capable of competing with cancer cells without becoming starved or exhausted even in a glucose-depleted environment. More specifically, the present disclosure provides a T cell having enhanced glucose uptake capacity, 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 and / or comprises a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

Description

Technology Field

[0001] The present disclosure relates to T cells having enhanced glucose uptake ability on demand, cell populations, and pharmaceutical compositions comprising such cells. Background Technology

[0002] Cancer cells reprogram their metabolic systems to be advantageous for their cell proliferation, actively utilize the glycolysis pathway which is inefficient for adenosine triphosphate (ATP) production even in the presence of oxygen, and increase glucose uptake and lactic acid production (Warburg effect).

[0003] Multiple factors exist in the tumor microenvironment that inhibit the metabolic activity and function of anti-tumor T cells, and in addition to immunosuppressive factors, metabolic competition between tumors and T cells is involved in the formation of the immunosuppressive environment. When T cells are stimulated via TCRs, intracellular Ca 2+ The concentration increases, and calcineurin is activated. NFAT, a transcription factor dephosphorylated by the activation of calcineurin, translocates into the nucleus and induces activation by promoting the transcription of genes such as IL-2 through interactions with other transcription factors.

[0004] Meanwhile, because cancer cells consume large amounts of glucose and cause glucose depletion, T, which requires glucose as an energy source eff Even if type 1 tumor-specific T cells receive signals via TCRs when infiltrating the tumor, intracellular Ca 2+ The concentration decreases and the nuclear potential of NFAT decreases. Consequently, T cells become dysfunctional, and cell proliferation and cytokine production are inhibited.

[0005] In addition to glucose, when nutrients such as amino acids and fatty acids are consumed in large quantities by cancer cells, T cells competing for these nutrients become dysfunctional. As mentioned above, the anti-tumor effector action of tumor-specific T cells in the tumor microenvironment is inhibited by metabolic mechanisms that support the active proliferation of cancer cells.

[0006] Therefore, cells capable of competing with cancer cells without becoming starved or exhausted even in such an environment are required.

[0007] [Means of Solving the Problem]

[0008] The present disclosure provides T cells having enhanced glucose uptake capacity that function without starvation or exhaustion even in a tumor environment, a cell population comprising such cells, and a pharmaceutical composition comprising such cells. In particular, a representative feature of the present disclosure is that glucose uptake capacity is enhanced in response to need (on demand).

[0009] Accordingly, the present disclosure provides the following.

[0010] [Item 1] T cells having enhanced glucose uptake capacity that are modified to express a glucose transporter in response to need (if necessary) or have enhanced glucose transporter expression in response to need (if necessary).

[0011] [Item 2] In the above item, the T cell is a T cell containing a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0012] [Item 2A] In any one of the above items, the T cell is a T cell containing a chimeric antigen receptor (CAR).

[0013] [Item 3] In any one of the above items, the T cell is a T cell having an effector function.

[0014] [Item 4] In any of the above items, the T cell is an effector T cell (effector T cell, T eff T cells, which are the precursor cells of ).

[0015] [Item 5] In any one of the above items, expression and / or enhancement, if necessary, are T cells specifically achieved in a tumor environment.

[0016] [Item 6] In any one of the above items, CAR is a T cell expressed in a T cell.

[0017] [Item 7] In any one of the above items, the glucose transporter is a T cell comprising GLUT1, GLUT2, GLUT3 and GLUT4.

[0018] [Item 8] In any one of the above items, the glucose transporter is a T cell that is GLUT3.

[0019] [Item 9] In any of the above items, the T cell is a human T cell.

[0020] [Item 10] A cell population comprising T cells having enhanced glucose uptake capacity in response to need (if necessary).

[0021] [Item 11] In any one of the above items, T cells are a group of cells comprising cells having the characteristic of exhibiting effector function when introduced into the body.

[0022] [Item 12] In any one of the above items, the T cells are a population of cells including a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0023] [Item 12A] In any one of the above items, the T cells are a cell population containing a chimeric antigen receptor (CAR).

[0024] [Item 13] In any one of the above items, T cells are modified to express glucose transporters in response to need (if necessary) or / or a population of cells in which the expression of glucose transporters is enhanced in response to need (if necessary).

[0025] [Item 14] In any one of the above items, T cells are a population of cells having effector functions.

[0026] [Item 15] In any one of the above items, the cell population is T eff A cell population containing

[0027] [Item 16] In any of the above items, the cell population is T eff A cell population containing precursor cells.

[0028] [Item 16A] In any one of the above items, cell population (or T eff The progenitor cell) is a cell population comprising at least one cell selected from the group consisting of Tnv, Tscm, Tcm, and Temra.

[0029] [Item 17] In any one of the above items, CAR is a population of cells expressed in T cells.

[0030] [Item 18] In any one of the above items, the glucose transporter is a cell population comprising GLUT1, GLUT2, GLUT3 and GLUT4.

[0031] [Item 19] In any one of the above items, a cell population in which the glucose transporter is GLUT3.

[0032] [Item 20] In any one of the above items, T cells are a population of cells that are human T cells.

[0033] [Item 21] A pharmaceutical composition comprising a T cell according to any one of the above items or a cell population according to any one of the above items.

[0034] [Item 22] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0035] [Item 22A] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the prevention or treatment of cancer.

[0036] [Item 23] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended to cure cancer.

[0037] [Item 24] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0038] [Item 25] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended to prevent or treat cancer so as not to cause cancer to recur.

[0039] [Item 26] To prevent or treat disease in the subject

[0040] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0041] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0042] (C) A pharmaceutical composition characterized by the step of administering to a subject a T cell or a cell population comprising T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0043] [Item 26A] In any one of the above items, the T cell is a pharmaceutical composition comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0044] [Item 26B] In any one of the above items, the T cell is a pharmaceutical composition comprising a chimeric antigen receptor (CAR).

[0045] [Item 26C] A pharmaceutical composition comprising, in any one of the above items, a disease including cancer, an autoimmune disease, an allergy or an infectious disease.

[0046] [Item 26D] In any one of the above items, the disease is a pharmaceutical composition comprising cancer.

[0047] [Item 27] ​​In any of the above items, T cells having enhanced glucose uptake capacity are not modified to express glucose transporters in response to need (when necessary), and T cells in which the expression of glucose transporters is not enhanced in response to need (when necessary) eff A pharmaceutical composition comprising cells modified to express a glucose transporter as needed (if needed) and / or cells in which the expression of a glucose transporter is enhanced as needed (if needed).

[0048] [Item 28] A composition in which, in any one of the above items, the glucose transporter is GLUT3.

[0049] [Item 29] In any one of the above items, the T cell or cell population is a T cell or cell population intended for use as a medicament.

[0050] [Item 30] In any one of the above items, the cell or cell population is a cell or cell population intended for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0051] [Item 30A] In any one of the above items, the cell or cell population is a cell or cell population intended for the prevention or treatment of cancer.

[0052] [Item 31] In any one of the above items, the cell or cell population is a cell or cell population intended to cure cancer.

[0053] [Item 32] In any one of the above items, the cell or cell population is a cell or cell population intended for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0054] [Item 33] In any one of the above items, the cell or cell population is a cell or cell population intended to prevent or treat cancer so as not to recur.

[0055] [Item 34] To prevent or treat disease in subjects

[0056] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0057] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0058] (C) A cell or cell population characterized by the step of administering to a subject a T cell or a cell population comprising T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0059] [Item 34A] In any one of the above items, the T cell is a cell or population of cells comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0060] [Item 34B] In any one of the above items, the T cell is a cell or population of cells containing a chimeric antigen receptor (CAR).

[0061] [Item 34C] In any one of the above items, the disease is a cell or population of cells including cancer, an autoimmune disease, an allergic or infectious disease.

[0062] [Item 34D] In any one of the above items, the disease is a cell or population of cells including cancer.

[0063] [Item 35] In any of the above items, T cells having enhanced glucose uptake capacity are not modified to express glucose transporters in response to need (when necessary), and T cells in which the expression of glucose transporters is not enhanced in response to need (when necessary) eff , and cells or a population of cells comprising cells modified to express a glucose transporter in response to need (if necessary) and / or cells in which the expression of a glucose transporter is enhanced in response to need (if necessary).

[0064] [Item 36] In any one of the above items, the glucose transporter is a cell or cell population that is GLUT3.

[0065] [Item 37] A method for treating or preventing in a subject, comprising the step of administering an effective amount of T cells according to any one of the above items or a cell population according to any one of the above items to a subject in need thereof.

[0066] [Item 38] In any one of the above items, the treatment or prevention of the subject is a method for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0067] [Item 38A] In any one of the above items, the treatment or prevention of the subject includes the prevention or treatment of cancer.

[0068] [Item 39] In any one of the above items, the treatment or prevention of the subject includes a method of curing cancer.

[0069] [Item 40] In any one of the above items, the treatment or prevention of the subject includes the prevention of cancer metastasis or the prevention of cancer recurrence.

[0070] [Item 41] In any one of the above items, the treatment or prevention of the subject includes a step of preventing or treating cancer so that the cancer does not recur.

[0071] [Item 42] To prevent or treat disease in subjects

[0072] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0073] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0074] (C) A method comprising the step of administering to a subject a T cell or a cell population containing T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0075] [Item 42A] A method in which, in any one of the above items, the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0076] [Item 42B] A method in which, in any one of the above items, the T cell comprises a chimeric antigen receptor (CAR).

[0077] [Item 42C] In any one of the above items, the disease includes cancer, autoimmune disease, allergy or infectious disease.

[0078] [Item 42D] In any one of the above items, the disease is a method including cancer.

[0079] [Item 43] In any of the above items, T cells having enhanced glucose uptake capacity are not modified to express glucose transporters in response to need (when necessary), and T cells in which the expression of glucose transporters is not enhanced in response to need (when necessary) effA method comprising cells modified to express a glucose transporter in response to need (if necessary) and / or cells in which the expression of a glucose transporter is enhanced in response to need (if necessary).

[0080] [Item 44] A method in which, in any one of the above items, the glucose transporter is GLUT3.

[0081] [Item 45] Use for producing a drug comprising a T cell or a cell population according to any of the above items, or a cell or cell population according to any of the above items.

[0082] [Item 46] In any one of the above items, the drug is used for the treatment or prevention of cancer, autoimmune diseases, allergies, or infectious diseases.

[0083] [Item 46A] In any one of the above items, the drug is intended for the prevention or treatment of cancer.

[0084] [Item 47] In any one of the above items, the drug is intended for use in curing cancer.

[0085] [Item 48] In any one of the above items, the drug is used for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0086] [Item 49] In any one of the above items, the drug is intended for use in preventing or treating cancer so as not to cause cancer to recur.

[0087] [Item 50] In any one of the above items, the drug is intended to prevent or treat a disease in a subject, and the drug is:

[0088] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0089] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0090] (C) Use characterized by the step of administering to a subject a T cell having enhanced glucose uptake ability or a cell population including T cells according to an appropriate administration method and dosage.

[0091] [Item 50A] In any one of the above items, the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0092] [Item 50B] In any one of the above items, the T cell is used for a chimeric antigen receptor (CAR).

[0093] [Item 50C] In any one of the above items, the use includes cancer, autoimmune disease, allergic or infectious disease.

[0094] [Item 50D] In any one of the above items, the disease is a use including cancer.

[0095] [Item 51] In any of the above items, T cells having enhanced glucose uptake capacity are not modified to express glucose transporters in response to need (when necessary), and T cells in which the expression of glucose transporters is not enhanced in response to need (when necessary) eff Use comprising cells modified to express a glucose transporter as needed (if needed) or cells in which the expression of a glucose transporter is enhanced as needed (if needed).

[0096] [Item 52] In any one of the above items, the glucose transporter is GLUT3.

[0097] The present disclosure also provides the following.

[0098] [Item A1] T cells having enhanced glucose uptake capacity in which the expression of glucose transporters in T cells is regulated in response to changes in tumor microenvironment conditions.

[0099] [Item A1A] T cells having enhanced glucose uptake capacity modified so that the expression of glucose transporters in T cells is regulated in response to changes in tumor microenvironment conditions.

[0100] [Item A2] In any one of the above items, the tumor microenvironment conditions include T cells, including the degree of TCR-related signaling.

[0101] [Item A3] In any one of the above Item A, the TCR-related signal is a T cell mediated by a molecule selected from the group consisting of a TCR signal or a part thereof, and a chimeric signal of an antigen different from the TCR signal (also called a CAR signal).

[0102] [Item A4] In any one of the above items, the TCR-related signal is a T cell mediated by a molecule containing a TCR signaling domain.

[0103] [Item A5] In any one of the above Item A, changes in tumor microenvironment conditions include the presentation of tumor antigens and / or costimulatory ligands and / or changes in cytokine conditions of T cells.

[0104] [Item A6] In any one of the above items, the degree of transmission of TCR-related signals is a T cell including the degree of TCR stimulation.

[0105] [Item A7] In any one of the above items, the degree of transmission of TCR-related signals comprises at least one selected from the group consisting of the degree of TCR stimulation, TCR co-stimulation, and humoral factor.

[0106] [Item A8] In any one of the above items, the degree of transmission of TCR-related signals comprises at least one selected from the group consisting of the degree of TCR stimulation and TCR co-stimulation and humoral factors.

[0107] [Item A9] In any one of the above items, the degree of transmission of TCR-related signals is T cells including the degree of TCR stimulation, TCR co-stimulation, and humoral factors.

[0108] [Item A10] In any one of the above Item A, the TCR stimulation is a T cell selected from the group consisting of the NFAT, NF-κB, and STAT transcription factor families that translocate into the nucleus in connection with the activation of the T cell.

[0109] [Item A11] In any one of the above items, the tumor microenvironment conditions include T cells with an oxygen concentration level.

[0110] [Item A12] A T cell comprising a nucleic acid sequence encoding a glucose transporter and operably linked to a tumor microenvironment-sensing element that detects changes in tumor microenvironment conditions.

[0111] [Item A13] In any one of the above claims, the tumor microenvironment sensing element is:

[0112] (i) an inducible activation motif that is activated in response to changes in tumor microenvironment conditions; and

[0113] (ii) T cells containing a nucleic acid sequence encoding a degradation factor for a glucose transporter that is inactivated in response to changes in tumor microenvironment conditions.

[0114] [Item A14] In any one of the above claims, the tumor microenvironment sensing element is:

[0115] (i) an inducible activation motif to which a nuclear translocation factor, which translocates into the nucleus in response to T cell activation, binds;

[0116] (ii) a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to a decrease in oxygen concentration binds; and

[0117] (iii) A T cell containing a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading glucose transporters is inactivated in response to a decrease in oxygen concentration.

[0118] [Item A15] In any one of the above items, a T cell comprising a nucleic acid sequence encoding a glucose transporter that comprises an inducible activation motif that is activated in response to a change in tumor microenvironment conditions and / or a nucleic acid sequence encoding a glucose transporter degradation factor that is inactivated in response to a change in tumor microenvironment conditions, and a construct operably linked to the nucleic acid sequence encoding the glucose transporter.

[0119] [Item A16] In any one of the above items, the nucleic acid sequence encoding a glucose transporter comprises a T cell comprising a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to activation of the T cell binds.

[0120] [Item A17] In any one of the above items, the nuclear translocation factor is a T cell selected from the group consisting of NFAT, NF-κB and STAT transcription factor families.

[0121] [Item A18] In any one of the above items, the nuclear translocation factor-binding inducible activation motif is a T cell selected from the group consisting of an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ No. 1), a STAT-binding sequence (TTCNNNGAA), and modified sequences thereof.

[0122] [Item A19] In any one of the above items, the combination of a nuclear transpotential factor and a nuclear transpotential factor-binding inducible activation motif is:

[0123] (1) A combination of NFAT and NFAT-combined domains;

[0124] (2) a combination of NF-κB and κB motif (GGGACTTTCC) (SEQ ID NO. 1); or

[0125] (3) T cells containing a combination of the STAT transcription factor family and the STAT-binding sequence (TTCNNNGAA).

[0126] [Item A20] In any one of the above items, a T cell comprising a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to a decrease in oxygen concentration binds, wherein the nucleic acid sequence encoding the glucose transporter comprises a nuclear translocation factor-binding inducible activation motif.

[0127] [Item A21] In any one of the above items, the nuclear translocation factor is a T cell selected from the group consisting of HIF-1.

[0128] [Item A22] In any one of the above items, the nuclear translocation factor-binding inducible activation motif is a T cell selected from the group consisting of HIF-RE.

[0129] [Item A23] In any one of the above items, the combination of a nuclear transpotential factor and a nuclear transpotential factor-binding inducible activation motif is:

[0130] (1) T cells selected from a group consisting of a combination of HIF-1 and HIF-RE.

[0131] [Item A24] In any one of the above items, a T cell comprising a nucleic acid sequence encoding a glucose transporter and a structure operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter is inactivated in response to a decrease in oxygen concentration.

[0132] [Item A25] In any one of the above items, the factor to be inactivated is a T cell selected from the group consisting of oxygen-dependent degradation factors (ODD).

[0133] [Item A26] In any one of the above items, the T cell is a T cell containing a chimeric antigen receptor (CAR).

[0134] [Item A27] In any one of the above items, the T cell is a T cell having effector function.

[0135] [Item A28] In any one of the above items, the T cell is an effector T cell (T eff T cells, which are the precursor cells of ).

[0136] [Item A29] In any one of the above items, a T cell in which the memory function of the precursor cell of the effector T cell is extended.

[0137] [Item A30] In any one of the above items, a T cell in which the memory T cell phenotype of the precursor cell of the effector T cell is maintained.

[0138] [Item A31] In any one of the above items, a T cell in which the effector T cell is activated or the effector T cell activity is maintained, and the memory function of the effector T cell precursor cell is extended or the memory T cell phenotype of the effector T cell precursor cell is maintained.

[0139] [Item A32] In any one of the above items, expression and / or enhancement is achieved specifically in a tumor environment in T cells.

[0140] [Item A33] In any one of the above items, CAR is a T cell expressed in a T cell.

[0141] [Item A34] In any one of the above items, the glucose transporter is a T cell comprising GLUT1, GLUT2, GLUT3 and GLUT4.

[0142] [Item A35] In any one of the above items, the glucose transporter is a T cell that is GLUT3.

[0143] [Item A36] In any one of the above items, the T cell is a human T cell.

[0144] [Item A37] A population of cells including T cells having enhanced glucose uptake capacity in response to need (when necessary).

[0145] [Item A38] A cell population containing T cells according to any one of the above items.

[0146] [Item A39] In any one of the above items, T cells are a group of cells comprising cells having the characteristic of exhibiting effector function when introduced into the body.

[0147] [Item A40] In any one of the above items, the T cells are a cell population containing a chimeric antigen receptor (CAR).

[0148] [Item A41] In any one of the above items, the T cells are modified so that the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions, and / or comprise a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0149] [Item A42] In any one of the above items, T cells are a group of cells having effector functions.

[0150] [Item A43] In any one of the above items, the cell population is T eff A cell population containing

[0151] [Item A44] In any one of the above items, the cell population is T eff A cell population containing precursor cells.

[0152] [Item A45] In any one of the above items, CAR is a population of cells expressed in T cells.

[0153] [Item A46] In any one of the above items, the glucose transporter is a cell population comprising GLUT1, GLUT2, GLUT3 and GLUT4.

[0154] [Item A47] In any one of the above items, a cell population in which the glucose transporter is GLUT3.

[0155] [Item A48] In any one of the above items, T cells are a group of cells that are human T cells.

[0156] [Item A49] A pharmaceutical composition comprising a T cell according to any one of the above items or a cell population according to any one of the above items.

[0157] [Item A50] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0158] [Item A51] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the prevention or treatment of cancer.

[0159] [Item A52] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended to cure cancer.

[0160] [Item A53] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0161] [Item A54] In any one of the above items, the pharmaceutical composition is a pharmaceutical composition intended to prevent or treat cancer so as not to recur.

[0162] [Item A55] To prevent or treat disease in subjects

[0163] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0164] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0165] (C) A pharmaceutical composition characterized by the step of administering to a subject a T cell or a cell population comprising T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0166] [Item A56] In any one of the above items, the T cell is a pharmaceutical composition comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0167] [Item A57] In any one of the above items, the T cell is a pharmaceutical composition comprising a chimeric antigen receptor (CAR).

[0168] [Item A58] A pharmaceutical composition comprising, in any one of the above items, a disease including cancer, an autoimmune disease, an allergy or an infectious disease.

[0169] [Item A59] In any one of the above items, the disease is a pharmaceutical composition comprising cancer.

[0170] [Item A60] In any one of the above items, T cells having enhanced glucose uptake capacity are not modified to regulate glucose transporter expression in response to changes in tumor microenvironment conditions and are not configured to regulate glucose transporter expression in response to changes in tumor microenvironment conditions. eff A pharmaceutical composition comprising a cell that is modified to regulate the expression of a glucose transporter in response to changes in tumor microenvironment conditions and / or configured to regulate the expression of a glucose transporter in response to changes in tumor microenvironment conditions.

[0171] [Item A60A] In any one of the above items, T cells having enhanced glucose uptake capacity are not modified to include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and T cells not including a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions eff A pharmaceutical composition comprising a cell modified to include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and / or a cell comprising a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0172] [Item A61] A composition in which, in any one of the above items, the glucose transporter is GLUT3.

[0173] [Item A62] In any one of the above items, the T cell or cell population is a T cell or cell population intended for use as a drug.

[0174] [Item A63] In any one of the above items, the cell or cell population is a cell or cell population intended for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0175] [Item A64] In any one of the above items, the cell or cell population is a cell or cell population intended for the prevention or treatment of cancer.

[0176] [Item A65] In any one of the above items, the cell or cell population is a cell or cell population intended to cure cancer.

[0177] [Item A66] In any one of the above items, the cell or cell population is a cell or cell population intended for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0178] [Item A67] In any one of the above items, the cell or cell population is a cell or cell population intended to prevent or treat cancer so as not to recur.

[0179] [Item A68] To prevent or treat disease in subjects

[0180] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0181] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0182] (C) A cell or cell population characterized by the step of administering to a subject a T cell or a cell population comprising T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0183] [Item A69] In any one of the above items, the T cell is a cell or population of cells containing a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0184] [Item A70] In any one of the above items, the T cell is a cell or population of cells containing a chimeric antigen receptor (CAR).

[0185] [Item A71] In any one of the above items, the disease is a cell or cell population including cancer, an autoimmune disease, an allergic or infectious disease.

[0186] [Item A72] In any one of the above items, the disease is a cell or group of cells including cancer.

[0187] [Item A73] In any one of the above items, T cells having enhanced glucose uptake capacity are not modified to regulate glucose transporter expression in response to changes in tumor microenvironment conditions and are not configured to regulate glucose transporter expression in response to changes in tumor microenvironment conditions. eff A cell or cell population comprising a cell that is modified to regulate glucose transporter expression in response to changes in tumor microenvironment conditions and / or configured to regulate glucose transporter expression in response to changes in tumor microenvironment conditions.

[0188] [Item A73A] In any one of the above items, T cells having enhanced glucose uptake capacity do not include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and T cells not including a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions effA cell or a population of cells comprising a cell modified to include a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and / or a cell comprising a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0189] [Item A74] In any one of the above items, the glucose transporter is a cell or cell population in which GLUT3 is the glucose transporter.

[0190] [Item A75] A method for treating or preventing in a subject, comprising the step of administering an effective amount of T cells according to any one of the above items or a cell population according to any one of the above items to a subject in need thereof.

[0191] [Item A76] A method in which, in any one of the above items, the treatment or prevention of the subject is for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease.

[0192] [Item A77] In any one of the above items, the treatment or prevention of the subject includes the prevention or treatment of cancer.

[0193] [Item A78] In any one of the above items, the treatment or prevention of the subject includes a method of curing cancer.

[0194] [Item A79] In any one of the above items, the treatment or prevention of the subject includes the prevention of cancer metastasis or the prevention of cancer recurrence.

[0195] [Item A80] In any one of the above items, the treatment or prevention of the subject includes preventing or treating cancer so that the cancer does not recur.

[0196] [Item A81] To prevent or treat disease in subjects

[0197] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0198] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0199] (C) A method comprising the step of administering to a subject a T cell or a cell population containing T cells having enhanced glucose uptake ability according to an appropriate administration method and dosage.

[0200] [Item A82] A method in which, in any one of the above items, the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0201] [Item A83] A method in which, in any one of the above items, the T cell comprises a chimeric antigen receptor (CAR).

[0202] [Item A84] In any one of the above items, the disease includes cancer, autoimmune disease, allergic or infectious disease.

[0203] [Item A85] In any one of the above items, the disease is a method including cancer.

[0204] [Item A86] In any one of the above items, T cells having enhanced glucose uptake capacity are not modified to regulate glucose transporter expression in response to changes in tumor microenvironment conditions and are not configured to regulate glucose transporter expression in response to changes in tumor microenvironment conditions. eff A method comprising a cell that is modified to regulate the expression of a glucose transporter in response to changes in tumor microenvironment conditions and / or configured to regulate the expression of a glucose transporter in response to changes in tumor microenvironment conditions.

[0205] [Item A86A] In any one of the above items, T cells having enhanced glucose uptake capacity do not include a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and T cells not including a nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions eff A method comprising a cell modified to include a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and / or a cell comprising a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0206] [Item A87] A method in which, in any one of the above items, the glucose transporter is GLUT3.

[0207] [Item A88] Use for producing a drug comprising a T cell or a cell population according to any of the above items, or a cell or cell population according to any of the above items.

[0208] [Item A89] In any one of the above items, the drug is used for the treatment or prevention of cancer, autoimmune diseases, allergies, or infectious diseases.

[0209] [Item A90] In any one of the above items, the drug is intended for the prevention or treatment of cancer.

[0210] [Item A91] In any one of the above items, the drug is intended for use in curing cancer.

[0211] [Item A92] In any one of the above items, the drug is intended for the prevention of cancer metastasis or the prevention of cancer recurrence.

[0212] [Item A93] In any one of the above items, the drug is intended for use in preventing or treating cancer so as not to cause cancer to recur.

[0213] [Item A94] In any one of the above items, the drug is intended to prevent or treat a disease in the subject, and the drug is:

[0214] (A) A step of collecting the value of glucose absorption capacity or the value of an indicator related thereto from a subject;

[0215] (B) a step of selecting a T cell or a cell population including T cells having enhanced glucose uptake ability and appropriate expression characteristics according to an indicator; and

[0216] (C) Use characterized by the step of administering to a subject a T cell having enhanced glucose uptake ability or a cell population including T cells according to an appropriate administration method and dosage.

[0217] [Item A95] In any one of the above items, the T cell is used for a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0218] [Item A96] In any one of the above items, the T cell is used for a chimeric antigen receptor (CAR).

[0219] [Item A97] In any one of the above items, the use includes cancer, autoimmune disease, allergic or infectious disease.

[0220] [Item A98] In any one of the above items, the disease is a use including cancer.

[0221] [Item A99] In any one of the above items, T cells having enhanced glucose uptake capacity are not modified to regulate the expression of glucose transporters in response to changes in tumor microenvironment conditions and are not configured to regulate the expression of glucose transporters in response to changes in tumor microenvironment conditions. eff Use comprising a cell modified to regulate glucose transporter expression in response to changes in tumor microenvironment conditions and / or configured to regulate glucose transporter expression in response to changes in tumor microenvironment conditions.

[0222] [Item A99A] In any one of the above items, T cells having enhanced glucose uptake capacity do not include a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and T cells not including a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions eff Use comprising a cell modified to include a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, and / or a cell comprising a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0223] [Item A100] In any one of the above items, the glucose transporter is GLUT3.

[0224] In addition to the combinations specified in this disclosure, it is intended that one or more of the above features may be provided in additional combinations. It should be noted that, where necessary, other embodiments and advantages of this disclosure will be recognized by those skilled in the art by reading and understanding the following detailed description.

[0225] It should be noted that features and notable functions and effects of the present disclosure other than those described above will be clearly apparent to those skilled in the art by referring to the embodiments and drawings of the invention below.

[0226] [Effect of the Invention]

[0227] According to the present disclosure, an effector T cell having enhanced glucose uptake ability (T eff It is possible to provide ) and by using these cells, it is possible to provide chimeric antigen receptor (CAR) T cells that function without starvation or exhaustion even in a tumor environment.

[0228] The cells of the present disclosure may be referred to as metabolism-enhanced T cells that apply the mechanism by which tumor cells survive in a tumor environment to T cells. According to the cells of the present disclosure, effective cytotoxic activity and cytokine production capabilities in low-glucose environments, such as the tumor microenvironment, which could not be achieved with conventional immune checkpoint inhibitor therapy, can be maintained, and high anti-tumor effects can be exerted in vivo. Furthermore, by using an on-demand glucose transporter expression system in which glucose transporters are expressed in response to increased glucose demand, T cell overdifferentiation and exhaustion due to excessive activation can be avoided, effects can be maintained for a long period, and memory phenotype T cells exhibiting significant effects in preventing recurrence can be induced. This on-demand type is achieved by the characteristic that glucose transporter expression in T cells is regulated in response to changes in tumor microenvironment conditions, or by including a nucleic acid sequence encoding a glucose transporter that is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. Brief explanation of the drawing

[0229] [Fig. 1] Fig. 1 illustrates the exemplary structure of a metabolically modified anti-EGFRvIII CAR (3C10 CAR_GLUT3) lentiviral vector plasmid. A single-stranded antibody (scFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) and the like was constructed, and the CAR was constructed by linking the single-stranded antibody to the CD8 hinge, CD28 transmembrane region (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BBICD, and CD3z. A high-affinity glucose transporter (GLUT3) was encoded downstream of the CAR via the P2A sequence. The figure illustrates an example of introducing 3C10 CARGLUT3 into a third-generation lentiviral vector plasmid having an EF-1a promoter. [Fig. 2] Fig. 2 illustrates T cell surface GLUT3 expression and enhanced glucose uptake. The top panel shows the structures of a conventional CAR (3C10 CAR) and a metabolically modified CAR (GLUT3 CAR). The bottom left shows fluorescence microscopy images of CAR and GLUT3 expression in T cells for 3C10 CAR and GLUT3 CAR. The bottom center shows GLUT3 expression quantified by fluorescence intensity. The bottom right shows the results of quantifying intracellular glucose uptake by the 2-NBDG method. CAR-T cells were obtained by introducing a conventional CAR (3C10 CAR) and a metabolically modified CAR (GLUT3 CAR) into healthy donor-derived T cells via gene transfer. Uniform GLUT3 expression was observed on the membrane of GLUT3 CAR-T cells through fluorescence microscopy. In addition, glucose uptake was enhanced in GLUT3 CAR-T cells compared to T cells and conventional CAR-T cells (3C10 CAR-T cells). [Fig. 3] Fig. 3 illustrates results showing that the T cell proliferation efficiency is equivalent to that of conventional CAR-T cells. The X-axis represents the number of days after introduction, and the Y-axis represents LOG2(FC). Peripheral blood mononuclear cells (PBMCs) containing T cells were stimulated with anti-CD3 / 28 beads, and then CAR was genetically introduced to analyze the T cell proliferation efficiency. 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). [Fig. 4] Fig. 4 illustrates the results showing the enhancement of T cell glycolysis by GLUT3 expression. The graph on the left shows the glycolysis of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu concentration 10 mM) and low glucose conditions (Glu concentration 0.5 mM). The graph below shows the glycolysis of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu concentration 10 mM). The left side of the right panel shows the glycolysis values ​​observed after glucose addition, and the right side shows the glycolysis values ​​observed after oligomycin addition. The metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a flux analyzer. In both low-glucose and normal glucose environments, GLUT3 CAR exhibited enhanced glycolysis compared to 3C10 CAR. [Fig. 5] Fig. 5 illustrates results indicating that GLUT3 CAR-T cells have a competitive advantage under low-glucose conditions. The left panel illustrates the experimental procedure. The center panel illustrates the cell number ratio of 3C10 CAR-T cells and GLUT3 CAR-T cells on days 0 and 3. The right panel illustrates 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. Under normal glucose conditions, similar survival was observed for both cells, whereas under low-glucose conditions, significant survival was observed for GLUT3 CAR-T cells. [Fig. 6] Fig. 6 illustrates the results showing the enhanced cytokine-producing ability of GLUT3 CAR-T cells. The left panel shows the intracellular IFNg and IL-2 analysis for CD8-positive cells in the upper section and the intracellular IFNg and IL-2 amounts for CD4-positive cells in the lower section. The right panel shows the ratio of positive cells to IFNγ, IL-2, and TNF-α. After antigen stimulation of 3C10 CAR-T cells and GLUT3 CAR-T cells, the ratio of cytokine-producing cells was analyzed by FCM. It was observed that the expression of cytokines (IFNγ, IL-2, TNF-α), which are important for the maintenance and activation of T cells and the exertion of anti-tumor activity, was significantly increased. [Fig. 7] Fig. 7 illustrates the results showing the function of GLUT3 CAR-T cells maintained even under low-glucose conditions. The panels from left to right show the ratios of IFNγ-, IL-2-, and TNF-α-producing cells under normal glucose conditions (10 mM) and low glucose conditions (0.5 mM), respectively. The cytokine production ability under normal glucose and low glucose conditions was compared. In conventional CAR-T cells (3C10 CAR-T cells), severe functional decline was observed under low glucose conditions. On the other hand, in GLUT3 CAR-T cells, high cytokine production ability was observed under normal glucose conditions, and even under low glucose conditions, GLUT3 CAR-T cells exhibited a function comparable to the cytokine production ability of 3C10 CAR-T cells under normal glucose conditions. [Fig. 8] Fig. 8 illustrates results showing the enhanced cytotoxic activity of GLUT3 CAR-T cells. The left panel shows the cytotoxic activity results 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 cytotoxic activity results 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 EGFRvIII-expressing cell lines as targets. Cytotoxic activity was analyzed under low-glucose and normal-glucose conditions, and GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells in both environments. [Fig. 9] Fig. 9 illustrates the results showing the characteristics conferred upon the T cell memory phenotype by GLUT3 CAR. The left panel shows CD45 and CD27 expression before and after co-culture with U87d tumor cell lines, and the right panel shows the ratio of CD45RA-positive / CD27-negative fractions before and after co-culture, starting from the left. The memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. In GLUT3 CAR-T cells, a stronger tendency to differentiate into effector T cells was observed compared to 3C10 CAR. [Fig. 10] Fig. 10 illustrates the inhibition of inhibitory molecule expression in GLUT3 CAR-T cells. Data for PD-1, LAG3, and TIM3 are shown from left to right. The expression of inhibitory molecules under stimulation by EGFRvIII antigen-positive cells was compared. In GLUT3 CAR-T cells, inhibition of PD-1, LAG3, and TIM3 expression was observed. [Fig. 11] Fig. 11 illustrates the mRNA analysis of GLUT3 CAR-T cells (evaluation of metabolism, exhaustion, activation, and differentiation). The upper left panel shows PDCD1 expression, LAG3 expression, and TIM3 expression from left to right, and the upper right panel shows the enrichment plots related to lactate metabolism and glucose metabolism from left to right. The lower panel shows the factors with increased expression based on gene ontology. Details of 3C10 CAR-T cells and GLUT3 CAR-T cells were analyzed based on mRNA expression. In GLUT3 CAR-T cells, as in FCM, a decrease in exhaustion-related molecules and an increase in lactate metabolism- and glycolysis-related factors were observed. These factors are associated with enhanced T cell effector functions, such as activation and cytokine production. [Fig. 12] Fig. 12 illustrates results demonstrating universality, that is, that similar effects are obtained not only with 3C10 CAR but also with CD19 CAR. The left panel illustrates experiments using 3C10 CAR, and the right panel illustrates experiments using CD19 CAR. In each panel, the upper left shows intracellular IL-2, IFNg, and TNF-α analysis of mock-introduced T cells, 3C10 CAR-T cells, and GLUT3 CAR-T cells; the upper right shows, from left, the proportion of IFNg-, IL-2-, and TNF-α-positive cells among 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (10 mM); and the lower left shows, from left, the proportion of IFNg-, IL-2-, and TNF-α-positive cells among 3C10 CAR-T cells and GLUT3 CAR-T cells under low glucose conditions (0.5 mM). The lower right shows, from left to right, the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against U87d cell line and U251MGd cell line under low-glucose conditions (0.5 mM). It was confirmed that a similar effect to that of 3C10 CAR was obtained when GLUT3 was introduced into CD19 CAR. In addition, it was found to be effective against pancreatic cancer cells obtained by expressing CD19 in pancreatic cancer cell lines. [Fig. 13] Fig. 13 illustrates that rapid tumor removal is achieved in an intracranial xenograft model. The upper left panel shows a simplified schematic diagram of the experiment. Briefly, 2.5 x 10⁶ U87d tumor cell lines were placed inside the cranium of an immunodeficient NSG mouse. 4 Transplant cells from dogs, and after 5 days, 1 x 10 CAR-T cells 6This is a model administered via the tail vein to dogs and quantifies tumor burden over time using bioluminescence imaging (BLI). The center left displays tumor images on days 0 and 11. The graph in the lower left shows luminescence intensity (reflecting tumor burden) over time. The central panel displays the Kaplan-Meier curves for each treatment group. Below the central panel, tumor images on days 34, 38, and 42 are shown in tumor challenge mice. The column on the right shows peripheral blood CD3 over time. + Indicates the number of T cells. Antitumor activity was observed in the U87Δ intracranial xenograft model. In the GLUT3 CAR-T cell therapy group, faster tumor removal, improved survival, and superior T cell proliferation were observed compared to the 3C10 CAR-T cell therapy group. Additionally, tumor rejection due to tumor re-challenge was confirmed in mice cured in the GLUT3 CAR-T cell group. [Fig. 14] Fig. 14 illustrates an overview of a CAR-T cell having GLUT3 expression when necessary. The figure schematically illustrates a system in which only CAR is expressed and GLUT3 is not expressed under normal conditions, and GLUT3 is expressed when the demand for glucose increases, such as during activation of the CAR-T cell or in a tumor environment. [Fig. 15] Fig. 15 illustrates the design of an inducible GLUT3 expression system. P represents the constitutive expression type, A represents NFATi-GLUT3, B represents HIF1i-GLUT3, and B' represents a hypoxia-sensing alternative version of HIF1i-GLUT3. The symbols in the schematic represent the EF-1 promoter (EF-1a), anti-EGFRvIII CAR (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), respectively, and an asterisk (*) represents a stop codon. A CAR construct was designed to express GLUT3 specifically in the tumor environment or upon activation. GLUT3 expression is possible in response to increased NFAT upon activation (A.) and tumor hypoxia (B. and B'). A CAR construct was designed to express GLUT3 specifically in the tumor environment or upon activation. GLUT3 expression is possible in response to increased NFAT upon activation (A.) and tumor hypoxia (B.). In A., GLUT3 is encoded via a minimal promoter (MP) under 8 repeat NFAT-RE, which binds to and induces transcription of NFAT that translocates into the nucleus upon T cell activation, independently of a CAR (e.g., EGFRvIII CAR) driven by and constitutively expressed by an EF-1a promoter. In B., GLUT3 is encoded via a minimal promoter under 8 repeat HIF-RE, which binds to and induces transcription of HIF-1a that translocates into the nucleus under hypoxic conditions of the T cell, also independently of the CAR. In B', the GLUT3-ODD element, in which GLUT3 is degraded in the presence of oxygen, is encoded via the P2A sequence immediately downstream of the CAR driven by the EF-1a promoter. [Fig. 16] Fig. 16 illustrates the structure of the above NFATi-GLUT3 CAR introduced 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 in which 3C10 CAR, constitutively driven by the EF-1a promoter, and GLUT3, driven in an NFAT nuclear potential-dependent manner by the minimal promoter and 8 repeat NFAT binding sites, are introduced into a single plasmid. [Fig. 17] Fig. 17 illustrates the operation and basic functions of the inducible GLUT3 CAR system. The upper left shows the structure of the NFAT-inducible GLUT3 CAR. The left center panel shows fluorescence microscopy images of CAR and GLUT3 expression in T cells under pre-stimulation and post-stimulation. The left lower panel shows IFN-γ, TNFα, and IL-2 after stimulation by the U87d cell line, starting from the right. The right center panel shows the expression intensity of intracellular IFN-γ, IL-2, and TNFα in each CAR-T cell under stimulation by the U87d cell line by flow cytometry. The right lower panel is a graph showing that the cytotoxic activity under glucose conditions (0.5 mM) is stronger than that of 3C10 CAR-T cells and equivalent to that of GLUT3 CAR-T cells. We constructed a system expressing GLUT3 (NFATi GLUT3 CAR-T) in relation to NFAT nuclear translocation and verified its operation. The cytotoxic activity of NFATiGLUT3 CAR-T cells (NFATi GLUT3 CAR-T) was equivalent to that of GLUT3 CAR-T cells with constitutive expression (GLUT3 CAR-T), and cytokine production was at an intermediate level between 3C10 CAR-T (without GLUT3) and GLUT3 CAR-T cells with constitutive expression (GLUT3 CAR-T). [Fig. 18] Fig. 18 illustrates 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 Fig. 15. The upper left shows the experimental schematic. The upper right shows the Kaplan-Meier survival curve. The lower panel shows the change in tumor burden over time as quantified by bioluminescence imaging (BLI). CR was obtained in four mice treated with GLUT3 CAR-T and two mice treated with NFATi GLUT3 CAR-T. [Fig. 19] Fig. 19 illustrates tumor challenge and tumor rechallenge experiments (sudden mouse death in the GLUT3 CAR-T cell population). The left panel shows the tumor burden measured by bioluminescent imaging in this order, representing survival at Day 1 after challenge, Day 5 after rechallenge, and Day 12 after rechallenge in mice treated with 3C10 CAR-T, GLUT3 CAR-T, and NFAT CAR-T. Areas without BLI images indicate mouse death. Tumors (U87d) were re-implanted into mice cured in the experiment of the previous figure, and rejection ability was evaluated (“surrogate indicator of memory formation”). 1 x 10 5 Tumor cells were re-implanted, and while tumor rejection was not achieved in 3C10 CAR-T, tumors were rejected in GLUT3 CAR-T and NFATiCAR-T. However, in the GLUT3 CAR-T group, 3 out of 4 mice died (deaths unrelated to tumor growth). [Fig. 20] Fig. 20 illustrates the inhibition of apoptosis after stimulation by NFATiGLUT3. The left panel shows the staining for 7-AAD and Annexin V as apoptosis and apoptosis markers in Mock T cells, 3C10 CAR-T cells, GLUT3 CAR-T cells, and NFATi CAR-T cells in the unstimulated state (top row of the left panel), 7 days after the first stimulation by the U87d cell line (middle row of the same panel), and 7 days after the second stimulation by the U87d cell line (bottom row of the same panel). The graph in the right panel shows the ratios of late-apoptotic cells, pre-apoptotic cells, and viable cells in each CAR-T cell from the top, 7 days after the first stimulation and 7 days after the second stimulation. CAR-T cells were repeatedly stimulated with U87d, and apoptosis of CAR-T cells was observed using Annexin V and 7-ADD. GLUT3 has an inhibitory effect on apoptosis (considered to be the sum of activation-induced apoptosis and apoptosis resulting from terminal differentiation). It is suggested that NFAT CAR-T has a higher inhibitory effect on apoptosis than GLUT3 CAR-T. [Fig. 21] Fig. 21 illustrates the enhancement of long-term survival after stimulation. Each CAR-T cell is observed over time under the above conditions. This figure illustrates the in vitro (CAR) T cell stimulation and culture test plan. Non-transgenic T cells (UTD), 3C10 CAR-T cells, GLUT3 CAR-T cells, and NFATi CAR-T cells are used as subjects for evaluation, and unstimulated and stimulated groups (stimulated by U87d cell line or stimulated by EGFRvIII magnetic beads) are provided. Additionally, each group is cultured under glucose-free conditions (0 mM), low-glucose conditions (0.5 mM), and normal glucose conditions (10 mM) (a total of 36 groups). For each group, the analysis shown in the schedule on the right is performed over time. Under low glucose conditions, conventional CAR-T (3C10) does not exhibit effective cytokine production, whereas GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibit effective cytokine production. However, due to excessive glucose uptake, GLUT3 CAR-T differentiates into CCR7-negative Tem or Temra over time (day 3 or day 7), undergoes terminal differentiation, loses stem cell characteristics, and undergoes apoptosis. On the other hand, in NFATi GLUT3 CAR-T cells, which are required cells, apoptosis is inhibited, and CCR7-positive Tcm and Tscm (memory cells, stem cells) are maintained. When CAR-T cells (established under normal glucose conditions) are suddenly (Day 0) placed under low-glucose conditions of 0 mM or 0.5 mM, glucose-dependent GLUT3 CAR-T cells are prone to apoptosis (⇔ NFATi GLUT-3 CAR-T cells exhibit resistance to low glucose). [Fig. 22] Fig. 22 illustrates the enhancement of long-term survival after stimulation. The antitumor effects, survival, and adverse events are investigated in a system similar to the mouse system described above. This figure illustrates the enhancement of long-term survival in vivo. The antitumor effects, survival, and adverse events are investigated in a system similar to the mouse system described above. U87d tumor cell lines were implanted into the cranial cavity of immunodeficient NSG mice, and after 5 days, Mock-introduced T cells (UTD), 3C10 CAR-T cells, GLUT3 CAR-T cells, or NFATi CAR-T cells were administered via the tail vein, and the antitumor effects and survival were analyzed by bioluminescent imaging. In Experiment 1, GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibited equivalent antitumor activity. In Experiment 2, GLUT3 CAR-T hyperactivation induced by glucose exposure leads to apoptosis and hyperdifferentiation, resulting in the loss of antitumor activity. In contrast, excessive activation is suppressed in NFATiCAR-T cells, thereby exhibiting an effective antitumor effect. Even upon retrying, NFATiCAR-T cells reject the tumor. In Experiment 4, superior CAR-T cell engraftment and intratumoral T-cell infiltration are observed in NFATiGLUT3 cells in proportion to the tumor effect. Experiment 3 is an exploratory experiment in a liver tumor model suggested to exhibit immunosuppression due to a low-glucose, high-lactic acid environment, and was conducted with the expectation that results similar to those of Experiment 2 would be obtained. [Fig. 23] Fig. 23 illustrates data (in vitro data) indicating the enhancement of long-term survival after stimulation. The three panels on the left show the results for Day 1, the three panels in the center show the results for Day 3, and the three panels on the right show the results for Day 7. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from the left, and the results for Mock, 3C10 CAR-T, and GLUT3 CAR-T are shown from the top. At each time point, staining data for 7-AAD and Annexin V, reflecting cell death and apoptosis, are shown. Cell death in each CAR-T cell was investigated using Annexin V and 7-AAD. Under unstimulated conditions, differences were observed according to glucose concentration, but no substantial differences were observed between the individual CAR-T cells. [Fig. 24] Fig. 24 illustrates in vitro data showing the enhancement of long-term survival after stimulation. The three panels on the left show the results for Day 1, the three panels in the center show the results for Day 3, and the three panels on the right show the results for Day 7. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from the left, and the results for Mock, 3C10 CAR-T, and GLUT3 CAR-T are shown from the top. At each time point, staining data for 7-AAD and Annexin V, reflecting apoptosis and cell death, are shown. The survival and apoptosis of each CAR-T cell after stimulation were evaluated in the same manner as in the previous figure. Compared to 3C10 CAR-T cells and GLUT3 CAR-T cells, survival was enhanced (apoptosis was inhibited) in NFATi GLUT3 CAR-T cells. [Fig. 25] Fig. 25 illustrates in vitro data showing the enhancement of long-term survival after stimulation. The three panels on the left show the results for Day 1, the three panels in the center show the results for Day 3, and the three panels on the right show the results for Day 7. In each panel, glucose concentrations of 0 mM, 0.5 mM, and 10 mM are shown from the left, and the results for Mock, 3C10 CAR-T, and GLUT3 CAR-T are shown from the top. The proportion of CAR-expressing cells is shown at each time point. The proportion of CAR-positive cells for each CAR-T cell population was analyzed after stimulation. After stimulation, the proportion of CAR-positive cells decreased, particularly in GLUT3 CAR-T cells with constitutive expression, and this fraction was considered to have undergone apoptosis. [Fig. 26] Fig. 26 illustrates the enhancement of long-term survival after stimulation. The number of T cells on day 7 is shown. As a model reflecting the kinetics following administration into the patient body (the dynamics of cells infiltrating from blood with sufficient glucose concentration into a tumor environment that is low-glucose to glucose-free after administration), cells were established and cultured under normal glucose conditions (10 mM glucose). After stimulation with EGFRvIII-binding beads, the cells were cultured under glucose-free (0 mM), low-glucose (0.5 mM), or normal glucose (10 mM) conditions, and the number of surviving cells after 7 days was evaluated. The graph shows the relative number of cells with the number of mock T cells set to 1. In GLUT3 CAR-T, almost all cells died under 0 mM. In contrast, many cells were maintained in NFATi CAR-T. It has been suggested that apoptosis may have resulted from hyperactivation induced by constitutive GLUT3 expression and glucose-dependent metabolism. [Fig. 27] Fig. 27 illustrates the expression of the inhibitory molecule (PD-1). The three panels on the left show the results of PD-1 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 panels on the right show the results of PD-1 expression on day 3 of culture. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from left to right, and the results for Mock, 3C10 CAR-T, and GLUT3 CAR-T are shown from top to bottom. In NFATi GLUT3 CAR-T cells, the expression of the inhibitory molecule (PD-1) was lower than in GLUT3 CAR-T cells. [Fig. 28] Fig. 28 illustrates the suppressed expression of the repressor molecule (TIM3). The three panels on the left show the TIM3 expression results on day 1 of culture under glucose-free (0 mM), low-glucose (0.5 mM), or normal glucose (10 mM) conditions, and the three panels on the right show the TIM3 expression results on day 3 of culture. In each panel, 0 mM, 0.5 mM, and 10 mM are shown from left to right, and the results for Mock, 3C10 CAR-T, and GLUT3 CAR-T are shown from top to bottom. In NFATi GLUT3 CAR-T cells, the expression of the repressor molecule (TIM3) was lower than in GLUT3 CAR-T cells. [Fig. 29] Fig. 29 illustrates that a low-glucose state impairs the function of CAR-T cells. (a) Interstitial fluid was collected by low-speed centrifugation from surgical or biopsy specimens of patients with glioblastoma (GBM), non-small cell lung cancer (lung cancer), or colorectal cancer (colon cancer) (n=5 each), and glucose concentrations were investigated. (a) Experimental schematic (b) Glucose concentrations in interstitial fluid of GBM specimens and paired serum specimens. Data are plotted 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 plotted as mean ± SEM. Statistical analysis by one-way ANOVA; *, P<0.05 (d,e) Mock-transduced T cells (Mock T cells) and conventional EGFRvIII CAR-T cells were prepared from PBMCs of healthy subjects (n=5). (d) Cytokine production in Mock-transduced T cells (Mock T cells) and conventional EGFRvIII CAR-T cells (conv CAR-T cells) was investigated by intracellular cytokine staining when stimulated by EGFRvIII-expressing tumor cells (U-87mGΔ) under low-glucose (0.5 mM) or high-glucose (10 mM) conditions. Representative contour plots (left) and a summary of cytokine (IFN-γ, IL-2, TNFα)-producing cell frequencies (right) are shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01 (e) Cytokine (IFN-γ, IL-2, TNFα) production in conventional EGFRvIII CAR-T cells co-cultured 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 plotted as mean ± SEM.Statistical analysis by Student's t-test; ***, P<0.001; ****, P<0.0001 (f) Gene expression of EGFRvIII CAR-T cells 24 hours after EGFRvIII bead stimulation under low-glucose (0.5 mM) or high-glucose (10 mM) conditions. Pathways enriched under low-glucose conditions compared to high-glucose conditions are plotted according to the molecular signature database. (g) Gene expression of selected genes is plotted as a heatmap. Gene expression was normalized row by row. All experiments were performed at least twice. [Fig. 30] Fig. 30 is a diagram illustrating that the overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells and increases cytotoxicity and cytokine production under low-glucose conditions. (a) Structures of a conventional EGFRvIII CAR (conv CAR, top) and an EGFRvIII CAR (GLUT3 CAR, bottom) that stably expresses GLUT3. (b) Immunofluorescence staining of a conventional EGFRvIII CAR-T cell (conv CAR-T) and a GLUT3 EGFRvIII CAR-T cell (GLUT3 CAR-T). The white dotted circle indicates a T cell that has not been transduced into a CAR. Katushka (red), 4,6-diamino-2-phenylindole (DAPI: blue), GLUT3 (green) (c) A summary of mean fluorescence intensity (MFI) of GLUT1 (left) and GLUT3 (right) expression levels in CAR-T cells is shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ****, P<0.0001 (d) A representative histogram (left) and a summary of mean fluorescence intensity (MFI) of CAR-T cells measured by glucose analog 2-NBDG uptake assay are shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; **, P<0.01 (e,f) Glycolysis (e) and metabolic phenotype (f) of CAR-T cells under low-glucose (0.5 mM) conditions analyzed by a flux analyzer. Data are plotted as mean ± SD. (g,h) CAR-T cells were prepared from PBMCs of healthy subjects (n=5). (g) Cytokine production upon stimulation by EGFRvIII-expressing tumor cells (U-87mGΔ) under low-glucose (0.5 mM) conditions was investigated by intracellular cytokine staining. Representative contour plots (left) and a summary of cytokine (IFN-γ, IL-2, TNFα)-producing cell frequencies (right) are shown. Data are plotted as mean ± SEM.Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01; ****, P<0.0001 (h) Cytokine (IFN-γ, IL-2, TNFα) production in CAR-T cells co-cultured with U-87mGΔ cells for 16 hours under low-glucose (0.5 mM) conditions was measured by ELISA. Data are plotted as mean ± SEM. Statistical analysis 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 co-cultured for 24 hours at the indicated effector-to-target (E:T) ratio, and specific lysis was calculated based on luciferase activity. Data are plotted 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 + A frequency summary of the CAR-T cell (k) population is shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ****, P<0.0001 [Fig. 31] Fig. 31 illustrates the 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 schematic: NSG mice (n=6) intracranially inoculated with U-87mGΔ-Luc cells were intravenously administered Mock T cells (Mock T), conventional EGFRvIII CAR-T cells (conv CAR-T), or GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) on day 5 after tumor inoculation. On day 35, the same tumor was re-challenged in mice in which the U-87mGΔ-Luc tumor had been eliminated by treatment with conventional EGFRvIII CAR-T cells or GLUT3 EGFRvIII CAR-T cells. 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. Cross marks indicate the cause of death. The numbers in the upper right panel indicate the number of mice that achieved a complete response (CR) and the number of mice that died by day 35. The horizontal dashed line shows the background BLI signal level obtained from mice not inoculated with tumors. (c) Kaplan-Meier curve. Statistical analysis by log-rank test for the Kaplan-Meier curve. (d) Change in body weight of each mouse during CAR-T cell therapy. Cross marks indicate random causes of death, and the colored dashed line indicates mice with treatment-related death (TRM). (e) Stacked bar graph showing results at the end of the observation period. TRM: Treatment-related death. (f,g) Pathological analysis of brain and lung tissues of mice in which severe body weight loss was observed on day 14. (f) Human CD3 in brain tissue. +IHC staining of cells (g) Hematoxylin and eosin (HE) staining of lung tissue (left: low magnification, center: high magnification) and human CD3 staining (right). Arrows indicate the hyaline membrane. (h) Phenotype of circulating human T cells (CAR-T cells) on day 14 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 plotted as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05 (i) Comparison of gene expression between conventional EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells on day 7 after stimulation with EGFRvIII beads under low glucose (0.5 mM) conditions. According to the molecular signature database, the pathway enriched in GLUT3 EGFRvIII CAR-T cells compared to conventional EGFRvIII CAR-T cells is shown. [Fig. 32] Fig. 32 illustrates that on-demand GLUT3 expression confers excellent effector activity to CAR-T cells. (a) A construct in which GLUT3 expression is driven by NFAT binding to the NFAT-binding domain (NFAT-BD), enabling on-demand GLUT3 expression (on-demand GLUT3 CAR) in CAR-T cells. BD: Binding domain (b) Immunofluorescence imaging of on-demand GLUT3 EGFRvIII CAR-T cells for CAR introduction markers Katushka (red), 4,6-diamino-2-phenylindole (DAPI: blue), and GLUT3 (green). (c) Time course of GLUT3 expression in conventional 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. Representative histogram (left) and MFI summary (right) are shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001 (e,f) Glycolytic ability (e) and metabolic phenotype (f) of CAR-T cells cultured under low-glucose (0.5 mM) conditions were investigated by flux analyzer on days 3 and 7 after stimulation with EGFRvIII beads as indicated. Data are plotted as mean ± SD. (g,h) CAR-T cells were prepared from PBMCs of healthy subjects (n=5). (g) Cytokine production associated with stimulation by EGFRvIII-expressing tumor cells (U-87mGΔ) under low glucose (0.5 mM) conditions was investigated by intracellular cytokine staining, and a representative contour plot (left) and a summary of cytokine (IFN-γ, IL-2, TNFα)-producing cell frequencies (right) are shown.Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001; ****, P<0.0001 (h) Cytokine (IFN-γ, IL-2, TNFα) production in CAR-T cells co-cultured with U-87mGΔ cells for 16 hours under low-glucose (0.5 mM) conditions was measured by ELISA. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ****, P<0.0001 (i) Cytotoxicity of CAR-T cells against U-87mGΔ and U-251mGΔ 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 or U-251mGΔ-Luc) were co-cultured for 24 hours under low-glucose (0.5 mM) conditions at the indicated effector-to-target (E:T) ratio, and specific lysis was calculated based on luciferase activity. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Fig. 33] Fig. 33 illustrates that when necessary, GLUT3 expression improves T cell survival by causing a reduction in differentiated T cells and avoiding overactivation. (a) Apoptotic status measured by Annexin V and 7-AAD staining of the indicated CAR-T cell population [Mock T cells (Mock T), conventional 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 plot (left) and survival (Live) (Annexin V - 7-AAD -), Pre-apoptosis (Annexin V + 7-AAD - ) and apoptosis (Annexin V + 7-AAD + (b) A summary of cell population frequencies (right) is shown. (b) Heatmap of gene expression related to apoptosis and T cell activation in CAR-T cells 72 hours after stimulation with EGFRvIII beads under low glucose (0.5 mM) conditions. Gene expression was normalized row by row. (c) Comparison of gene expression between conventional 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 glucose (0.5 mM) conditions. The pathways enriched in GLUT3 EGFRvIII CAR-T cells compared to conventional EGFRvIII CAR-T cells are shown along with their interrelationships. (d,e) CAR-T cells were prepared from PBMCs of healthy subjects (n=5) and stimulated with EGFRvIII-expressing tumor cells (U-87mGΔ) under low-glucose (0.5 mM) conditions. (d) T cell phenotype (CD62L) of the indicated CAR-T cell population + CD45RA + : Naive(na ve) / stem cell memory; CD62L + CD45RA - : Central memory; CD62L - CD45RA - : Effector memory; and CD62L - CD45RA + : Final differentiated effector memory). Representative contour plot (left) and naive / stem cell memory T cells (CD62L + CD45RA +A summary of population frequencies (right) is shown. Data are plotted 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 CAR-T cell population frequencies is presented. Data are plotted 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 GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) as needed after stimulation with EGFRvIII beads under low-glucose (0.5 mM) conditions. Principal component analysis of RNA-seq transcriptome profiles of GLUT3 EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells as needed on day 3 or 7 after stimulation. (g) Heatmap of CAR-T cell metabolic scores analyzed by METAFlux using RNA-seq data on day 7 after stimulation. Gene expression was normalized row by row. FA: Fatty acid [Fig. 34] Fig. 34 is a diagram illustrating GLUT3 expression as needed by CAR-T cells. GLUT3 expression as needed by CAR-T cells induces a sustained antitumor effect with excellent survival benefits in an intracranial human GBM cell xenograft model. (a) Experimental schematic diagram. On the 5th day after tumor inoculation, NSG mice (n=10, except for the Mock T cell group, n=5) inoculated with U-87mGΔ-Luc cells intracranially were administered Mock T cells (Mock T), conventional EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), or as needed GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T). Mice in which U-87mGΔ-Luc tumors had been re-challenged by conventional EGFRvIII CAR-T cell, GLUT3 EGFRvIII CAR-T cell, or GLUT3 EGFRvIII CAR-T cell treatment as needed 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 cell. Cross marks indicate the cause of death. The numbers in the upper right panel indicate the number of mice that achieved a complete response (CR) and the number of mice that died by day 21. The horizontal dashed line shows the background BLI signal level obtained from mice not inoculated with the tumor. (c) Change in body weight of each mouse during CAR-T cell treatment. Cross marks indicate any cause of death, and colored dashed lines indicate mice with treatment-related death (TRM). (d) Mouse body weight on day 42 after CAR-T cell injection. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05 (e) Kaplan-Meier curve for survival of U-87mGΔ tumor-bearing mice (n=10, except for the mock T cell group n=5) treated with labeled CAR-T cells.Statistical analysis by log-rank test for Kaplan-Meier curves; ns, not significant; *, P<0.05 (f) Phenotype of circulating human T cells on day 14 after CAR-T cell administration Representative contour plot (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 +- : Frequency summary of final differentiated effector memory (EMRA) is shown. (g) Kaplan-Meier curves for survival of U-251mGΔ cell-bearing mice (n=10, except for the mock T cell group, n=5) treated with the indicated CAR-T cells. Statistical analysis by log-rank test for Kaplan-Meier curves; ns, not significant; *, P<0.05 (h) Serum cytokine (TNFα and GM-CSF) concentrations on Day 14 after CAR-T cell administration. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05 (i) Reduction rate of serum TNFα and GM-CSF on Day 14 compared to Day 7 after CAR-T cell administration. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05 From the results of Figure 34, it can be said that constitutively expressed GLUT3 enhances the antitumor effect (i.e., effector function) of EGFRvIII CAR-T cells. Furthermore, when necessary, GLUT3 expression not only enhances the effector function of EGFRvIII CAR-T cells but also extends memory function and maintains the memory T cell phenotype. Additionally, it can be concluded that adverse events such as weight loss and treatment-related death induced by constitutively expressed GLUT3 were prevented, and as a result, significant improvement in survival, including cure, was achieved. [Fig. 35] Fig. 35 is a diagram illustrating that low-glucose conditions impair the function of CAR-T cells. (a) Volcano plot showing differentially expressed genes in conventional EGFRvIII CAR-T cells cultured under low-glucose (0.5 mM) conditions compared to high-glucose (10 mM) conditions. The X-axis represents log2 (fold change) (log2FC), and the Y-axis represents -log 10 (Adjusted p-value)[(-log 10 [Padj] is plotted. The dashed lines represent the cutoff values ​​x=±1.4 and -log for log2 (multiple change). 10 Indicates the cutoff value y=0.1 for (adjusted p-value). The top 5 genes and bottom 5 genes are highlighted in green and purple, respectively. [Fig. 36] Fig. 36 illustrates that the overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells. (a) Representative histograms of GLUT1 (left) and GLUT3 (right) expression in conventional EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), U-87mGΔ cells, and U-251mGΔ cells are shown. (b) Glycolytic parameters of CAR-T cells cultured under low-glucose (0.5 mM) conditions measured by a glycolytic stress test. Data are plotted as mean ± SD. Statistical analysis by Student's t-test; ns, not significant; ***, P<0.001 (c,d) Competitive analysis of CAR-T cells under low-glucose (0.5 mM) or high-glucose (10 mM) conditions (72-hour culture) (c) Experimental schematic (n=3) (d) Representative contour plot (left) and summary of proportions for each CAR-T cell population (right) are shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001 [Fig. 37] Fig. 37 illustrates that stable expression of GLUT3 improves CAR-T cell function under low-glucose conditions. (a) Cytokine production in Mock T cells (Mock T), conventional EGFRvIII CAR-T cells (conv CAR-T), and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) (n=5) stimulated by EGFRvIII-expressing tumor cells (U-87mGΔ) under high-glucose (10 mM) conditions was investigated by intracellular staining. (a) Cytokine (IFN-γ, IL-2, TNFα)-producing CD3 + A summary of CAR-T cell frequencies is shown. Data are plotted 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 + A summary of CAR-T cell frequencies (right) is shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ***, P<0.001 (c) Representative contour plot (left) and cytokine (IFN-γ, IL-2, TNFα)-producing CD4 +A summary of CAR-T cell frequencies (right) is shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; **, P<0.01. (d,e) Cytotoxicity of CAR-T cells against U-87mGΔ and U-251mGΔ cells under low-glucose (0.5 mM) or high-glucose (10 mM) conditions was determined by luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc and U-251mGΔ-Luc) were co-cultured for 24 hours at the indicated effector-to-target (E:T) ratio, 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 plotted 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 cells or U-251mGΔ-Luc cells under high glucose (10 mM) conditions. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001 Representative contour plots of the expression of exhaustion markers (PD-1, f; PD-1 and Tim-3, g) by the indicated CAR-T cell population are shown. [Fig. 38] Fig. 38 illustrates that the improvement in CAR-T cell function by stable expression of GLUT3 does not depend on the type of scFv. (a,b) Mock T cells (Mock T), conventional CD19 CAR-T cells (conv CD19 CAR-T), and GLUT3 CD19 CAR-T cells (GLUT3 CD19 CAR-T) were prepared from PBMCs of healthy subjects (n=5). (a) Cytokine production by the indicated CAR-T cells upon stimulation by CD19-expressing tumor cells (NALM6) under low-glucose (0.5 mM) conditions was determined by intracellular cytokine staining, and a representative contour plot (left) and a summary of cytokine (IFN-γ, IL-2, TNFα)-producing cell frequencies (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 in conventional CD19 CAR-T cells and GLUT3 CD19 CAR-T cells co-cultured with NALM6 for 16 hours under low-glucose (0.5 mM) conditions was measured by ELISA. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; ***, P<0.001; ****, P<0.0001 (c) The cytotoxicity of conventional 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 luciferase-based assays. CAR-T cells and luciferase-expressing target cells (NALM6-Luc and CD19-expressing AsPC-1-Luc) were co-cultured for 24 hours at the indicated effector-to-target (E:T) ratios, and specific lysis rates were evaluated based on luciferase activity. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01 [Fig. 39] Fig. 39 illustrates that when necessary, GLUT3 expression enhances metabolic fitness and improves cytokine production and cytotoxicity of CAR-T cells. (a) Glycolytic parameters (measured by glycolytic stress tests) of conventional EGFRvIII CAR-T cells (conv CAR-T) and when necessary GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) cultured under low glucose (0.5 mM) conditions are shown. Data are plotted as mean ± SD. Statistical analysis by Student's t-test; **, P<0.01; ***, P<0.001; ****, P<0.0001 (b,c) Conventional EGFRvIII CAR-T cells (conv CAR-T) and, if necessary, GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) were prepared from PBMCs of healthy subjects (n=5). (b) Cytokine production by the indicated CAR-T cells upon stimulation by EGFRvIII-expressing tumor cells (U-87mGΔ) under high glucose (10 mM) conditions was investigated by intracellular cytokine staining. A summary of the cytokine (IFN-γ, IL-2, TNFα)-producing cell frequencies is plotted. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ***, P<0.001 (c) The cytotoxicity of CAR-T cells against U-87mGΔ and U-251mGΔ cells under high glucose (10 mM) conditions was determined by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87mGΔ-Luc or U-251mGΔ-Luc) were co-cultured for 24 hours under high glucose (10 mM) conditions at the indicated effector-to-target (E:T) ratio, and specific lysis was calculated based on luciferase activity. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01; ****,<0.0001. [Fig. 40] Fig. 40 illustrates that GLUT3 expression improves CAR-T cell survival when necessary. (a,b) CD3 after 24 hours of stimulation by anti-EGFRvIII beads under low-glucose (0.5 mM) conditions + T cell proliferation and CAR among cells + T cell frequency; Mock T cells (Mock T), conventional EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T as needed) were prepared from PBMCs of healthy subjects (n=3). (c) Phenotype of CAR-T cells on day 7 after stimulation by U-87mGΔ cells. Representative contour plot (left) and central memory (CD45RA CCR7 -+ A summary of T cell frequencies (right) is shown. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ****, P<0.0001 (d) Expression of bacterial shedding markers (PD-1 and Tim-3) by the indicated CAR-T cell population 72 hours after stimulation by EGFRvIII-expressing tumor cells (U-87mGΔ) is shown. Representative contour plots are shown. [Fig. 41] Fig. 41 is a diagram illustrating GLUT3 expression as needed by CAR-T cells. GLUT3 expression as needed by CAR-T cells increases survival while maintaining a sustained antitumor effect in an intracranial human GBM cell xenograft model. (a) BLI of mice with U-87mGΔ-Luc tumors (b,c) NSG mice (n=10, except for the Mock CAR-T cell group, n=5) intracranially inoculated with U-251mGΔ-Luc cells, were administered Mock T cells (Mock T), conventional EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), or as needed GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) intravenously on the 5th day after tumor inoculation. On day 21, the same tumor was re-challenged in mice in which the U-87mGΔ-Luc tumor had been removed by EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, or, if necessary, GLUT3 EGFRvIII CAR-T cells. Tumor burden was monitored by BLI. (b) Tumor burden measured by BLI. Each colored line represents an individual mouse. The cross marks indicate the cause of death. The numbers in the upper right indicate the number of mice that showed a complete response (CR) and the number of mice that died by day 21. The horizontal dashed line represents the baseline BLI signal level obtained from mice not inoculated with the tumor. (c) Changes in body weight of mice during CAR-T cell therapy. Cross marks indicate any cause of death, and colored dashed lines indicate mice that experienced treatment-related death (TRM). (d) Serum cytokine and chemokine concentrations (IL-5, IP-10, and MDC) on Day 14 after CAR-T cell administration were investigated by ELISA. Data are plotted as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01 Specific details for implementing the invention

[0230] The present disclosure is described below in its best form. Throughout this specification, singular expressions should be understood to include the concept of the plural form unless otherwise specified. Accordingly, singular articles (e.g., English “a,” “an,” and “the,” etc.) should be understood to include the concept of their plural forms unless otherwise specifically stated. Furthermore, terms used in this specification should be understood to be used in the sense commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as generally understood by those skilled in the art to which this disclosure pertains. In the event of a conflict, this specification (including definitions) shall prevail.

[0231] Hereinafter, definitions of terms particularly used in this specification and / or basic technical details are appropriately explained.

[0232] In this specification, “about” means ±10% of the following number. For example, “about 20” is intended to include “18 to 22”. A numerical range includes all numbers between both endpoints and numbers of both endpoints. “About” regarding a range applies to both endpoints of the range. Thus, for example, “about 20 to 30” is intended to include “18 to 33”.

[0233] In this specification, the term “T cell” is used in the broad sense as used in the art and refers to lymphocytes that are generated in the bone marrow, migrate to the thymus, and mature. T cells may be CD45-positive and CD3-positive cells among the normal fractions of mononuclear cells derived from peripheral blood and bone marrow. As T cells used, T cells isolated from a donor, particularly a human donor, may be used, but are not limited thereto. 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 being immortalized, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions. It is known that multiple types of T cells exist according to function, examples of which are effector memory T cells (T eff These include naive T cells (Tnv), stem cell memory T cells (Tscm), central memory T cells (Tcm), and finally differentiated RA-positive T cells (Temra). In this specification, the term “peripheral T cells” refers to T cells located outside the thymus and may be obtained from peripheral blood, lymph nodes, or other tissues. In this specification, when “peripheral T cells” are referred to, it is sufficient that peripheral T cells are included in a cell population, and it is not necessary for the T cells to be isolated. Cell fractions containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMC), may be used.

[0234] In this specification, the term “cell population” refers to a group comprising two or more cells, and may be, for example, a mass of cells formed by cells being gathered together in a planar manner or by cells being attached to each other in three dimensions. Additionally, the “cell population” may be formed of a single type of cell or may include multiple types of cells. When referring to a cell population of T cells in this disclosure, it is sufficient for the cell population to include at least one cell, such as Tnv, Tscm, Tcm, or Temra, which can have an effector function at the site where the cell is to function (typically in vivo).

[0235] In this specification, the term “flow cytometry” refers to a technique for measuring the number of particles and individual physical, chemical, and biological properties of cells, organisms, and other biological particles suspended in a liquid. An apparatus using this technique is referred to as a “flow cytometer.” In this disclosure, “positive” and “negative” results for cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are determined by flow cytometry as is commonly used in the art. More specifically, in flow cytometry, cells are aligned and flowed in a single column, and the number of cells is counted by spectroscopic techniques. For example, the number of target cells is counted by irradiating cells labeled with a fluorescent or luminescent enzyme with laser light and detecting the fluorescent or luminescent signal emitted from the cells with a detector such as a photodiode. Additionally, the detection results from the detector can be imported into a computer to generate and display a two-dimensional plot. This allows for easy identification of the presence or absence of target cells, their number, etc.

[0236] In this specification, the term “effector function” refers to biological activities such as cytotoxic activity, cytokine production, and division / proliferation induced by signaling via TCR or CAR in T cells, and biological activities induced by the Fc region of an antibody in other immune cells. Examples of effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells. In this disclosure, whether 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 methods exemplified in the examples, etc.

[0237] In this specification, the terms “effector cell” or “effector T cell” refer to an immune cell that is a T cell having an effector function and exerting cytotoxic effects on target cells, and T eff It may also be referred to as such. In this specification, whether a cell is an “effector” cell can be determined by determination methods such as cytotoxicity analysis, surface antigen analysis by flow cytometry, and intracellular cytokine staining. Effector cells are immune cells that perform effector functions and mediate antibody-dependent cytotoxicity (ADCC), etc. Effector cells include, for example, peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, neutrophils, etc., and can be isolated from natural tissues such as blood.

[0238] In this specification, the term “effector T cell (T effProgenitor cells of the T cell type (also referred to as “effector T cell precursors”) refer to cells that are not effector cells but acquire effector function upon being delivered to a site of function (e.g., in vivo) or receiving other stimuli. These cells include lymphoid progenitor cells, Tnv, Tscm, Tcm, and Temra. Effector T cell precursors are activated during the early stages of the immune response and mature into effector T cells through proliferation and differentiation, whereas some precursor cells possess the ability to differentiate into memory T cells. These precursor cells exhibit specific gene expression patterns and metabolic states, which contribute to the extension of memory function. Several molecular and metabolic characteristics are involved in this extension of memory function. First, effector T cell precursors possess specific characteristics in their metabolic programs. In these cells, oxidative phosphorylation predominates over the glycolysis pathway characteristic of effector T cells; this metabolic pathway is energy-efficient and enables long-term cell survival. This metabolic characteristic is a key factor supporting the long-term maintenance of function as a memory T cell. Next, the role of transcription factors is also important. Transcription factors such as T-bet and Eomes are expressed in effector T progenitor cells; these promote differentiation into memory T cells, enabling their long-term survival and function. Furthermore, these progenitor cells, which express surface markers such as CD62L and CCR7, possess the ability to migrate to secondary lymphoid tissues. Through this homing mechanism, progenitor cells receive necessary survival signals in secondary lymphoid tissues and contribute to the maintenance of memory function. Moreover, the inhibition of antigenic stimulation is also involved in the extension of memory function. Some progenitor cells are freed from antigenic stimulation, thereby inhibiting complete differentiation into effector T cells and instead retaining their characteristics as memory T cells. Through this mechanism, excessive effector responses are suppressed, while memory function is simultaneously maintained. Additionally, the cytokine environment surrounding the cells plays a crucial role.In particular, cytokines such as IL-7 and IL-15 provide survival signals necessary for the maintenance of memory T cells, which significantly contributes to the extension of memory function. Due to these factors, effector T progenitor cells survive for longer periods compared to normal effector T cells and maintain memory functions that enable rapid and strong secondary immune responses. This characteristic is considered to play a crucial role not only in immune defense against infectious diseases but also in cancer immunotherapy and vaccine development. Therefore, understanding the characteristics of effector T progenitor cells holds great significance for both basic immunological research and clinical applications.

[0239] In effector T cell precursors, the phrase “maintaining the memory T cell phenotype” refers to the phenomenon in which some precursor cells retain phenotypes characteristic of memory T cells (e.g., surface markers, metabolic state, expression of transcription factors, etc.) during the differentiation process into effector T cells. These precursor cells survive even after the immune response is completed and possess the ability to provide immune memory over a long period. This phenomenon is considered a crucial mechanism for achieving both the process of T cells exercising effector function and the memory function that enables long-term survival and rapid secondary immune responses. The characteristics of this phenomenon are explained below. First, effector T cell precursors retain surface markers characteristic of memory T cells. They contain homing molecules such as CD62L and CCR7, and the expression of these molecules enables the precursor cells to maintain the ability to migrate to secondary lymphoid tissues. Next, these precursor cells are characterized by the maintenance of their metabolic state. Specifically, long-term survival is made possible by maintaining a metabolic program in which oxidative phosphorylation is dominant over glycolysis and which is highly energy-efficient. This metabolic characteristic is a key factor in maintaining function as a memory T cell. Furthermore, progenitor cells also maintain a characteristic balance in the expression of transcription factors. The proper expression of transcription factors such as T-bet and Eomes plays a role in aligning differentiation into effector T cells with the maintenance of the memory T cell phenotype. Moreover, progenitor cells are responsive to cytokine signaling and can receive survival signals such as IL-7 and IL-15. This responsiveness enables progenitor cells to survive for extended periods while maintaining memory T cell characteristics. Finally, these progenitor cells can be maintained in an antigen-independent manner. That is, memory T cell progenitor cells possess the ability to maintain their phenotype and remain prepared for a rapid immune response even in the absence of antigens.Due to the above characteristics, the maintenance of the memory T cell phenotype by effector T cell precursors plays a very important role in the flexibility of the immune system and the establishment of long-term immune memory. These characteristics are considered to have great significance in defense against infectious diseases, cancer immunotherapy, and vaccine design.

[0240] In this specification, the term “antigen receptor” refers to any molecule that specifically binds to a target antigen and transmits a signal into a cell expressing the receptor. In this disclosure, the antigen receptor may be introduced into a T cell and may be a natural molecule or an artificial molecule. For example, in addition to T cell receptors (TCRs) and B cell receptors (BCRs), chimeric antigen receptors (CARs) are also included.

[0241] In this specification, the term “Chimeric Antigen Receptor (CAR)” refers to a modified receptor capable of conferring antigen specificity to a cell (e.g., immune cell). A CAR is also known as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. A CAR is a modified receptor that conveys antigen specificity to immune system cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof, NK cells, macrophages, etc.) and may comprise, for example, an antigen-specific targeting region, an extracellular domain, a transmembrane domain, a co-stimulating domain, and / or an intracellular signaling domain. Additionally, a CAR may also include a CAR that uses multiple antigen-specific targeting regions (often two types) (a bispecific CAR). Preferably, the CAR of the present disclosure comprises at least one extracellular domain, at least one transmembrane domain, and at least one intracellular domain capable of binding to an antigen.

[0242] In this specification, the term “T cell receptor (TCR)” refers to a receptor present on T cells. A TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains and includes an αβ-type TCR expressed by normal T cells and a γδ-type TCR having a specific function. The α-chain and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens replicated intracellularly in connection with viral infection and oncogenes derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Additionally, antigens derived from foreign microorganisms are absorbed by antigen-presenting cells via endocytosis and, after processing, are presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. Co-stimulatory molecules such as CD28, ICOS, and OX40 are also known to be important for stimulation mediated by TCR molecules. With respect to αβ type TCRs, which are one of the main focuses of this specification, the gene products of α and β, respectively, are believed to express specificity through unique combinations.

[0243] In this specification, the term “glucose uptake capacity” refers to the ability to absorb glucose into a cell per unit time. Glucose uptake into a cell can be evaluated by methods such as 2-NBDG uptake assay.

[0244] In this specification, the term “enhanced glucose uptake capacity” refers to an improvement in glucose uptake capacity by any method, and an improvement in glucose uptake capacity may be achieved by modifying immune cells (e.g., T cells) to express one or more types of glucose transporters, etc., or by modifying immune cells to enhance the expression of glucose transporters. Such genetically modified immune cells are expected to exhibit high glucose uptake, for example, in a low-glucose environment (e.g., a tumor microenvironment). Accordingly, immune cells co-expressing one or more types of glucose transporters and chimeric receptor polypeptides may exhibit superior physiological activities, such as cell proliferation, activation (e.g., increased cytokine production, e.g., IL-2 or IFNγ production), cytotoxicity, and / or in vivo antitumor activity (e.g., under a tumor microenvironment such as low-glucose conditions, selectively in the presence of therapeutic antibodies).

[0245] In this specification, the term “glucose transporter” (GLUT) refers to a transporter that performs facilitated diffusion-type transport of glucose (dextrose). Regarding GLUT, molecular species GLUT1 to GLUT14 have been reported. Among these, for example, GLUT1 is expressed in a wide range of tissues and is responsible for constitutive basal glucose transport, GLUT2 operates when glucose concentration increases to enhance glucose uptake, GLUT3 plays a central role in the central nervous system and exhibits transport function even at low glucose concentrations, and GLUT4 operates in response to insulin stimulation.

[0246] In this specification, the term “tumor microenvironment” is used interchangeably with “tumor environment.” The tumor microenvironment (TME) refers to the local biological environment surrounding tumor tissue and is a concept that includes various factors involved in tumor progression, proliferation, metastasis, drug resistance, etc. 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. Furthermore, humoral factors such as the extracellular matrix (ECM), cytokines, chemokines, and growth factors are also important components. Moreover, the tumor microenvironment is also influenced by physical factors such as oxygen concentration, pH, and trophic status. Changes in intercellular interactions and physical factors within the tumor microenvironment are believed to play a significant role in tumor malignancy and therapeutic responsiveness. In particular, the immunosuppressive microenvironment is receiving attention as a factor that reduces the efficacy of immunotherapy. In this invention, the term “tumor microenvironment” refers to a set of biological and physical factors including these elements and their interactions, and is an important target in tumor treatment and diagnosis.

[0247] In this specification, the term “tumor microenvironment conditions” refers to the tumor microenvironment or equivalent conditions. Accordingly, “tumor microenvironment conditions” refers to a broad concept that includes similar or equivalent environmental conditions in which a tumor is situated within the body, in addition to the conditions constituting the tumor microenvironment. Specifically, tumor microenvironment conditions include not only the local biological environment surrounding the tumor tissue but also endogenous and exogenous factors that affect the tumor when it is present in the body. This environment includes interactions between tumor cells and non-tumor 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, trophic status, and physical pressure. Furthermore, “tumor microenvironment conditions” also include situations in which the tumor interacts with the broad physiological environment within the body and is therefore not limited to the local environment surrounding the tumor. In the present invention, “tumor microenvironment conditions” comprehensively represents the broad environmental conditions faced by a tumor within the body and includes all factors that influence tumor progression and therapeutic responsiveness. Therefore, tumor microenvironment conditions include the tumor interior, TCR-related signaling, stroma and surrounding (external) environments, and similar conditions.

[0248] In this specification, the term “modification” broadly refers to any structural, functional, or expression-related change applied to a gene (including the corresponding nucleic acid and protein) and includes naturally occurring or artificial changes. Specifically, modification includes sequence changes by substitution, deletion, insertion, addition, or combinations thereof of nucleotide sequences; changes in the amino acid sequence or characteristics of an encoded protein; or chemical modifications to add or improve functionality (e.g., glycosylation, phosphorylation, acetylation, etc.). Additionally, “modification” includes the introduction of genes from external sources (e.g., transfection or introduction via viral vectors), knockout or knock-in of specific genes using transformation or genome editing technologies, and repression of gene expression (e.g., repression by RNA interference or CRISPR technology). Furthermore, epigenetic modifications (e.g., DNA methylation, histone modification, etc.) and modifications of expression regulatory elements are also included. In this specification, the term “modification” comprehensively refers to any form of change affecting the structure, expression, function, or biological behavior of a gene, including not only endogenous changes but also changes caused by external introduction or manipulation.

[0249] In this specification, the expression “in response to need (when needed)” means that a target gene is expressed when needed. Need refers to, for example, when glucose is about to be depleted (e.g., when T cells infiltrate a low-glucose environment such as a tumor environment or bone marrow, when glucose consumption increases due to metabolic changes to support the exercise of cytotoxic activity, cytokine production, division and proliferation, etc., by T cell activation, or both), and the duration is optional. For example, expression may occur for only a few hours, a few days, or a few weeks, but is not limited thereto, and cases where the duration of need is longer than a few days may also be included in the definition of when needed. The phrase “in response to need (when needed)” may refer to a characteristic in which the expression of a glucose transporter in T cells is regulated in response to changes in tumor microenvironment conditions, or may include a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. In some cases, the target gene is expressed in the cell without being integrated into the host genome. In other cases, the target gene is integrated into the host genome and expressed in the cell. As a method used for expression when necessary, methods known to those skilled in the art may be used, and transfection or transduction may be performed using kits provided by the supplier. Screening may be performed to select cells capable of achieving expression when necessary, and, for example, drug screening or magnetic bead screening widely known to those skilled in the art may be used. If the constructed structure already possesses the characteristics of the structure when necessary, the structure may be used as is. As an example, the expression of glucose transporters in T cells is regulated in response to changes in tumor microenvironment conditions.

[0250] In this specification, the term “tumor microenvironment conditions” refers to any conditions associated with a tumor, including conditions within the tumor, conditions of the stroma and surrounding (external), and similar conditions.

[0251] In this specification, the phrase “expression of glucose transporters (in cells such as T cells) is regulated in response to changes in tumor microenvironment conditions” means that when tumor microenvironment conditions deteriorate (when the cancer condition worsens), the expression or level of glucose transporters is reduced or their degradation is promoted, and when tumor microenvironment conditions improve (when the health condition improves), the expression or level of glucose transporters is enhanced or their 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 the cells, such as by introducing these gene constructs.

[0252] Here, the phrase “deterioration of tumor microenvironment conditions” refers to changes in the tumor microenvironment (TME) that promote tumor growth or progression, as well as equivalent conditions, that make the TME more favorable to the tumor. This tumor microenvironment consists not only of tumor cells but also surrounding blood vessels, immune cells, fibroblasts, the extracellular matrix (ECM), signaling molecules, etc. The “deterioration” of this environment includes the following specific changes.

[0253] 1) Progression of hypoxic state

[0254] As a tumor grows, blood vessels become insufficient and oxygen supply decreases, leading to a hypoxic state around the tumor. This condition acts as a factor that promotes the proliferation and metastasis of tumor cells. Therefore, the deterioration of tumor microenvironmental conditions can be detected by examining oxygen levels.

[0255] 2) Activation of abnormal angiogenesis

[0256] Tumor growth promotes angiogenesis (the formation of new blood vessels), but these new vessels are often structurally abnormal, and an imbalance in the supply of oxygen and nutrients accelerates tumor progression. Therefore, deterioration of tumor microenvironmental conditions can be detected by examining the state of angiogenesis.

[0257] 3) Formation of an immunosuppressive environment

[0258] Tumors suppress anti-tumor immunity and reduce the immune system's ability to eliminate tumors by increasing immunosuppressive cells (such as regulatory T cells and myeloid-derived suppressor cells). Therefore, deterioration of tumor microenvironmental conditions can be detected by investigating the state of the immunosuppressive environment.

[0259] 4) Progression of fibrosis

[0260] Activation of fibroblasts around the tumor and abnormal accumulation of the extracellular matrix increase the migration ability and drug resistance of tumor cells. Therefore, deterioration of tumor microenvironmental conditions can be detected by examining the state of fibrosis.

[0261] 5) Activation of chronic inflammation

[0262] Persistent chronic inflammatory responses in the tumor microenvironment promote tumor cell proliferation, angiogenesis, and metastasis. Therefore, deterioration of tumor microenvironmental conditions can be detected by investigating the state of (chronic) inflammation.

[0263] 6) Metabolic abnormalities

[0264] Tumor cells consume excessive glucose and activate anaerobic metabolism (Warburg effect). As a result, lactic acid accumulates, the microenvironment becomes acidic, and the function of immune cells decreases. Therefore, deterioration of tumor microenvironment conditions can be detected by examining metabolic states (e.g., glucose consumption, lactic acid accumulation, etc.).

[0265] The effects induced by the deterioration of tumor microenvironment conditions are diverse, and the deterioration of the tumor microenvironment affects tumor characteristics, including the following non-limiting examples: 1) promotion of tumor growth (tumor cells become activated and division and proliferation are accelerated), 2) promotion of metastasis (tumor cells spread more easily to other organs through blood vessels or lymphatic vessels), 3) increase in treatment resistance (changes in the tumor microenvironment may reduce the effectiveness of chemotherapy or immunotherapy), etc.

[0266] In this specification, the phrase “modifies the expression of glucose transporters (in cells such as T cells) to be regulated in response to changes in tumor microenvironment conditions” means that the expression of glucose transporters in cells is regulated in response to changes in tumor microenvironment conditions by introducing new glucose transporters in cells or by modifying existing glucose transporters. Such modifications can be realized by operably linking a factor to the nucleic acid sequence encoding the glucose transporter that enables the regulation of the expression or degradation of glucose transporters in cells in response to changes in tumor microenvironment conditions. Alternatively, this can be achieved by using genetic engineering techniques, such as gene manipulation or genome editing, to modify existing glucose transporters or factors that directly or indirectly regulate them so that the expression or degradation of such glucose transporters is regulated.

[0267] In this specification, the term “TCR-related signal” refers to any signal associated with a T cell receptor (TCR) and includes not only signals directly associated with the TCR (e.g., naturally expressed non-transforming T cell receptors) but also signals associated with a chimeric antigen receptor (CAR), which is a complex of a part of the TCR and a co-stimulating factor, the expression of a tumor antigen (referring to an antigen recognized by the TCR or CAR) or a co-stimulating ligand, changes in cytokine conditions, etc. For example, a TCR-related signal may be mediated by any factor involved in TCR signaling and may include a TCR signal or a part thereof, a chimeric signal of an antigen different from the TCR signal (also referred to as a CAR signal), etc.

[0268] In this specification, the term “TCR signaling domain” refers to any domain involved in TCR signaling, and representative examples thereof include the immune receptor tyrosine-based activation motif (ITAM) present in CD3 molecules (CD3ε, CD3δ, CD3γ) and ζ chains (CD247, zeta chain) constituting the CR complex. ITAM has characteristics such as the presence of tyrosine residues (ITAM contains two tyrosine residues, and signaling is initiated when they are phosphorylated), kinase recruitment (phosphorylation of tyrosine residues recruits signaling kinases (mainly Lck and ZAP-70) to activate the signaling chain reaction), and initiation of the signaling chain reaction (after phosphorylation of ITAM, calcium signaling, the MAPK pathway, the NF-κB pathway, etc. are activated, and immune responses such as T cell proliferation, differentiation, and cytokine secretion are induced). Examples of TCR signaling domains include the CD3ζ chain (zeta chain; the CD3ζ chain is the major signaling molecule of the TCR complex and has three ITAMs, enabling amplification of the TCR signal), the CD3ε domain (epsilon domain; CD3ε has one ITAM and contributes to initial signaling via Lck after antigen recognition), modified TCR signaling domains (applications in CAR-T cells), and chimeric antigen receptor (CAR) therapy (artificially designing TCR signaling domains and achieving stronger signaling by combining co-stimulating molecule domains such as CD28 and 4-1BB in addition to the ITAMs of the CD3ζ chain).

[0269] In this specification, the term “tumor antigen,” also referred to as “cancer antigen,” refers to a molecule expressed by tumor cells and recognized by the immune system. Tumor antigens are primarily classified into those specifically expressed in tumor cells and those expressed in normal cells but abnormally overexpressed 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 the following two categories: tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are antigens specifically expressed in tumor cells and rarely expressed in normal cells. They are typically generated by the following causes: genetic mutations (mutant peptides (neoantigens) generated by genetic mutations such as RAS and p53), and viral-derived antigens (antigens expressed in tumors caused by viral infections (e.g., human papillomavirus (HPV)-derived E6 / E7 antigens)). TAAs are antigens that are expressed in normal cells but are abnormally overexpressed in tumor cells. Their key characteristics are as follows: embryonic antigens (antigens expressed during the fetal stage but barely expressed in adult tissues (e.g., CEA, AFP)) and overexpressed antigens (antigens expressed at low levels in normal tissues but at high levels in tumor cells (e.g., HER2, EGFR)). Examples of tumor antigens include: examples of neoantigens (antigens generated by mutations in tumor cells): KRAS mutant peptide; examples of oncolytic virus-derived antigens (antigens expressed in virus-infected tumors): HPV-derived E6 / E7 (cervical cancer); examples of embryonic antigens (expressed only during the fetal stage in normal cells and silenced in adults): Carcinoembryonic antigen (CEA), α-fetoprotein (AFP); examples of overexpressed antigens: HER2 (breast cancer), EGFR (lung cancer).

[0270] In this specification, the term “co-stimulating ligand” refers to a ligand that binds to a co-stimulating molecule to complement antigen presentation (recognition by the T cell receptor (TCR) or B cell receptor (BCR)), which is a key signal in the activation of T cells and other immune cells. Co-stimulating ligands are expressed on the surface of antigen-presenting cells (APCs), tumor cells, and other cells, and play a role in enhancing or regulating immune responses. The interactions between these ligands and co-stimulating molecules are important for T cell activation, proliferation, cytokine secretion, and immune memory formation, and contribute to the proper regulation of immune responses. Co-stimulating ligands are molecules that play a significant role in immune responses and are involved in the activation and regulation of immune cells, including T cells. As for their characteristics, first, co-stimulating ligands provide a second signal following antigen recognition via the T cell receptor (TCR) (the first signal), thereby enabling the full activation of T cells. Furthermore, the expression of co-stimulating ligands and their interactions play a role in regulating the intensity and nature of immune responses and maintaining the proper functioning of the immune system. Furthermore, since the absence of co-stimulating ligands can cause T cells to become unresponsive (anergy) and induce immune tolerance, co-stimulating ligands are also important for maintaining a balance between the immune response and immune tolerance.

[0271] Specific examples of co-stimulating ligands include those that promote activation and those that have inhibitory roles. Co-stimulating ligands that promote activation include B7-1 (CD80) and B7-2 (CD86), which bind to CD28 receptors on T cells to promote the initial activation of T cells. Furthermore, OX40L (CD252) binds to OX40 (CD134) and promotes the formation of immune memory by supporting the proliferation and survival of T cells. 4-1BBL (CD137L) binds to 4-1BB (CD137) to support the proliferation and sustained activation of activated T cells, and ICOSL (CD275) binds to ICOS receptors and is involved in the activation of helper T cells (Tfh cells) and B cell assistance. Meanwhile, inhibitory co-stimulatory ligands include Programmed Death-Ligand 1 (PD-L1) and PD-L2, which bind to PD-1 receptors on T cells to suppress T cell function and induce immune tolerance. Additionally, B7-H3 (CD276) and B7-H4 are also believed to possess inhibitory functions and are thought to be involved in immunosuppression and the induction of inhibitory T cells. The application of co-stimulatory ligands holds great potential in the fields of immunotherapy and disease treatment. In cancer immunotherapy, therapies using 4-1BBL, OX40L, etc., to enhance T cell activation 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, designs utilizing adjuvants expressing co-stimulatory ligands to enhance the immune response are advancing. In addition, research is being conducted on the use of inhibitory co-stimulatory ligands to control excessive immune responses in the treatment of autoimmune diseases.

[0272] In this specification, the term “degree of TCR-related signaling” refers to the strength or quality of signaling 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-related signaling is an important factor determining T cell activation and immune responses and is regulated by various factors. Specifically, the degree of signaling is enhanced as the binding strength (affinity) between the peptide-MHC complex and the TCR increases. Additionally, signaling via the TCR is further enhanced when the density of peptide-MHC complexes present on the surface of antigen-presenting cells is high. Furthermore, TCR signaling is amplified when ligands of co-stimulating molecules such as CD28 and ICOS (such as B7 family molecules) are present, whereas signaling is inhibited when immunosuppressive molecules such as PD-1 and CTLA-4 are involved. Furthermore, the phosphorylation status and expression levels of downstream signaling molecules (such as Lck, ZAP-70, LAT, and SLP-76) are also important factors influencing signal intensity. Additionally, metabolic environments such as cytokines, oxygen concentration, and glucose concentration contribute to the enhancement or inhibition of the signal. The degree of TCR-related signaling is critical in determining the quality of the immune response. For example, strong signals induce the activation of effector T cells and cytotoxicity, whereas weak signals are involved in immune tolerance and the induction of regulatory T cells (Tregs). Utilizing these characteristics, the regulation of TCR-related signaling is applied in immunotherapy and disease treatment. In cancer immunotherapy, attempts have been made to induce T cell responses that effectively attack cancer cells by increasing TCR signal intensity. Moreover, in the treatment of autoimmune diseases, approaches to control excessive immune responses by inhibiting TCR signaling are being investigated.Furthermore, in T-cell engineering, ensuring an appropriate level of signal transduction when designing chimeric antigen receptors (CARs) or artificial TCRs is key to enhancing therapeutic efficacy and safety.

[0273] In this specification, the term “degree of TCR stimulation” refers to the intensity or quality of stimulation that occurs when a T cell receptor (TCR) recognizes a peptide-MHC complex on an antigen-presenting cell (APC) or a target cell. This degree of stimulation is an important factor in determining the outcome of T cell activation and immune response and is defined by the intensity, duration, and frequency of the signal.

[0274] In this specification, the term “TCR co-stimulation” refers to additional signaling required to induce full T cell activation when a T cell recognizes a peptide-MHC complex on an antigen-presenting cell (APC) via a T cell receptor (TCR). Antigen recognition by the TCR (first signal) alone often results in incomplete activation or an unresponsive (honergy) state of the T cell. Therefore, TCR co-stimulation is important for effectively inducing an adaptive immune response, including T cell proliferation, cytokine production, effector function, and the formation of immune memory. TCR co-stimulation is provided by the interaction between co-stimulating molecules on the surface of the T cell and co-stimulating ligands on the antigen-presenting cell. This amplifies the TCR signal and modulates the immune response.

[0275] In this specification, the term “humoral factor(s)” refers to soluble molecules present in body fluids that are involved in intercellular communication and the maintenance of homeostasis in vivo. 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 play a significant role primarily in the immune system, and representative examples include molecules such as cytokines and complement components. These molecules bind to receptors on cell surfaces to initiate signal transduction and regulate the response of target cells.

[0276] In this specification, the term “transcription factor translocating into the nucleus in connection with T cell activation” refers to a transcription factor that is activated and translocates into the nucleus as a result of the initiation of a series of signaling cascades when a T cell recognizes a peptide-MHC complex presented by an antigen-presenting cell (APC) via a T cell receptor (TCR). These transcription factors play important roles in the activation, proliferation, differentiation, and expression of effector functions of T cells. For example, major transcription factors translocating into the nucleus in connection with T cell activation include NFAT, NF-κB, AP-1, and the STAT family. Nuclear factor of activated T-cells (NFAT) is activated by calcium signaling, translocates into the nucleus after dephosphorylation by calcineurin, and promotes the expression of interleukin-2 (IL-2) and other cytokine genes. Meanwhile, NF-κB (nuclear factor kappa-light chain-enhancer in activated B cells) is activated through the degradation of IκB by TCR signaling and controls the transcription of inflammatory cytokines and genes associated with cell proliferation. Additionally, Activator Protein-1 (AP-1) is activated by MAPK signaling pathways such as ERK and JNK and contributes to the expression of IL-2 and interferon γ (IFN-γ). Furthermore, the STAT family (signaling and transcriptional activators) (including at least seven types: STAT1-4, STAT5A, STAT5B, and STAT56) is activated by signaling through cytokine receptors. For example, STAT3 is activated via IL-6 and IL-10 signaling and regulates the expression of genes associated with inflammatory responses and immunosuppression. STAT5 is activated in response to signals from IL-2 and IL-7 and plays a crucial role in T cell proliferation and survival. As described above, the NFAT, NF-κB, AP-1, and STAT families translocate into the nucleus through different pathways and function cooperatively in the regulation of T cell responses.

[0277] In this specification, the term “degree of oxygen concentration” is an indicator representing the amount of oxygen present in a specific environment or tissue, typically measured as oxygen partial pressure (unit: mmHg) or oxygen saturation (%). In vivo, oxygen concentration is a critical factor that directly influences 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. First, normoxia refers to a state where the oxygen partial pressure is within the normal physiological range; generally, under atmospheric pressure, the oxygen concentration is about 21% and the oxygen partial pressure is about 100 mmHg. This state represents the typical oxygen concentration observed in healthy tissues and organs; for example, the oxygen partial pressure in arterial blood is typically between 80 and 100 mmHg. Within this range, cells efficiently generate energy and physiological functions are maintained normally. Meanwhile, hypoxia refers to a state where the partial pressure of oxygen is below the normal range. For example, in the tumor microenvironment, oxygen supply may be insufficient, and the partial pressure of oxygen can drop to 20 mmHg or lower. Such hypoxic conditions promote tumor growth and metastasis, and also induce resistance to radiation therapy. Furthermore, hypoxia is known to occur at high altitudes (above 3,000 meters above sea level) and cause acute mountain sickness. Next, hyperoxia is a state where the partial pressure of oxygen exceeds the normal range. In hyperbaric oxygen therapy, this hyperoxic state is utilized, and oxygen supply to tissues is increased by raising the partial pressure of oxygen. This method can be used for wound healing and the treatment of infectious diseases, and as a specific example, it is expected to be effective in the treatment of diabetic foot ulcers.Finally, extreme hypoxia refers to a state where oxygen is almost or completely absent. For example, in cases of cardiac arrest or severe ischemia (insufficient blood flow), oxygen supply is completely cut off, depriving tissues of oxygen and ultimately leading to necrosis. Such extreme oxygen deficiency has a severe impact on life. As mentioned above, the degree of oxygen concentration is deeply involved in physiological and pathological processes, and understanding the impact of each condition on bodily function and disease progression is crucial for effective treatment and management.

[0278] In one embodiment, the term “tumor microenvironment sensing element” refers to an element that senses changes in tumor microenvironment conditions, and refers to a molecule or structure that senses specific conditions or stimuli of the tumor microenvironment (TME) (e.g., hypoxic conditions, acidification, nutrient deficiency, inflammatory cytokines, changes in metabolites, etc.) and transmits the information to cells. These elements function as molecular mechanisms for tumor cells, surrounding immune cells, fibroblasts, vascular endothelial cells, etc., to adapt to the tumor microenvironment and contribute to tumor progression, metastasis, and additionally, therapeutic resistance. The tumor microenvironment condition sensing element plays a role in sensing specific environmental conditions and inducing an adaptive response of tumor cells in response. Examples of key conditions and corresponding sensing elements are shown below.

[0279] First, under oxygenated conditions (hypoxia), Hypoxia-Inducible Factor (HIF) functions as a sensing element. This element detects the hypoxic environment and induces tumor cell metabolism and the expression of vascular endothelial growth factor (VEGF). In particular, HIF-1α plays a central role in tumor cells adapting to hypoxic conditions. Next, under acidification (pH decrease), pH sensors such as proton-sensitive ion channels and carbonic anhydrase IX (CAIX) function as sensing elements. They detect the acidic environment of the tumor and promote tumor cell survival by regulating intracellular and extracellular pH balance. Under nutrient-deficient conditions (deficiency of glucose or amino acids), the mTOR pathway and AMPK (AMP-activated protein kinase) function as sensing elements. They play a role in maintaining cell survival by detecting nutrient deficiencies and regulating energy metabolism. Regarding inflammatory cytokines (e.g., IL-6, TNF-α), cytokine receptors (e.g., IL-6R, TNF receptors) and the NF-κB signaling pathway act as sensing factors. These detect the inflammatory environment, promote tumor cell proliferation, and induce the expression of immunosuppressive factors. Additionally, regarding changes in metabolites (e.g., accumulation of lactate), monocarboxylic acid transporters (MCTs) function as sensing factors. This enables the detection of metabolites such as lactate and promotes the survival and proliferation of tumor cells. Furthermore, in response to oxidative stress (increased ROS), Nuclear factor erythroid 2-related factor 2 (NRF2) functions as a sensing factor. NRF2 plays a role in protecting tumor cells by detecting reactive oxygen species (ROS) and activating antioxidant responses. These condition-sensing factors provide important mechanisms for tumor cells to adapt to the microenvironment and are deeply involved in tumor progression and treatment resistance.

[0280] A nucleic acid sequence encoding a glucose transporter operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, having this configuration, plays a role in responding to changes in tumor microenvironment conditions and regulating glucose transport by including the following important components. First, the sequence of the sensing element has a function that responds to specific conditions of the tumor microenvironment (e.g., hypoxia, acidification, nutrient deficiency). Specifically, the sequence includes a portion encoding a HIF-binding domain corresponding to hypoxic conditions and a protein region that functions as a pH sensor detecting acidification. This sequence enables the detection of specific changes in the tumor microenvironment. Next, the sequence of the glucose transporter encodes glucose transporters (e.g., GLUT1, GLUT3) that serve to transport glucose across the cell membrane. This sequence enables efficient glucose transport between the inside and outside of the cell and meets the metabolic demands of the tumor cell. Furthermore, the sequence of the linkage region encodes a linker region that functionally links the sensing element and the glucose transporter. This linker region provides flexibility and stability, enabling the sensing element and glucose transporter to work cooperatively. However, metabolic adaptation of tumor cells can be blocked by targeting the function of the protein encoded by this nucleic acid sequence. Specifically, to inhibit tumor adaptation, the energy supply to tumor cells is cut off by detecting changes in the tumor microenvironment, such as hypoxia or acidification, and inhibiting glucose transport based on this. Additionally, to block environmental responsiveness, tumor cells are prevented from adapting to harsh environmental conditions by preventing the detection of changes in the tumor microenvironment through the sensing element. Through such functional inhibition, therapeutic strategies that effectively inhibit the survival and growth of tumor cells can be realized.

[0281] Examples of tumor microenvironment sensing elements include, but are not limited to, nucleic acid sequences encoding (i) an inducible activation motif (HIH-RE, NFAT-RE, etc.) that is activated in response to changes in tumor microenvironment conditions (deterioration, etc.) and (ii) a glucose transporter degradation factor (ODD, etc.) that is inactivated in response to changes in tumor microenvironment conditions (deterioration, etc.).

[0282] In this specification, the term “inducible activation motif responsive to changes in the tumor microenvironment” refers to a molecular motif that controls signaling that is selectively activated in response to changes in specific conditions of the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deficiency, inflammatory signals, increased oxidative stress, etc.). This motif is involved in the regulation of metabolism, growth, and immune responses in tumor cells and surrounding cells, and influences tumor progression, metastasis, and treatment resistance. Inducible activation motifs function in specific processes of intracellular signaling pathways and alter gene expression or metabolic pathways in response to the deterioration of tumor microenvironment conditions (e.g., progression of hypoxia or nutrient deficiency). This enhances the ability of tumor cells to adapt to harsh environmental conditions.

[0283] In this specification, “a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to a decrease in oxygen concentration binds (hypoxia-responsive nuclear translocation and activation motif)” refers to a DNA sequence or protein motif to which a factor that is activated in response to a decrease in oxygen concentration (hypoxic state, hypoxia) and translocates into the nucleus binds, as a specific factor (e.g., HIF-1α). This motif functions as a molecular switch regulating gene expression under hypoxic conditions and plays a crucial role in the adaptation of tumor cells and normal cells to hypoxic environments. This inducible activation motif controls the transcription of hypoxia-responsive genes (e.g., angiogenesis factors, metabolic enzymes, and oxidative stress tolerance factors), enhances cellular adaptive capacity, and is deeply involved in cancer progression and treatment resistance.

[0284] In this specification, the term “inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to T cell activation binds (inducible activation motif bound by a nuclear translocation factor 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 by T cell receptor (TCR) stimulation or signaling of a co-stimulator molecule specifically binds. This motif serves to receive signals related to T cell activation in the nucleus and to induce gene expression and transcriptional regulation. The inducible activation motif functions as a central molecular mechanism controlling T cell proliferation, differentiation, cytokine production, and the expression of effector functions.

[0285] In this specification, the term “tumor microenvironment-responsive glucose transporter inactivation or degradation factor (glucose transporter degradation factor responding to changes in the tumor microenvironment)” refers to a molecule or motif that serves to inactivate or degrade glucose transporters (GLUTs) in response to changes in specific conditions of the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deficiency, oxidative stress, etc.). These degradation factors play a role in inhibiting the metabolic activity of tumor cells and hindering tumor growth and progression, particularly by controlling excessive glucose transport and metabolic adaptation. Specific examples include oxygen-dependent degradation domains (ODDs), which regulate the stability of target proteins according to environmental conditions.

[0286] In this specification, the term “nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to T cell activation binds (inducible activation motif bound by a nuclear translocation factor in response to T cell activation)” refers to a DNA sequence or protein motif to which a transcription factor, which is activated in response to T cell receptor (TCR) stimulation or signaling by a co-stimulator, binds and translocates into the nucleus, as a specific transcription factor (e.g., NFAT, NF-κB, AP-1, etc.). This motif plays a role in controlling gene expression related to T cell activation and regulating T cell proliferation, differentiation, cytokine production, and the expression of effector functions. This inducible activation motif is a central molecular mechanism regulating the immune response and is an important transcriptional regulatory element in T cell responses. It primarily comprises the following components. First, the motif includes a nuclear translocation factor-binding site. Transcription factors that translocate into the nucleus upon T cell activation (e.g., NFAT, NF-κB, AP-1) specifically bind to this site and convert signals into gene transcription. Second, the motif includes an environment-responsive region that is dynamically activated in response to TCR stimulation or signals from co-stimulatory molecules. This region is characterized by not being activated in the resting state. Third, the motif possesses transcriptional regulatory functions. The motif promotes or inhibits the transcription of target genes through the binding of nuclear translocation factors. This motif has various functions. First, the motif induces gene expression. The motif regulates the gene expression of cytokines and effector molecules such as IL-2, IFN-γ, and TNF-α. Next, the motif regulates immune responses and determines the quality and intensity of adaptive immune responses by controlling T cell proliferation, differentiation, and effector functions. Furthermore, the motif is involved in the promotion or inhibition of inflammatory responses and the formation of immune memory, and regulates the function of the entire immune system.Specific examples include the NFAT-binding motif present in the IL-2 promoter region, the NF-κB response element in the TNF-α promoter region, and the region to which AP-1, activated via the MAPK pathway, binds. These motifs each regulate important gene expression associated with T cell activation. Various applications for these motifs are being considered. In cancer immunotherapy and the treatment of autoimmune diseases, the development of drugs targeting this inducible activation motif is anticipated. Furthermore, in gene therapy and synthetic biology, it is possible to design artificial systems that regulate gene expression under T cell-specific conditions using this motif. Moreover, analysis of the activation state of the motif is also 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 No. 1)), the STAT-binding sequence (TTCNNNGAA), or modified sequences thereof.

[0287] In this specification, the term “nuclear translocation factor” is a collective term for molecules that are activated in response to extracellular stimuli or intracellular signals and function to regulate the transcription of target genes by translocating from the cytoplasm into the nucleus. These factors are activated by specific signaling pathways, cross the nuclear membrane via nuclear localization signals (NLS), and play a role in regulating gene expression by binding to DNA sequences or other transcription factors. Examples of nuclear translocation factors that may be used in this specification include NFAT, NF-κB (nuclear factor-kappa B), and the STAT transcription factor family (seven types: STAT1-4, STAT5A, STAT5B, and STAT56).

[0288] In this specification, the term “combination of nuclear translocation factors and nuclear translocation factor-binding inducible activation motifs” refers to a combination of a transcription factor (nuclear translocation factor) that is activated and translocates into the nucleus in response to extracellular or intracellular stimuli (e.g., T cell activation, hypoxic conditions, inflammatory signals, etc.) and a DNA sequence or protein motif to which the transcription factor specifically binds (nuclear translocation factor-binding inducible activation motif). This combination serves to precisely regulate gene expression in response to specific stimuli and to control cellular function and adaptation.

[0289] In this specification, this combination is responsible for the coordination between signaling pathways and the control of gene expression, and is important in biological processes such as immune responses, inflammation, metabolic regulation, and cell differentiation. It is also known to play a pathological role in various pathological conditions, including cancer and autoimmune diseases. Examples of key combinations are as follows.

[0290] NFAT-binding motif of NFAT and IL-2 promoter

[0291] Nuclear Translocation Factor: NFAT (Nuclear Factor of Activated T Cells)

[0292] Binding motif: NFAT response element of the IL-2 promoter

[0293] Function: In response to T cell activation, NFAT translocates into the nucleus, induces transcription of the IL-2 gene, and promotes T cell proliferation and immune response.

[0294] NF-κB response motifs of NF-κB and TNF-α promoters

[0295] Nuclear translocation factor: NF-κB (nuclear factor kappa-light chain-enhancer in activated B cells)

[0296] Binding motif: NF-κB response element of the TNF-α promoter (e.g., κB motif (GGGACTTTCC))

[0297] Function: In response to inflammatory signals (e.g., cytokine stimulation), NF-κB translocates into the nucleus, induces the expression of TNF-α, and regulates the inflammatory response.

[0298] HIF-1α and HRE (hypoxia-response factor)

[0299] Nuclear translocation factor: HIF-1α (hypoxia-inducible factor-1α)

[0300] Binding motif: HRE (Hypoxia-reactive element)

[0301] Function: In response to hypoxic conditions, HIF-1α translocates into the nucleus and induces the transcription of angiogenic factors (e.g., VEGF).

[0302] STAT Family and Cytokine Response Factors

[0303] Nuclear translocation factor: STAT3 (Signal transduction and transcriptional activator factor 3)

[0304] Binding motif: STAT-binding site of cytokine-responsive gene (e.g., STAT-binding sequence (TTCNNNGAA))

[0305] Function: In response to stimulation by cytokines (e.g., IL-6), STAT3 translocates into the nucleus and induces immunomodulation and inflammatory responses.

[0306] AP-1 and growth factor response motifs

[0307] Nuclear translocation factor: AP-1 (activated protein-1)

[0308] Binding motif: AP-1 response element within the promoter of a gene related to cell proliferation or differentiation

[0309] Function: AP-1 activated via the MAPK pathway translocates into the nucleus and promotes cell proliferation and differentiation.

[0310] In this specification, the term “factor whose activity of degrading glucose transporters is inactivated in response to a decrease in oxygen concentration (factor whose activity of degrading glucose transporters is inactivated in response to hypoxia)” refers to a factor that inhibits the activity of a system inducing the degradation of glucose transporters (e.g., GLUT1, GLUT3) in response to a decrease in oxygen concentration (hypoxic state, hypoxia) and plays a role in increasing the stability of glucose transporters. This factor acts through a structure such as an oxygen-dependent degradation domain (ODD) to inhibit the degradation of glucose transporters under hypoxic conditions, thereby enabling cells to maintain the energy supply required by the cell. Furthermore, in the tumor microenvironment, this factor plays an important role in promoting metabolic adaptation of tumor cells and contributing to their survival and proliferation. The following is a description of the components and characteristics of the factor whose activity of degrading glucose transporters is inactivated in response to a decrease in oxygen concentration. First, a hypoxia-responsive sensor region may be mentioned. This region plays a role in detecting a decrease in oxygen concentration and emits degradation-inhibitory signals under hypoxic conditions. A representative example is Hypoxia-Inducible Factor-1α (HIF-1α), which stabilizes under hypoxic conditions and regulates the activity of degradation factors. Next, the Oxygen-Dependent Degradation Domain (ODD) is discussed. This region possesses the characteristic of regulating degradation depending on oxygen concentration. Typically, when oxygen concentration is high, ubiquitination is induced through this region, and degradation proceeds. On the other hand, under hypoxic conditions, degradation activity is inactivated, and the stability of glucose transporters increases. Additionally, degradation-inhibitory factors are also important components. These factors inhibit degradation activity mediated by ubiquitination or the proteasome system and help stabilize glucose transporters. Specific examples include the regulation of pVHL (von Hippel-Lindau protein) activity. Furthermore, transcriptional and translational regulatory functions are also included.This factor plays a role in preventing degradation and ensuring stability at the cell membrane by regulating the transcription and translation of glucose transporters under hypoxic conditions. These components cooperate with each other, enabling the inhibition of degradation activity in response to changes in oxygen concentration and playing an important role in allowing the cell to maintain energy supply even in a hypoxic environment.

[0311] As described above, the phrase “expression and / or enhancement as needed is specifically achieved in the tumor environment (expression and / or enhancement as needed specifically achieved in the tumor microenvironment)” refers to a phenomenon in which the expression of a specific gene or protein and / or the enhancement of its function are induced in response to characteristic conditions of the tumor microenvironment (TME) (e.g., hypoxia, acidification, nutrient deficiency, presence of inflammatory cytokines, etc.), and expression in normal tissues outside the tumor is controlled to be minimal or absent. This phenomenon is important for developing tumor-specific therapeutic targets and designing therapeutic strategies with reduced side effects. Tumor environment-specific regulatory mechanisms enable the enhancement of therapeutic effects while minimizing impact on normal tissues. The key characteristics of the phenomenon in which expression and / or enhancement as needed is specifically achieved in the tumor environment are explained below. First, one characteristic of this phenomenon is environment-dependent regulation. This refers to a mechanism in which the expression of a gene or protein is induced or enhanced in response to specific conditions of the tumor microenvironment, such as hypoxic conditions, acidic pH, nutrient deficiency, or inflammatory signals. Specific examples include cases where gene expression is induced by HIF-1 (hypoxia-response factor) under hypoxic conditions. Next, this phenomenon is characterized by tumor specificity. This indicates that while these genes or proteins are rarely or only expressed at extremely low levels in normal tissues outside the tumor, their expression or function is specifically activated within the tumor microenvironment. Furthermore, the characteristic of being on demand is also important. Expression or enhancement is regulated as needed when specific conditions are met due to changes in the tumor environment, such as a decrease in oxygen concentration or an increase in inflammatory cytokines. This characteristic enables a mechanism in which function is efficiently exerted only within the tumor environment. Finally, this phenomenon possesses suitability as a therapeutic target. Genes or proteins that are specifically expressed or enhanced in tumors can be used as targets for cancer therapy.By utilizing these properties, therapeutic strategies can be designed to enhance the therapeutic effect on tumors while minimizing side effects on normal tissues. Due to these characteristics, the mechanism by which expression and / or enhancement are specifically achieved within the tumor environment when needed is a highly promising element in novel approaches to cancer therapy. By leveraging these properties, the development of safer and more effective therapies is anticipated.

[0312] In this specification, with respect to genes (nucleic acids, proteins, etc.), the term “modification” refers to inserting a nucleotide sequence of nucleic acid within a cell, changing a nucleotide sequence of nucleic acid, partially deleting a nucleotide sequence of nucleic acid within a cell, or a combination thereof.

[0313] In this specification, the term “disease” is interpreted in its broadest sense and refers to a state in which discomfort or unease occurs in the mind or body of a human or animal, and refers to any state that cannot be considered healthy, such as diseases, illnesses, and various symptoms, even if not specifically defined. Diseases that may be the target of this disclosure include, but are not limited to, diseases in which an immune response may be involved, e.g., cancer, autoimmune diseases, allergies, and infectious diseases.

[0314] In this specification, the phrase “the subject has an immune response” to a specific component or substance means that some immune response occurs to said component or substance. In the subject or a biological component derived from the subject (e.g., cells, etc.), this may be determined by observing changes in various immune cells or increases and decreases in immune-related substances (e.g., cytokines, etc.), judged by objective indicators, or judged subjectively based on the experience of a physician, etc.

[0315] Whether a subject possesses “immune memory” in relation to a specific component or substance can be assessed by measuring whether the component or substance in the subject or a biological component derived from the subject (e.g., cells, etc.) (i) antigen-dependently enhances cytokine production or promotes proliferation in memory CD4-positive T cells, (ii) alters the expression of surface antigens on memory regulatory T cells, (iii) alters the Treg-to-Th1 ratio, (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, (vii) alters the ability to produce TNF-α, or (viii) the subject possesses antibodies specific to the component or substance in the blood, and confirming that at least one of these results in a positive outcome.

[0316] In this specification, “infectious disease” may be any infectious disease including any type of infectious disease such as viral infectious disease (including any viral form such as single-stranded or double-stranded DNA virus or RNA virus), bacterial infectious disease, protozoal infectious disease or mycoplasma infectious disease, e.g. tuberculosis, coronavirus infection, malaria, yellow fever virus, smallpox virus, vaccineinia, measles / rubella, polio, epidemic parotitis / mumps, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcus infection, whooping cough, Japanese encephalitis, meningococcal infection, salmonella infection, pathogenic E. coli E. coli It may be ), toxoplasma, Zika virus, herpes virus type 1, EBV / Epstein-Barr virus (herpes virus type 4), CMV / cytomegalovirus (herpes virus type 5), influenza, MARS, rabies, and diphtheria.

[0317] In this specification, the term “immune abnormality” refers to any disease, condition, or state that is at least partially induced or suspected to be induced by an abnormality of the immune system. An immune abnormality refers to a state in which an abnormality in immunity occurs due to some cause, making the subject susceptible to infectious diseases or causing allergic reactions. Examples of immune abnormalities include, but are not limited to, allergies and autoimmune diseases. When it concerns self-antigens, it is generally referred to as an autoimmune disease, and when it concerns external antigens, it is referred to as an allergy. When the immune response is strong, it can be said that an autoimmune disease state occurs regarding self-antigens and an allergic state occurs regarding non-self-antigens, whereas when the immune response is weak, it can be said that an cancer state occurs regarding self-antigens and an infectious disease occurs regarding non-self-antigens.

[0318] In this specification, the term “autoimmune disease” refers to an immune response that occurs excessively against specific autoantigens. An autoimmune disease can be defined as a disease caused by a breakdown of immune tolerance in which the immune system, which is responsible for recognizing and eliminating foreign substances, overreacts and attacks its own normal cells and tissues to cause symptoms. Examples of autoimmune diseases or conditions include, but are not limited to: inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel diseases (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 (SLE) (including, but not limited to, lupus nephritis and cutaneous lupus);Diabetes (e.g., type I diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome (Sj; Gren's syndrome); juvenile-onset diabetes; immune responses associated with acute and delayed-type hypersensitivity mediated by cytokines and T lymphocytes, typically observed in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte leakage; inflammatory diseases of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; Antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disorder; Reiter's disease; stiff-man syndrome; Behcet's disease et's disease); giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP), autoimmune uveitis, or autoimmune thrombocytopenia.

[0319] In this specification, the term “allergy” refers to an immune response that occurs excessively to a specific non-autoantigen and is a disease in which an immune response occurs to an “allergen.” “Allergen” refers to an antigen capable of reacting with antibodies of a subject having an allergic disease. Examples of this include allergens derived from tree pollen (acacia, alder, velvet ash, European beech, birch, maple, mountain cedar, red cedar, poplar, cypress, American elm, Chinese elm, Douglas fir, rubber tree, eucalyptus, hackberry, hickory, American linden, sugar maple, mesquite, paper mulberry, oak, olive, pecan, black pepper, pine, privet, Russian olive, American plane tree, leatherleaf, black walnut, black willow, etc.), and pollen from grasses and plants (cotton, Bermuda grass, Kentucky bluegrass, bromegrass, corn, meadow fescue, Johnson grass, oats, orchardgrass, redtop, ryegrass, rice, fragrant grass, timothy, amaranth, goosefoot, burdock, dock, goldenrod, kochia, white goosefoot, calendula, Allergens derived from nettle, barnyard grass, plantain, Japanese knotweed, ragweed, false ragweed, Russian thistle, mugwort, broom grass, small sorrel, etc.), allergens derived from insects (silkworms, mites, honeybees, wasps, ants, cockroaches, etc.), fungi (alternaria ( Alternaria ), Aspergillus( Aspergillus ), botulinum ( Botulinum ), Candida( White), Cephalosporium( Cephalosporium ), curbularia( Curved ), Epicocum( Epicoccum ), epidermophytone( Epidermophyton ), Fusarium( Fusarium ), Helminthosporium( Helminthosporium ), Cladosporium herbarum( Cladosporium herbarium ), Mucor( I am sick. ), Penicillium( Penicillium ), Poma( Food ), Flularia fluluance ( Sprouting sprout ), Rhizopus( Rhizopus Allergens derived from (e.g., ) etc.), allergens derived from animal hair (dogs, cats, birds, etc.), allergen proteins derived from house dust, food allergens (OVA, etc.), etc., are included but not limited thereto. Representative diseases of “allergy” include atopic dermatitis, allergic rhinitis (pollinosis, etc.), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergy, drug allergy, urticaria, etc.

[0320] (Desirable embodiment)

[0321] Preferred embodiments of the present disclosure are 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 descriptions below. Accordingly, it is evident that those skilled in the art may make appropriate modifications within the scope of the present disclosure in consideration of the descriptions in this specification. Furthermore, the following embodiments of the present disclosure may be used alone or in combination.

[0322] In one aspect, the present disclosure provides immune cells, such as T cells, having enhanced glucose uptake capacity when necessary, a cell population comprising such cells, a pharmaceutical composition comprising such cells or a cell population, and other related technologies. As such immune cells, such as T cells, having enhanced glucose uptake capacity when necessary, and as a cell population comprising such cells, typically T cells having enhanced glucose uptake capacity are used, and the T cells have or are modified to have a characteristic in which the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions, or comprise a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0323] In another aspect, the present disclosure provides immune cells such as T cells having enhanced glucose uptake capacity in which the expression of glucose transporters in T cells is regulated in response to changes in tumor microenvironment conditions, cell populations comprising such cells, pharmaceutical compositions comprising such cells or cell populations, and other related technologies.

[0324] In another aspect, the present disclosure provides a T cell comprising a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0325] In one aspect of the present disclosure, a T cell having enhanced glucose uptake capacity (T) is modified to express a glucose transporter when necessary and / or has enhanced expression of a glucose transporter when necessary. effT cells modified to express a glucose transporter when necessary and / or to enhance the expression of a glucose transporter when necessary, T cells in which the expression of a glucose transporter is regulated in response to changes in tumor microenvironment conditions, or T cells comprising a nucleic acid sequence encoding a glucose transporter and in which the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions have not been previously considered. In T cells with constitutively enhanced glucose uptake capacity, differentiation into effector memory T cells and exhaustion were observed, and significant T cell death was induced when the T cells were activated under glucose-free to low-glucose conditions. In contrast, T cells in which the expression of a glucose transporter is enhanced when necessary are distinguished in that T cell death under glucose-free to low-glucose conditions is avoided while equivalent cytotoxic activity is maintained, and the maintenance of a memory phenotype and resistance to exhaustion are induced, which are expected to maintain effects for a longer period. It is advantageous for the cells of the present disclosure to have enhanced glucose uptake capacity. These cells, whose glucose absorption capacity is enhanced when needed, exhibit remarkable effects against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied in the form of CAR-T cells, TCR-T cells, etc.

[0326] Furthermore, the cells of the present disclosure exhibited a significantly high relapse prevention effect when used in CAR-T therapy, etc. Additionally, sudden mouse death due to toxicity caused by non-tumor factors observed in glucose-constitutively enhanced T cells was avoided in cells in which glucose transporters are expressed or enhanced when necessary, in cells in which glucose transporter expression is regulated in response to changes in tumor microenvironment conditions, or in cells containing a nucleic acid sequence encoding a glucose transporter and in which the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. These findings were unexpected and, without being bound by theory, can be explained by the avoidance of host toxicity caused by excessive T cell activation or excessive glucose uptake, a significant reduction in exhaustion, nutrient supply meeting nutritional requirements, inhibition of apoptosis caused by appropriate activation, and induction of memory formation. Additionally, it is desirable for the cells of the present disclosure to have effector functions at the site of function, particularly after introduction into the body. An example of such cells is T eff It includes, but is not limited to, lymphoid progenitor cells, Tnv, Tscm, Temra, etc.

[0327] In one embodiment, in one aspect of the cell of the present disclosure, tumor microenvironment conditions include the degree of transmission of TCR-related signals. In a preferred embodiment, examples of TCR-related signals include a TCR signal or a part thereof, and a chimeric signal of an antigen different from the TCR signal (also called a CAR signal).

[0328] In a preferred embodiment, in one aspect of the cell of the present disclosure, a TCR-related signal is mediated by a molecule comprising a TCR signaling domain. Such a molecule may be a nucleic acid molecule.

[0329] In a preferred embodiment, changes in tumor microenvironment conditions include the presentation of tumor antigens and / or co-stimulating ligands and / or changes in cytokine conditions.

[0330] In one embodiment, the degree of TCR-related signaling used in this disclosure comprises at least one selected from the group consisting of a degree of TCR stimulation, a TCR co-stimulation, and a humoral factor. In a specific embodiment, the degree of TCR-related signaling comprises at least one selected from the group consisting of a degree of TCR stimulation, a TCR co-stimulation, and a humoral factor. In another embodiment, the degree of TCR-related signaling comprises a degree of TCR stimulation. In another embodiment, the degree of TCR-related signaling comprises a degree of TCR stimulation, a TCR co-stimulation, and a humoral factor. One or more of the intensity of TCR stimulation, the influence of co-stimulating molecules, and the action of humoral factors may be combined. TCR stimulation is initiated when a T cell receptor recognizes an antigen peptide bound to the major histocompatibility complex (MHC) on an antigen-presenting cell (APC). The intensity and duration of such recognition significantly influence the degree of signaling. Strong TCR stimulation promotes T cell activation and proliferation, as well as the exercise of effector functions, whereas weak TCR stimulation can lead to partial activation or anergy. Furthermore, the involvement of co-stimulatory molecules is essential for TCR-mediated signaling. Co-stimulatory molecules play a role in providing secondary signals necessary for T cell activation and amplifying TCR signals. For example, CD28 is a stimulating co-stimulator that enables complete T cell activation, whereas the inhibitory CTLA-4 acts to suppress the immune response. The presence of appropriate co-stimulatory signals enhances the immune response and enables the formation of memory T cells and a sustained immune response. On the other hand, in the absence of co-stimulation, T cells enter a state of immune tolerance, and excessive immune responses to autoantigens are suppressed. Additionally, humoral factors, particularly cytokines, play a crucial 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 act to suppress the immune response.Furthermore, specific cytokines secreted in an inflammatory environment, such as IL-12 and IL-4, induce differentiation into Th1 and Th2 cells, respectively, and determine the nature of the immune response. As described above, humoral factors fine-tune the immune response and enable an effective response adapted to pathogens. The immune response is adaptively and flexibly regulated by TCR stimulation, co-stimulation, or humoral factors alone, or through the interaction of multiple such factors. This enables rapid and specific immune defense against pathogens while simultaneously maintaining the overall balance of the immune system, such as preventing autoimmune diseases and preserving immune tolerance. This mechanism is an essential element for T cells to exhibit optimal responses to the environment and stimuli, and underpins the diversity and functionality of the immune system.

[0331] In certain embodiments, TCR stimulation may be considered to include NFAT, NF-κB (nuclear factor-kappa-B), STAT transcription factor family (including but not limited to seven types of STAT1-4, STAT5A, STAT5B, and STAT56) that translocate into the nucleus in relation to T cell activation.

[0332] In another embodiment, tumor microenvironment conditions include the degree of oxygen concentration. The degree of oxygen concentration in the tumor microenvironment is a critical factor that significantly influences tumor progression and therapeutic response. In tumor tissue, oxygen supply is often insufficient compared to normal tissue due to the abnormal formation of blood vessels and structural heterogeneity; this condition is referred to as "hypoxia." Hypoxia has various effects on the metabolism, proliferation, immune evasion, and treatment resistance of tumor cells. First, tumor cells induce the activation of hypoxia-inducible factors (HIFs) to adapt to the hypoxic state. HIFs promote the expression of angiogenic factors (such as VEGF) in tumor cells and induce the formation of new blood vessels; however, these new vessels possess abnormal structures and become factors that cause additional hypoxia. HIFs also alter the metabolic pathways of tumor cells and promote the "Warburg effect," in which glycolytic pathways that do not require oxygen are prioritized. These metabolic changes not only maintain tumor cell proliferation but also play a role in creating an acidic environment and suppressing the activity of immune cells.

[0333] In one aspect of using a glucose transporter operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions, in one embodiment, a glucose transporter connected to a tumor microenvironment sensing element that detects characteristics such as oxygen concentration, pH, redox status, and inflammatory cytokines in the tumor microenvironment is an innovative subject of research utilizing tumor-specific metabolic properties. Such a transporter can be used for treatment or diagnosis by responding to changes in the tumor microenvironment and specifically engaging in the energy supply and metabolic pathways of tumor cells. For example, by connecting a response element of hypoxia-inducible factor (HIF-1), which is specifically activated in a hypoxic environment, to the glucose transporter, a mechanism is designed in which glucose uptake increases in a hypoxic environment. Such a transporter can be applied to the development of drug delivery or diagnostic probes that target hypoxic conditions. Furthermore, since the tumor environment is characterized by a decrease in pH, a transporter that is specifically activated in the acidic environment where tumor cells are present can be constructed by introducing an element that detects an acidic environment into the glucose transporter. This mechanism contributes to the design of therapies specialized for acidic environments. In addition, designs in which redox sensing elements are linked to glucose transporters are considered to respond to the redox state of the tumor microenvironment. Since tumor cells are often exposed to higher oxidative stress than normal cells, these transporters function specifically in high-oxidative-stress environments and are useful as therapeutic or diagnostic tools that target tumor cells. Furthermore, by introducing inflammatory cytokine (e.g., IL-6 or TNF-α) response elements, it is possible to develop transporters that sense inflammatory states in the tumor microenvironment and regulate glucose transport in response. This is expected to enable the identification of inflammatory tumors and improve therapeutic efficacy.As described above, a glucose transporter connected to a tumor microenvironment sensing element is a technology that can serve as a basis for performing diagnosis and treatment more effectively and specifically by utilizing a tumor-specific metabolic environment.

[0334] In one embodiment, the tumor microenvironment sensing element comprises a nucleic acid sequence encoding (i) an inducible activation motif (e.g., HIH-RE, NFAT-RE) that is activated in response to a change (deterioration, etc.) in the tumor microenvironment conditions, and (ii) a degradation factor for a glucose transporter (e.g., ODD) that is inactivated in response to a change (deterioration, etc.) in the tumor microenvironment conditions.

[0335] In one embodiment, the tumor microenvironment sensing element comprises: (i) an inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to T cell activation binds (e.g., NFAT-RE); (ii) a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus binds in response to a decrease in oxygen concentration; and (iii) a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading glucose transporters is inactivated in response to a decrease in oxygen concentration.

[0336] In another embodiment, the nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nucleic acid sequence encoding an inducible activation motif that is activated in response to changes in tumor microenvironment conditions and / or a degradation factor for the glucose transporter that is inactivated in response to changes in tumor microenvironment conditions.

[0337] In another embodiment, the nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to T cell activation binds.

[0338] In another embodiment, nuclear translocation factors that can be used include NFAT, NF-κB (nuclear factor-kappa-B), STAT transcription factor family (STAT1-4, STAT5A, STAT5B, STAT56, etc.).

[0339] In certain embodiments, the nuclear translocation factor-binding inducible activation motif may be an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ No. 1), a STAT-binding sequence (TTCNNNGAA), or a modified sequence thereof. The modified sequence may include one, two, or three or more substitutions (interchanges of ATGC) compared to the original sequence.

[0340] In a specific embodiment, the combination of a nuclear transpotential factor and a nuclear transpotential factor-binding inducible activation motif comprises the following:

[0341] (1) A combination of NFAT and NFAT-combined domains;

[0342] (2) A combination of NF-κB (nuclear factor-kappa-B) and κB motif (GGGACTTTCC (SEQ No. 1)); or

[0343] (3) A combination of STAT transcription factor family (7 types of STAT1-4, STAT5A, STAT5B and STAT56) and STAT-binding sequence (TTCNNNGAA).

[0344] In a specific embodiment, the nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to a decrease in oxygen concentration binds. In this embodiment, the nuclear translocation factor may be HIF-1, etc. In this embodiment, the nuclear translocation factor-binding inducible activation motif may be HIF-RE, etc.

[0345] In a specific embodiment, the combination of the nuclear translocation factor and the nuclear translocation factor-binding inducible activation motif may be, for example, a combination of HIF-1 and HIF-RE.

[0346] In another embodiment, a nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter is deactivated in response to a decrease in oxygen concentration. In this embodiment, the factor being deactivated may be, for example, an oxygen-dependent degradation factor (ODD).

[0347] In a specific embodiment, the T cell of the present disclosure comprises a chimeric antigen receptor (CAR).

[0348] In another embodiment, the T cell has an effector function. Alternatively, the cell is an effector T cell (T eff It is a precursor cell of ).

[0349] In a specific embodiment, the memory function of the effector T cell precursor cells of the present disclosure is extended. The characteristic of extending the memory function in the effector T cell precursor cells has a significant advantage in improving the sustainability and efficacy of the immune response. This characteristic is described below. The effector T cell precursor cells possess the ability to perform effector functions in response to antigen stimulation, while also having the potential to differentiate into memory T cells. Therefore, immune memory regarding the antigen can be maintained for a long period even after the initial immune response has subsided. In particular, the extension of memory function is advantageous in that it enhances the ability to induce a rapid and strong immune response upon reinfection. First, the extension of memory function strengthens immune defense against tumors, etc. 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, etc., will be eliminated before they spread within the body and can prevent severe diseases. Furthermore, the extension of memory function also serves as a factor that enhances the effectiveness of vaccines. If effector T progenitor cells formed after vaccination maintain long-term memory function, the duration of immune memory is extended, making it possible to reduce the frequency of additional vaccinations (booster vaccinations). This also contributes to improving the efficiency of vaccination strategies and reducing the burden on patients. In the field of tumor immunology, the memory function of effector T progenitor cells is important. While tumor cells often hide antigens or create an immunosuppressive environment, the presence of T cells with long-term memory is expected to maintain immune surveillance against tumor cells and reduce the risk of recurrence. In particular, when combined with immune checkpoint inhibitors or CAR-T cell therapy, a more sustained and potent anti-tumor effect can be achieved. Furthermore, even in conditions such as chronic infections and autoimmune diseases, effector T progenitor cells with memory function contribute to maintaining appropriate immune responses and regulating pathological conditions.It is expected that long-term monitoring and regulation of specific pathogens or abnormal cells will become possible, while unnecessary inflammation and tissue destruction will also be suppressed. Overall, the characteristic of effector T cell precursors to extend memory function offers significant advantages in the treatment of infectious diseases and tumors, as well as in vaccine development. By utilizing these characteristics, it becomes possible to induce more sustained and effective immune responses, which is expected to contribute to the advancement of medicine and biotechnology.

[0350] In another embodiment, the memory T cell phenotype is maintained in effector T cell precursors. There are many advantages to effector T cell precursors maintaining the memory T cell phenotype. These characteristics are crucial for improving the efficiency of long-term immune memory and adaptive immune responses. First, when effector T cell precursors maintain the memory T cell phenotype, a rapid and efficient immune response becomes possible upon reinfection. Memory T cells maintain antigen-specific memory and are rapidly activated to perform effector functions when the antigen re-enters the body. As a result, tumors and other pathogens are effectively eliminated before they spread, and severe diseases can be prevented. Maintaining the memory T cell phenotype improves the sustainability of the immune response. While effector T cells have a short lifespan, memory T cells remain in the body for a long time, so immune memory formed after vaccination or natural infection is maintained for an extended period. These characteristics are particularly advantageous in terms of reducing the frequency of additional vaccinations (booster vaccinations) and providing continuous immune defense. In tumor immunity, maintaining the memory T cell phenotype is important. While tumor cells often evade the surveillance of the immune system by utilizing antigenic mutations or immunosuppressive environments, T cells possessing long-term immune memory continuously maintain a surveillance function against tumor cell recurrence and metastasis. This reduces the risk of recurrence after treatment and increases the potential for improved patient prognosis. Furthermore, this contributes to the control of chronic infections. For example, regarding pathogens such as tuberculosis and HIV, sustained immune memory is crucial for inhibiting infection progression and managing the pathology. Maintaining a memory T cell phenotype enables long-term surveillance and control against pathogens. Additionally, applications in the management of autoimmune diseases and immunotherapy are anticipated. If the immune response can be appropriately regulated while maintaining the memory T cell phenotype, it is possible to induce a selective immune response against pathogens or abnormal cells while suppressing excessive immune responses.Overall, the maintenance of the memory T cell phenotype by effector T cell progenitors offers significant advantages in the treatment or prevention of infectious diseases, tumors, and chronic diseases. By understanding and utilizing these characteristics, the development of more effective and sustainable immunotherapy and vaccine strategies is expected.

[0351] In a specific embodiment, effector T cells are activated or the activation of effector T cells is maintained, and the memory function of the effector T cell precursor is extended or the memory T cell phenotype of the effector T cell precursor is maintained. Both the extension of memory function and the maintenance of the memory T cell phenotype can be achieved. In this case, the effect can be synergistically enhanced.

[0352] The present disclosure is effective against all tumors, including solid tumors. Although clinical trials of CAR-T cells against solid tumors have not demonstrated efficacy to date, the inventors have discovered that CAR-T cell dysfunction in solid tumors is caused by glucose deficiency in the tumor microenvironment (TME) or equivalent tumor microenvironment conditions (also referred to as TMEC), and have shown that metabolic supplementation as needed, rather than continuous metabolic supplementation, significantly improves the anti-tumor effects of CAR-T cells. Mass consumption of glucose by cancer cells reduces glucose levels in the TME of solid tumors, and consequently impairs CAR-T cell function. The inventors have discovered that when a glucose transporter (GLUT, e.g., GLUT3), a high-affinity glucose transporter, is stably expressed in CAR-T cells, cytokine production and cytotoxic activity are restored. However, while CAR-T cells stably expressing GLUT induced tumor reduction in solid tumor models, its overactivation resulted in adverse events and death in mice. CAR-T cells to which GLUT was introduced as needed exhibited improved metabolic compatibility and increased antitumor effects, thereby preventing adverse events. Furthermore, Activated T cell nuclear factor (NFAT), which translocates into the nucleus upon activation by stimulation by the target antigen used as an example, binds to tumor microenvironment sensing elements to induce the transcription of GLUT or inhibit the degradation of GLUT, resulting in organ tumor control in an intracranial human GBM cell xenograft model. The inventors believe that stable metabolic compatibility as needed, such as when exposed to tumor antigens, is a novel concept for maximizing the antitumor effects of CAR-T cells against solid tumors.

[0353] In certain embodiments, the present disclosure indicates that dysfunction of CAR-T cells in GBM is caused by glucose deficiency in the tumor microenvironment (TME), and that metabolic supplementation as needed, rather than continuous metabolic supplementation, significantly improves the antitumor effect of CAR-T cells. Mass consumption of glucose by cancer cells reduces glucose levels in the TME of GBM and consequently impairs the function of CAR-T cells. When GLUT3, a high-affinity glucose transporter, was stably expressed in CAR-T cells, cytokine production and cytotoxic activity were restored. However, while CAR-T cells stably expressing GLUT3 induced tumor reduction in a GBM model, its overactivation resulted in adverse events and death of mice. When necessary, GLUT3 transcription was induced in GLUT3 CAR-T cells by nuclear translocation of activating T cell nuclear factor (NFAT) due to target antigen stimulation, and the cells showed improved metabolic compatibility and increased antitumor effects, resulting in organ tumor control in an intracranial human GBM cell xenograft model while preventing adverse events. The inventors propose that stable metabolic compatibility when necessary, such as when exposed to tumor antigens, is a novel concept for maximizing the antitumor effects of CAR-T cells against solid tumors.

[0354] In one aspect, glioblastoma (GBM), targeted by the present disclosure, is a fatal malignant brain tumor with an annual incidence of 3.19 per 100,000 people. Since the introduction of combination therapy consisting of temozolomide and radiation in 2005 and the development of the tumor treatment regimen (Novo TTF) for primary GBM, the prognosis of GBM patients in clinical practice has improved. However, the 5-year overall survival rate for GBM remains less than 20% [References 1 and 2]. In light of 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 have been actively studied [References 3 to 10]. In clinical trials, some patients treated showed promising results, such as CAR-T cell infiltration into the tumor microenvironment (TME) and reduction of tumor burden, but most patients did not respond to CAR-T cell therapy [Reference 3].

[0355] GBMs possess a metabolically harsh TME, impairing the function of effector T cells, including CD8+ T cells, and exposing adoptive CAR-T cells to the TME. In the brain, nervous system cells such as neurons and glial cells require glucose as an energy source, and the brain consumes 25% of systemic glucose-derived energy [Ref. 11]. Furthermore, aggressive GBM cells consume three times more glucose than normal neurons due to aerobic glycolysis, a metabolic characteristic of cancer cells known as the Warburg effect [Ref. 12]. This leads to glucose depletion in the TME of the GBM [Ref. 13–16]. Meanwhile, naive T cells switch energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to meet increased energy demands. Consequently, strong glucose-mediated metabolic competition occurs between activated T cells and cancer cells in the TME of the GBM. 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 [References 16 to 18]. As a result, GBM cells expressing GLUT3 can survive and proliferate continuously while absorbing sufficient glucose from the TME, whereas activated T cells fail in metabolic competition and their function is impaired [References 19 and 20].

[0356] Considering the significant glucose uptake by GBM cells mediated by high-affinity GLUT3, equivalent or higher GLUT3 expression by CAR-T cells can enable full competition with GBM cells. In the present disclosure, a construct in which SLC2A3 is linked to a CAR was developed to induce GLUT3 expression in CAR-T cells. However, stable expression of GLUT3 induced hyperactivation of CAR-T cells, indicating that effector T cells require sophisticated energy replenishment. Therefore, GLUT3 expression was modified to be induced by glucose requirements (expressed as needed) by utilizing the activation of CAR-T cells associated with target antigen exposure in the TME. In CAR-T cells with GLUT3 expression as needed, GLUT3 transcription was induced by the nuclear translocation of Activated T Cell Nuclear Factor (NFAT) upon target antigen stimulation, thereby restoring metabolic fitness; consequently, sustained antitumor effects were enhanced and survival was extended in the harsh TME of a GBM model with low glucose levels. In summary, the concept of restoring T cell function in the TME by enhancing metabolic compatibility needs to be studied as a necessary approach rather than as a stable supplement.

[0357] (Cell population)

[0358] In one aspect of the present disclosure, a cell population comprising immune cells, such as T cells, having enhanced glucose uptake capacity as needed is provided. Alternatively, a cell population comprising T cells having enhanced glucose uptake capacity is provided, 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 comprising T cells having enhanced glucose uptake capacity is provided, 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 comprising T cells having a nucleic acid sequence encoding a glucose transporter is provided, wherein the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. When at least some of the T cells included in such a cell population are modified to express a glucose transporter and / or enhanced, it has been unexpectedly shown that significant effects against diseases such as cancer are achieved when the cell population is used as CAR-T cells, etc. (Fig. 19). It is advantageous for the cell population of the present disclosure to have enhanced glucose uptake capacity. In addition, it is preferable that the cell population of the present disclosure comprises cells having effector functions at a site of function, particularly after being introduced into the body. Examples of such cells are T eff It includes, but is not limited to, lymphoid progenitor cells, Tnv, Tscm, Temra, etc.

[0359] In cell populations containing T cells with constitutively enhanced glucose uptake capacity, differentiation into effector memory T cells and exhaustion were observed. In contrast, cell populations containing cells in which glucose transporter expression is enhanced when needed are distinguished in that they induce resistance to exhaustion and the maintenance of a memory phenotype, which is expected to maintain effects for a longer period while maintaining equivalent cytotoxic activity. It is advantageous for the cell population of the present disclosure to be a cell population containing cells having enhanced glucose uptake capacity. Such cell populations containing cells in which glucose uptake capacity is enhanced when needed exert significant effects against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when used as CAR-T cells, TCR-T cells, etc.

[0360] Furthermore, the cell population of the present disclosure exhibited a significantly high relapse prevention effect when used as CAR-T cells, etc. Additionally, sudden mouse death due to toxicity caused by non-tumor factors observed in glucose-constitutively enhanced T cells was avoided in cells in which glucose transporters are expressed or enhanced when necessary, or in cells in which glucose transporter expression in T cells is regulated in response to changes in tumor microenvironment conditions, and which contain a nucleic acid sequence encoding a glucose transporter and in which the nucleic acid sequence is operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. These findings were unexpected and, without being bound by theory, can be explained by the avoidance of host toxicity caused by excessive T cell activation or excessive glucose uptake, a significant reduction in exhaustion, nutrient supply meeting nutritional requirements, inhibition of apoptosis caused by appropriate activation, and induction of memory formation. Additionally, it is desirable for the cell population comprising the cells of the present disclosure to have an effector function at a site of function, particularly after being introduced into the body. An example of such cells is T effIncludes, but not limited to, T eff It may be a precursor cell, for example, lymphoid progenitor cell, Tnv, Tcm, Temra, etc.

[0361] In one embodiment of the present disclosure, in a cell population comprising immune cells such as the T cells described above, it is advantageous for the cell population to comprise T cells having the features of the present disclosure (e.g., enhancement or expression of GLUT, effector function, or both, as needed) in an amount of about 10% or more of the cell population. In one embodiment, in the cell population of the present disclosure, the proportion of T cells having the features of the present disclosure may be 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.

[0362] In one embodiment, the cell population of the present disclosure comprises T cells having characteristics that exhibit effector functions when introduced into the body.

[0363] In one embodiment, in the cell population of the present disclosure, the T cells comprise a chimeric antigen receptor (CAR).

[0364] In one embodiment, in the cell population of the present disclosure, T cells are modified to express a glucose transporter in response to (if necessary) or the expression of a glucose transporter is enhanced in response to (if necessary). Alternatively, the cells in the cell population of the present disclosure may be T cells having enhanced glucose uptake capacity in which the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions; T cells having enhanced glucose uptake capacity modified to regulate the expression of a glucose transporter in the T cells in response to changes in tumor microenvironment conditions; or T cells comprising a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions.

[0365] In one embodiment, in the cell population of the present disclosure, T cells have an effector function.

[0366] Alternatively, in T cells within the cell population of the present disclosure, tumor microenvironment conditions include the degree of transmission of TCR-related signals, and in one embodiment, the TCR-related signal is mediated by a molecule selected from the group consisting of a TCR signal or a part thereof, and a chimeric signal of an antigen other than the TCR signal (also called a CAR signal). Preferably, the TCR-related signal is mediated by a molecule comprising a TCR signal domain.

[0367] Alternatively, changes in tumor microenvironment conditions include changes in the presentation of tumor antigens and / or co-stimulating ligands and / or cytokine conditions. Preferably, the degree of TCR-related signaling includes the degree of TCR stimulation. Alternatively, in another embodiment, the degree of TCR-related signaling includes at least one selected from the group consisting of the degree of TCR stimulation, TCR co-stimulation, and humoral factors.

[0368] In another embodiment, in cells within the cell population of the present disclosure, the degree of transmission of a TCR-related signal comprises at least one selected from the group consisting of a degree of TCR stimulation, a TCR co-stimulation, and a humoral factor. In one embodiment, the degree of transmission of a TCR-related signal comprises a degree of TCR stimulation, a TCR co-stimulation, and a humoral factor. Preferably, the TCR stimulation is selected from the group consisting of the NFAT, NF-κB, and STAT transcription factor families that translocate into the nucleus in connection with T cell activation. Alternatively, tumor microenvironment conditions comprise a degree of oxygen concentration.

[0369] In another embodiment, in a cell population comprising T cells comprising a nucleic acid sequence encoding a glucose transporter operably connected to a tumor microenvironment sensing element of the present disclosure for sensing a change in tumor microenvironment conditions, for each T cell, the tumor microenvironment sensing element comprises: (i) an inducible activation motif that is activated in response to a change in tumor microenvironment conditions; and (ii) a nucleic acid sequence encoding a glucose transporter degradation factor that is inactivated in response to a change in tumor microenvironment conditions. Herein, the tumor microenvironment sensing element preferably comprises: (i) an inducible activation motif to which a nuclear translocation factor translocates into the nucleus in response to activation of the T cell binds; (ii) a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor 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 a decrease in oxygen concentration.

[0370] In another embodiment, with respect to T cells in a cell population of the present disclosure, a nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nucleic acid sequence encoding an inducible activation motif that is activated in response to a change in tumor microenvironment conditions and / or a degradation factor for the glucose transporter that is inactivated in response to a change in tumor microenvironment conditions. In one embodiment, the nucleic acid sequence encoding the glucose transporter comprises a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor that translocates into the nucleus in response to activation of the T cell binds. In a preferred embodiment, the nuclear translocation factor is selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families (e.g., TAT1-4, STAT5A, STAT5B, STAT5, etc.), and the nuclear translocation factor-binding inducible activation motif is selected from the group consisting of an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ No. 1), a STAT-binding sequence (TTCNNNGAA), and modified sequences thereof. In a preferred embodiment, the combination of the nuclear translocation factor and the nuclear translocation factor-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 No. 1)), or (3) a combination of a STAT transcription factor family and a STAT-binding sequence (TTCNNNGAA).

[0371] In another embodiment, with respect to T cells in a cell population of the present disclosure, a nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nuclear transposition factor-binding inducible activation motif to which a nuclear transposition factor translocating into the nucleus in response to a decrease in oxygen concentration binds. Preferably, the nuclear transposition factor is selected from the group consisting of HIF-1, and the nuclear transposition factor-binding inducible activation motif is selected from the group consisting of HIF-RE, or the combination of the nuclear transposition factor and the nuclear transposition factor-binding inducible activation motif is selected from the group consisting of (1) combinations of HIF-1 and HIF-RE.

[0372] In another embodiment, with respect to T cells in a cell population of the present disclosure, a nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter is deactivated in response to a decrease in oxygen concentration, and preferably, the factor deactivated is selected from the group consisting of oxygen-dependent degradation factors (ODDs).

[0373] In another embodiment, with respect to T cells in a cell population of the present disclosure, the T cells have an effector function, and the T cells are effector T cells (T eff It may include or be composed of ) progenitor cells. Here, the memory function of the effector T progenitor cells is extended and / or the memory T cell phenotype is maintained. Alternatively, the effector T cells are activated or the activation of the effector T cells is maintained, the memory function of the effector T progenitor cells is extended or the memory T cell phenotype of the effector T progenitor cells is maintained.

[0374] In another embodiment, with respect to T cells in the cell population of the present disclosure, expression and / or enhancement are achieved specifically in a tumor environment.

[0375] In one embodiment, the cell population of the present disclosure is T eff and / or Teff It includes precursor cells of. Preferably, a cell population (or T eff The progenitor cells) include at least one type of cell such as lymphoid progenitor cells, Tnv, Tscm, Tcm, or Temr.

[0376] In one embodiment, in the cell population of the present disclosure, CAR is expressed in T cells.

[0377] In one embodiment, in the cell population of the present disclosure, the glucose transporter comprises GLUT1, GLUT2, GLUT3 and GLUT4, preferably GLUT3.

[0378] In one embodiment, in the cell population of the present disclosure, the T cells are human T cells.

[0379] In one embodiment, the cell population of the present disclosure is provided as a pharmaceutical or pharmaceutical composition. Its use may be for the prevention or treatment of cancer, the cure of cancer, the prevention of cancer metastasis, or the prevention of cancer recurrence. In a specific embodiment, the cell population of the present disclosure may be for the prevention or treatment of cancer so as not to cause cancer to recur.

[0380] Glucose Transporter

[0381] Cancer cells reprogram their metabolic systems to favor their proliferation, actively utilize the glycolysis pathway—which has low ATP generation efficiency even in the presence of oxygen—and increase glucose uptake and lactic acid production (Warburg effect). Consequently, because cancer cells consume and deplete large amounts of glucose, T, which requires glucose as an energy source eff Even if type 1 tumor-specific T cells receive signals via TCRs when infiltrating a tumor, intracellular Ca 2+ The concentration decreases, leading to a state of starvation and exhaustion.

[0382] Accordingly, in one embodiment of the present disclosure, T cells and cell populations are provided that are genetically engineered to express a glucose transporter to promote aerobic glycolysis in a glucose-depleted tumor microenvironment (TME) and to enhance the competitiveness and / or fitness of immune cells.

[0383] In one embodiment, examples of glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, and among these, the glucose transporter having high affinity for glucose is GLUT3. Accordingly, in a preferred embodiment, the glucose transporter used in the cell of the present disclosure is GLUT3.

[0384] GLUT3 was first identified in the mouse brain and was initially defined as a neuronal glucose transporter. Subsequently, GLUT3 was shown to be expressed in other cells requiring glucose, such as mouse sperm for securing energy necessary for motility and blastocysts crucial for post-implantation development. Additionally, GLUT3 is expressed in immune cells such as lymphocytes, monocytes, macrophages, and platelets; it is typically stored in intracellular vesicles and, upon activation, translocates to the cell surface to maintain metabolic switching. More recently, T cells, particularly CD8 + It has been suggested that T cells rely on GLUT3 as well as GLUT1 for glucose uptake because GLUT3 is highly expressed during differentiation and activation.

[0385] In one embodiment, the T cells and T cells included in the cell population of the present disclosure may be modified to express a glucose transporter as needed and / or the expression of the glucose transporter may be enhanced as needed. In T cells with constitutively enhanced glucose uptake capacity, differentiation into effector memory T cells and exhaustion were observed, and significant T cell death was induced when the T cells were activated under glucose-free to low-glucose conditions. In contrast, T cells with enhanced glucose transporter expression as needed are significant in that T cell death under glucose-free to low-glucose conditions is avoided while maintaining equivalent cytotoxic activity, and the maintenance of a memory phenotype and resistance to exhaustion are induced, which are expected to maintain effects for a longer period.

[0386] When glucose transporters such as GLUT3 are activated in T cells, enhancement of effector functions, such as cytotoxic activity and cytokine production capacity, is observed due to glucose uptake; however, at the same time, when T cells are activated for a long period, differentiation into short-acting effector memory cells and T cell exhaustion are significantly induced. Accordingly, in one embodiment of the present disclosure, an NFAT-induced glucose expression system induced by a glucose derivative or a hypoxia-induced glucose expression system applying the mechanism of HIF1a may be used, which is triggered by activation where the glucose demand of T cells increases or by entry into a tumor environment, which is a hypoxic environment (e.g., FIG. 12). This makes it possible to realize transient expression in response to increased demand specifically in a tumor environment. Thus, the cells of the present disclosure can maintain memory phenotype T cells while maintaining cytotoxic activity and avoid exhaustion.

[0387] 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.

[0388] In one embodiment of the present disclosure, the T cells and the T cells included in the cell population of the present disclosure may advantageously be human T cells.

[0389] Chimeric Antigen Receptor (CAR)

[0390] In another aspect of the present disclosure, T cells having enhanced glucose uptake capacity, wherein the T cells comprise a chimeric antigen receptor (CAR) and the T cells are modified to express a glucose transporter when necessary and / or have enhanced expression of a glucose transporter when necessary, or a cell population comprising such cells is provided. The T cells or cell populations of the present disclosure may comprise the CAR as a protein or may comprise a nucleic acid molecule expressing the CAR, insofar as the CAR can function as a CAR. In one embodiment, the CAR of the present disclosure may be expressed in T cells.

[0391] The CAR disclosed in this specification comprises at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0392] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain of an antibody (e.g., a single-strand variable fragment (scFv)) connected to a T cell signaling domain through a transmembrane domain. A characteristic of CARs is that they redirect the specificity and responsiveness of T cells to a selected target by utilizing the antigen-binding properties of monoclonal antibodies independently of MHC. MHC-independent antigen recognition can provide T cells expressing CARs or T cells within a cell population with the ability to recognize antigens independently of antigen processing, thereby avoiding tumor immune evasion.

[0393] The intracellular T cell signaling domain of the CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulation signaling domain, or both. The T cell receptor signaling domain refers to a portion of the CAR that includes an intracellular domain of a T cell receptor, for example, an intracellular portion of the CD3 zeta protein. The co-stimulation signaling domain refers to a portion of the CAR that includes an intracellular domain of a co-stimulation molecule, which is a cell surface molecule other than the antigen receptor or its ligand, necessary for the efficient response of lymphocytes to an antigen.

[0394] <T 세포 수용체(TCR)>

[0395] In another aspect of the present disclosure, T cells having enhanced glucose uptake capacity, wherein the T cells comprise a T cell receptor (TCR) and the T cells are modified to express a glucose transporter when necessary and / or the expression of the glucose transporter is enhanced when necessary, or a cell population comprising such cells is provided. As long as the TCR can function as a TCR, the T cells or cell populations of the present disclosure may comprise the TCR as a protein or may comprise a nucleic acid molecule expressing the TCR. In a representative embodiment, the TCR of the present disclosure may be expressed in the T cells.

[0396] In this specification, a TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains, comprising an αβ-type TCR expressed by normal T cells and a γδ-type TCR having a specific function. The α-chain and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens replicated intracellularly in relation to viral infection and cancer antigens derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Additionally, antigens derived from foreign microorganisms are absorbed by antigen-presenting cells via endocytosis and, after processing, are presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. Co-stimulatory molecules such as CD28, ICOS, and OX40 are also known to be important for stimulation mediated by TCR molecules. With respect to αβ type TCRs, the respective gene products of α and β can exhibit specificity through unique combinations. TCRs can be appropriately modified and may include a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain includes the intracellular domain of the T cell receptor, for example, the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain includes the intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand, necessary for the efficient response of lymphocytes to an antigen.

[0397] (Extracellular domain)

[0398] In one embodiment, a CAR used on T cells included in the T cells or cell population disclosed herein comprises an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof may be appropriately selected depending on the type and number of ligands on the surface of the target cell, for example, the antigen-binding domain may be selected to recognize a ligand acting as a cell surface marker on the target cell associated with a specific disease state. Accordingly, examples of cell surface markers that may act as ligands for the antigen-binding domain in the CAR of the present disclosure include tissue-specific markers, cell type-specific markers, and those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0399] The extracellular binding domain of the CAR can consist of a single-chain variable fragment (scFv) obtained by fusing the variable heavy and light chain regions of a mouse or humanized monoclonal antibody. Alternatively, Fab-derived scFvs (e.g., obtained from a Fab library rather than an antibody) may be used. The scFv can be fused to the transmembrane domain and then to the intracellular signaling domain.

[0400] In one embodiment, the antigen-binding domain portion of the CAR of the present disclosure may target antigens including: (1) alloantigens including MHC class I, MHC class II, etc.; (2) extracellular autoantigens including thyroid-stimulating hormone receptor (TSHR), desmoglein 3 (DSG3), cytokeratin 8, etc.; (3) foreign antigens including gliadin, Ara h2, etc.; and (4) targeting molecules including CD4, CD8, CD19, BCMA, CD68, mesothelin (MSLN), mucosal vascular addressin cell adhesion molecule 1 (MadCam1), etc., but the antigens that can be targeted by the antigen-binding domain portion of the CAR of the present disclosure are not limited thereto.

[0401] In one embodiment, the CAR of the present disclosure may be modified to include an antigen-binding domain specific to the target antigen target, depending on the target antigen to be targeted. For example, if CD19 is the target antigen, an antibody against a cancer antigen such as CD19 may be used as the antigen-binding domain within 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 A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, midkine, 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, ART1, 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; Includes 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.

[0402] (Membrane-penetrating domain)

[0403] A CAR used for T cells or T cells included in a cell population disclosed in this specification may include one or more transmembrane domains fused to an extracellular domain.

[0404] In one embodiment, a linker domain derived from an extracellular domain may be linked to a transmembrane domain. The transmembrane domain may be natural or synthetic, and the natural transmembrane domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain particularly used in this disclosure may be derived from the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0405] In one embodiment, in a CAR used in effector T cells disclosed herein, a spacer domain may also be positioned between an extracellular domain and a transmembrane domain or between an intracellular domain and a transmembrane domain. The spacer domain may preferably have a sequence that promotes binding to the antigen of the CAR and enhances signal transduction into the cell.

[0406] (Intracellular domain)

[0407] The cytoplasmic signaling domain (or intracellular signaling domain) of the CAR is involved in the activation of at least one normal effector function of immune cells expressing the CAR. The intracellular signaling domain refers to a portion of a protein that transmits effector function signals and directs cells expressing the CAR to perform specific functions. The intracellular signaling domain may include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit signals that induce or block immune cell effector function.

[0408] In one embodiment, examples of intracellular signaling domains used in CAR include cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that induce signaling after binding to antigen receptors.

[0409] (Alloantigens, allergens, and haptens related to rejection reactions)

[0410] The CAR used in the T cells or T cells included in the cell population disclosed in this specification may include alloantigens, allergens, and haptens associated with rejection reactions.

[0411] <T 세포 또는 세포 집단에 포함된 T 세포의 약제학적 용도>

[0412] In one aspect of the present disclosure, a pharmaceutical composition is provided comprising T cells having enhanced glucose uptake capacity, wherein the T cells are modified to express a glucose transporter when necessary and / or the expression of a glucose transporter is enhanced when necessary, or a cell population comprising such T cells. In another aspect of the present disclosure, a pharmaceutical composition is provided comprising T cells having enhanced glucose uptake capacity, wherein the T cells include a chimeric antigen receptor (CAR), a T cell receptor (TCR), etc., wherein the T cells are modified to express a glucose transporter when necessary and / or the expression of a glucose transporter is enhanced when necessary, or a cell population comprising such T cells. In one embodiment of the present disclosure, the T cells included in the T cells or cell population of the present disclosure may have one or more of the characteristics of the effector T cells described above.

[0413] The cells, cell populations, etc. of the present disclosure may be used in immunotherapy. Immunotherapy is considered effective for diseases in which antigenic lesions occur (e.g., cancer, autoimmune diseases, allergies, infectious diseases, etc.) and for 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. of the present disclosure may be used for the treatment, therapy, or prevention of autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD), rejection, or engraftment failure upon 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 1 diabetes mellitus, Graves' disease, Hashimoto's disease, idiopathic Addison's disease, autoimmune hepatitis, Goodpasture 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. Additionally, treatment or prevention by the cells, cell populations, etc. of the present disclosure may be used for diseases in which an abnormal immune response to a specific antigen is involved in the onset or progression of the pathological condition.

[0414] In certain embodiments, the present disclosure provides such immunotherapy, and the immunotherapy comprises TCR-T therapy or CAR-T therapy. In this specification, the term “TCR-T therapy” refers to a cell therapy using a modification of a T cell receptor (TCR) and, for example, a therapy used for cancer treatment. In this specification, the term “CAR-T therapy” refers to a gene and cell therapy in which a chimeric antigen receptor (CAR) (e.g., genetically engineered to overcome tumor immune evasion mechanisms) is genetically introduced into a patient’s T cells, and the T cells are proliferated and cultured in vitro and then injected into the patient.

[0415] The cells, cell populations, etc. of the present disclosure may be appropriately combined with other cancer treatments and used as combination therapy. Typically, they may be administered in combination with one or more additional drugs. Alternatively, the combination therapy may be a combination with radiation therapy. The one or more additional drugs may be any chemotherapy agent or may include immune checkpoint inhibitors. Alternatively, other cancer treatments used in combination therapy include, but are not limited to, other cancer immunotherapies (e.g., immune checkpoint inhibitors), hyperthermia, surgical procedures, etc.

[0416] In one aspect, a therapeutic agent comprising the T cells or cell population of the present disclosure is provided, wherein a disease of the subject is diagnosed and an appropriate CAR, TCR, etc. included in the T cells or cell population is selected based on the diagnosis.

[0417] In another aspect, the present disclosure provides a pharmaceutical composition comprising any immune cell described herein (e.g., T cells such as effector T cells) or a cell population comprising such cells, and a pharmaceutically acceptable carrier. Where the immune cell expresses 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 may bind to a target antigen, such as an antigen on the immune cell specific to a tumor antigen, a pathogen antigen, or an autoantigen. The pathogen antigen may be a bacterial antigen, a viral antigen, or a fungal antigen.

[0418] In one embodiment, the Fc-containing therapeutic agent is adalimumab, ado-trastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, brentuximab, canakinumab, cetuximab, sertolizumab, daclizumab, denosumab, dinutuximab, eculizumab, epalizumab, efratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumomab, infliximab, ipilimumab, labetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tocitumomab, It may be a therapeutic antibody including, but not limited to, ustekinumab, mogamulizumab, and vedolizumab.

[0419] Additionally, the present disclosure provides: (i) a first pharmaceutical composition comprising any T cell or a cell population comprising such T cell as described herein, and a pharmaceutically acceptable carrier; and (ii) a kit comprising an Fc-containing therapeutic agent and a pharmaceutically acceptable carrier as described herein.

[0420] In another aspect, the present disclosure provides 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 inhibiting cell activity), said method comprising the step of administering the cells, cell population and / or pharmaceutical composition of the present disclosure to a subject. In one embodiment, at least some of the cells expressing the target antigen may be in a low-glucose environment.

[0421] In one embodiment, the subject treated by the method of the present disclosure may be a human patient suffering from cancer, e.g., carcinoma, lymphoma, sarcoma, blastoma, or leukemia. Examples of 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. Examples of B-cell derived cancers include B-cell acute lymphoblastic leukemia, B-cell chronic lymphoblastic leukemia, and B-cell non-Hodgkin lymphoma.

[0422] In addition to the treatment of target diseases or conditions such as cancer or infectious diseases, the use of the T cells or cell populations of the present disclosure for producing agents for intended treatment is also within the scope of the present disclosure.

[0423] In one embodiment, a pharmaceutical composition comprising T cells or a cell population comprising the chimeric antigen receptor (CAR) or T cell receptor (TCR) of the present disclosure, etc., may be used in cell therapy. In cell therapy, T cells comprising the chimeric antigen receptor (CAR) or T cell receptor (TCR) of the present disclosure, etc., may be injected into a subject in need of such cells as a pharmaceutical composition or as a formulation of a therapeutically effective cell population expressing the CAR of the present disclosure. The T cells injected into the subject or the cell population comprising such T cells may treat a target disease or condition of the subject, such as cancer or an infectious disease. The subject may be the same subject from which the cells were obtained (autologous cell therapy), or the cells may be derived from another subject of the same species (allogeneic cell therapy).

[0424] In one embodiment, T cells comprising the CAR, TCR, etc. of the present disclosure, or a cell population comprising such T cells, may 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, TCR, etc. of the present disclosure, or a cell population comprising such T cells, may comprise pharmaceutically acceptable excipients (carriers or diluents). Excipients included in the formulation have different purposes, for example, depending on the nature of the antigen-binding domain of the CAR of the present disclosure. Non-limiting examples of commonly used excipients include: saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonic agents, extenders, and lubricants.

[0425] In this specification, when gene names and their products are denoted, unlike in ordinary usage, if they are denoted in all capital letters, they may refer to both the gene and the protein. For example, the FOXP3 gene and the FOXP3 protein can be distinguished from each other, and when denoted as FoxP3, the notation refers to both the concept and the substance (the whole) of the gene or the protein.

[0426] Formulations comprising therapeutically effective T cells, such as the CAR, TCR, etc. of the present disclosure, or cell populations comprising such T cells, may be administered to a subject using methods and techniques known to those skilled in the art. Examples thereof include, but are not limited to, intravenous injection. Other methods include, but are not limited to, administration into a tumor, intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial, intramedullary, intracardiac, intra-articular (joint), intramuscular (synovial area), intracranial, intraspinal, and intradural (cerebrospinal fluid) areas.

[0427] In one embodiment, the present disclosure is provided as a pharmaceutical composition for preventing or treating cancer in a subject, wherein the pharmaceutical composition is characterized by: (A) collecting a value of glucose uptake capacity or a value of an indicator related thereto in a subject; (B) selecting a cell population comprising T cells having enhanced glucose uptake capacity or T cells having appropriate expression characteristics according to the indicator; and (C) administering the cell population comprising T cells having enhanced glucose uptake capacity or T cells to a subject according to an appropriate administration method and dose, wherein the T cells comprise chimeric antigen receptors (CARs).

[0428] In one embodiment, T cells having enhanced glucose uptake capacity are not modified to express a glucose transporter in response to need (when necessary), and T cells in which the expression of a glucose transporter is not enhanced in response to need (when necessary) eff, and includes cells modified to express a glucose transporter in response to need (if necessary) and / or cells in which the expression of a glucose transporter is enhanced in response to need (if necessary).

[0429] In one embodiment, the glucose transporter is GLUT3.

[0430] (General Technology)

[0431] The molecular biological, biochemical, 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 third edition (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene 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 Update; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Experimental Medicine Special Issue, “Experimental Methods for Gene Introduction & Expression Analysis”, YODOSHA CO., LTD., 1997, etc., and relevant parts (may be the whole) of these are incorporated herein by reference.

[0432] For DNA synthesis techniques and nucleic acid chemistry to generate artificial synthetic genes, gene synthesis or fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) may also be used. Additionally, these techniques and chemistry are described, for example, in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., and their relevant parts are incorporated herein by reference.

[0433] In this specification, “or” is used where “at least one” of the items listed in the text may be adopted. The same applies to “or”. Where this specification explicitly states “within a range” of “two values”, the range includes the two values ​​themselves.

[0434] References cited in this specification, such as scientific literature, patents, and patent applications, are incorporated herein by reference in their entirety to the same extent as specifically described therein.

[0435] The present disclosure has been described above with reference to preferred embodiments for ease of understanding. Hereinafter, the present disclosure is described based on embodiments, but the above description and the following embodiments are provided merely for illustrative purposes and are not intended to limit the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the claims.

[0436] Examples

[0437] In this embodiment, various functions of T cells with enhanced glucose uptake capacity were investigated. As a reagent, the product specifically described in the embodiment was used, but it can be replaced with an equivalent product from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R&D Systems, USCN Life Science INC, etc.).

[0438] (Example 1; T cells expressing GLUT3)

[0439] (Method and materials)

[0440] Preparation of GLUT3 3C10-CAR construct and preparation of viral vector

[0441] The SLC2A3 sequence (GLUT3), with the P2A sequence positioned immediately upstream, was inserted into an anti-EGFRvIII CAR backbone plasmid (3C10 CAR-Katushka2S) having the structure CD8 leader sequence-anti-EGFRvIII single-strand variable region (scFv)-CD8 hinge-CD8 transmembrane region (TM)-CD28 intracellular domain (ICD)-4-1BB ICD-CD3z-P2A-Katushka2S, and the 3C10 CAR-GLUT3-Katushka2S lentiviral vector was constructed (Fig. 1). The lentiviral vector was transfected into 293T cells along with the packaging vectors psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259), and lentiviruses were obtained from the supernatant. This was introduced into PBMCs containing T cells to obtain T cells expressing CAR and GLUT3.

[0442] Construction of GLUT3-expressing 3CAR constructs if necessary

[0443] NFAT-induced GLUT3-CAR

[0444] An NFAT-inducible GLUT3 construct containing an 8-repeated NFAT binding site, a minimal promoter (minP), and an SLC2A3 (GLUT3) sequence was inserted into the GLUT3 3C10-CAR plasmid described above to construct an NFAT-inducible GLUT3 CAR plasmid (NFATiGLUT3 CAR: see Fig. 15; identical lentivirus plasmid: see Fig. 16). In this system, the CAR is induced by the EF1a promoter and constitutively expressed, and GLUT3 is expressed by detecting the nuclear translocation of NFAT associated with the activation of CAR-T cells.

[0445] Hypoxia-Inducible GLUT3-CAR

[0446] A HIF-inducible GLUT3 construct containing xx-repeated HIF1a binding sites, minP, and SLC2A3(GLUT3) sequences was inserted into the GLUT3 3C10-CAR plasmid described above to construct a hypoxia-inducible GLUT3 CAR plasmid (Fig. 15). Alternatively, the 2A sequence was placed downstream of 3C10-CAR, and a conjugate of GLUT3 and the HIF-1α oxygen-dependent degradation domain (ODD) was inserted. In both cases, the CAR was induced by the EF1a promoter and constitutively expressed. In the former case, GLUT3 expression was induced by detecting the nuclear translocation of NFAT associated with CAR-T cell activation, while in the latter case, GLUT3 continued to degrade under normal oxygen conditions, but under hypoxia, the degradation of GLUT3 by ODD was inhibited, resulting in the induction of expression.

[0447] Gene delivery

[0448] Peripheral blood mononuclear cells from healthy subjects were isolated from heparin-treated whole blood by the Ficoll method (Ficoll-Paque PLUS, GE Healthcare), stimulated with anti-CD3 / 28 beads (Dynabeads T-Activator CD3 / CD28, Veritas), and CAR gene delivery was performed at MOI=4 on day 1 after stimulation. Cells were cultured in the presence of IL-2 30 U / ml and either cryopreserved or used directly for each evaluation on days 8 to 12.

[0449] Immunostaining

[0450] 1 × 10 610 GLUT3 3C10 CAR-T cells were fixed with 4% PFA and permeated with methanol. Blocking was performed with 3% BSA for 1 hour, and the cells were incubated with the anti-GLUT3 antibody (ab15311) for 2 hours. The cells were washed with 0.05% PBST and incubated with the anti-rabbit IgG antibody (Alexa flour 488) for 1 hour. The cells were washed with 0.05% PBST, and nuclear staining was performed with DAPI. After staining, cells were attached to a slide glass using Cytospin (Thermo Fisher Scientific), mounted using VECTASHIELD (VECTOR LABORATORIES), and observed using a Keyence XZ-800.

[0451] Phenotype Analysis (FCM)

[0452] The antibodies used for FCM analysis are presented in Table 1.

[0453] [Table 1]

[0454]

[0455] Cells were washed with 4% FBS-PBS. Cell staining and surface staining were performed, and after washing, data were acquired using FACSymphony A3 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0456] Analysis of in vitro CAR-T cell survival and function (Fig. 21)

[0457] The long-term survival, memory formation, and maintenance of function of each CAR-T cell were evaluated. CAR-T cells were stimulated with a stimulant (medium, EGFRvIII stimulating beads, or U87d cell line as negative controls) in a 1:1 ratio. After 24 hours, the stimulant was removed by ceramic beads or sorting, and the CAR-T cells were divided into 0 mM, 0.5 mM, and 10 mM groups and cultured serially. Before stimulation and on days 1, 3, and 7 after stimulation, cell counts were performed, along with the analysis of memory phenotypes such as Annexin V / 7AAD, CD27, CCR4, and CD45RA to evaluate cell survival and apoptosis, inhibitory factors such as PD-1, TIM3, and LAG3 (Figs. 23 to 28), and other parameters (transcription factors, activation / aging markers). Additionally, RNA was extracted from each sample, and detailed analyses such as RNA sequencing were performed. After a series of analyses, some of the Day 7 cells were re-stimulated in the same group, and cell survival and function analyses after repeated stimulation were performed in the same manner.

[0458] Cytokine Analysis (FCM)

[0459] 1 × 10 5 10 CAR-T cells and 5 × 10 5CAR-T cells were stimulated by co-culturing 1 EGFRvIII-expressing U87 cell line (U87Δ) in 1 ml of medium for 12 hours, followed by incubation in the presence of 5 μg / ml Monensin (BD 554724, BD Bioscience) for 6 hours. As the medium, glucose-free RPMI1640 (Wako) supplemented with 10% dialyzed FBS (Cytiva) and adjusted to glucose concentrations of 10 mM and 0.5 mM using glucose solution (Gibco) was used. Fixation / permeabilization was performed with Fixation / Permeabilization Diluent (Invitrogen); intracellular cytokines were stained with the antibodies shown in Table 1; data were acquired using LSRFortessa X20; and data were analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0460] Analysis of cytotoxic activity

[0461] Target cells (luciferase-expressing U87Δ cells) 2 × 10 per well 5 The cells were seeded into 96-well plates 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, viable cells were induced to glow using VivoGlo Luciferin (Promega); luciferase activity was measured using Cytation (Promega); and cytotoxic activity was calculated.

[0462] Metabolic analysis

[0463] Cellular metabolic capacity was measured according to product protocols using the XFe24 Cell Flux Analyzer (Bioscience) and the XF Glycolysis Stress Test Kit (Agilent Technologies). The day before analysis, 24-well flat-bottom plates for analysis were coated with poly-D-lysine (0.1 mg / ml), and sensor cartridges were hydrated in a CO2-free incubator. On the day of analysis, cells for analysis were washed with analysis medium (Seahorse XF RPMI medium, 2 mM L-glutamine), and 2 × 10⁶ cells were placed on the poly-D-lysine-coated analysis plates. 5 The samples were seeded and incubated at 37°C for 60 minutes under CO2-free conditions. Glucose at a final concentration of 10 mM or 0.5 mM, oligomycin at a final concentration of 1 μM, 2-DG at a final concentration of 50 mM, and, appropriately, oligomycin and FCCP at final concentrations of 20 μM and 10 μM, respectively were loaded into the sensor cartridge port, after which analysis was performed using a flux analyzer and metabolic function was evaluated based on OCR and ECAR.

[0464] Evaluation of glucose absorption capacity

[0465] 2 × 10 4 100 GLUT3 CAR-T cells were seeded into 96-well plates and cultured in 200 μl of glucose-free RPMI1640 (Wako) for 5 hours. 5 minutes before analysis, 1 μl of 2-NDBG (abcam) was added to each well, and after washing, data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0466] Intracranial tumor xenograft model

[0467] 2.5 × 10⁶ from luciferase-expressing U87Δ tumor cell line (U87Δ-luc) 4Dog cells were intracranially injected into the right basal cerebrum of NSG mice, and after engraftment, 2 × 10⁶ Mock cells, 3C10 CAR-T cells, or GLUT3 CAR-T cells were each injected. 6 They were administered individually via the tail vein. Tumor burden was monitored over time and survival was observed through in vivo imaging. In a tumor rechallenge study to verify the long-term survival, memory formation, and recurrence prevention effects of CAR-T cells, 2.5 × 10⁶ of the same tumor cells (U87Δ-luc) were administered to mice that achieved CR after the above treatment. 4 Piece (Fig. 13) or 1.0 × 10 5 Dogs (Fig. 19) were administered intracranially again, and BLI and survival were monitored.

[0468] (method)

[0469] Figure 1 illustrates the structure of a metabolically modified CAR (EGFRvIII_CAR_GLUT3). A single-strand antibody (asFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) was constructed and linked to the CD8 hinge, CD28 transmembrane region (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BB ICD, and CD3z to construct the CAR. A high-affinity glucose transporter (GLUT3) was encoded downstream of the CAR via a P2A sequence.

[0470] (result)

[0471] The results are illustrated in Fig. 2 and subsequent figures. As illustrated in Fig. 2, the expression of GLUT3 and the enhancement of glucose uptake on the surface of T cells were shown. CAR-T cells were obtained by introducing a conventional CAR (3C10 CAR) and a metabolically modified CAR (GLUT3 CAR) into healthy donor-derived T cells via gene transfer. Uniform GLUT3 expression was observed on the membranes of GLUT3 CAR-T cells through fluorescence microscopy. Furthermore, glucose uptake was enhanced in GLUT3 CAR-T cells compared to T cells and conventional CAR-T cells (3C10 CAR-T cells).

[0472] Next, T cell proliferation efficiency equivalent to that of conventional CAR-T cells is shown in Fig. 3. As shown in Fig. 3, CAR was genetically introduced after stimulation with anti-CD3 / 28 beads, and T cell proliferation efficiency was analyzed. GLUT3 CAR-T cells had establishment efficiency equivalent to that of conventional CAR-T cells (3C10 CAR-T cells).

[0473] Next, the enhancement of T cell glycolysis by GLUT3 expression is illustrated in Figure 4. As shown, the metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Flax analyzer. GLUT3 CAR enhanced glycolysis in both low-glucose and normal-glucose environments.

[0474] Next, it was shown that GLUT3 CAR-T cells have a competitive advantage under low glucose conditions (Fig. 5). As shown in Fig. 5, 3C10 CAR-T cells and GLUT3 CAR-T cells were co-cultured under normal glucose and low glucose conditions. While the survival of both was observed to be equal under normal glucose, significant survival of GLUT3 CAR-T cells was observed under low glucose conditions, suggesting that GLUT3 CAR-T cells can perform advantageous glucose uptake.

[0475] Next, the enhancement of the cytokine-producing ability of GLUT3 CAR-T cells is illustrated in Fig. 6. As shown in Fig. 6, after antigen-stimulating 3C10 CAR-T cells and GLUT3 CAR-T cells, the ratio of cytokine-producing cells was analyzed by FCM. It was observed that the expression of cytokines (IFN-γ, IL-2, TNF-α) important for the maintenance and activation of T cells and the exertion of anti-tumor activity was significantly increased.

[0476] Next, the function of GLUT3 CAR-T cells maintained even under low glucose conditions is illustrated in Fig. 7. As shown, cytokine production ability was compared under normal glucose and low glucose conditions. In conventional CAR-T cells (3C10 CAR-T cells), severe functional decline was observed under low glucose conditions. On the other hand, high cytokine production ability was observed in GLUT3 CAR-T cells under normal glucose conditions, and even under low glucose conditions, GLUT3 CAR-T cells exhibited a function comparable to the cytokine production ability of 3C10 CAR-T cells under normal glucose conditions.

[0477] Next, the enhanced cytotoxic activity of GLUT3 CAR-T cells is illustrated in Fig. 8. As shown, cytotoxic activity was analyzed using EGFRvIII-expressing cell lines as targets. Cytotoxic activity was analyzed under low-glucose and high-glucose conditions, and GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells in both environments.

[0478] Next, the characteristics conferred by GLUT3 CAR on the memory phenotype are illustrated in Fig. 9. As shown, the memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. A tendency for GLUT3 CAR-T cells to differentiate into effector T cells was observed.

[0479] Next, the inhibition of inhibitory molecule expression in GLUT3 CAR-T cells is illustrated in Fig. 10. The expression of inhibitory molecules was compared under non-stimulated conditions and under stimulation by EGFRvIII antigen-positive cells. Inhibition of PD-1, LAG3, and Tim3 expression was observed in GLUT3 CAR-T cells.

[0480] Next, mRNA analysis of GLUT3 CAR-T cells (assessment of metabolism, exhaustion, activation, and differentiation) is shown in Fig. 11. Details of 3C10 CAR-T cells and GLUT3 CAR-T cells were analyzed based on mRNA expression. In GLUT3 CAR-T cells, as in FCM, a decrease in exhaustion-related molecules and an increase in factors related to lactate metabolism and glycolysis were observed. Overall, the data indicated an enhancement of T cell effector functions, such as activation and cytokine production.

[0481] Next, Figure 12 illustrates that the same effect was obtained when GLUT3 was introduced into CD19 CAR as well as 3C10 CAR, thus proving its universality. It was confirmed that when GLUT3 was introduced into CD19 CAR, an effect similar to that of 3C10 CAR was obtained. In addition, CD19 was expressed in pancreatic cancer cell lines, and it was shown that an effect was obtained against pancreatic cancer as well.

[0482] As shown in Fig. 13, rapid tumor removal was achieved in an intracranial xenograft model. Antitumor activity was observed in the U87Δ intracranial xenograft model. In GLUT3 CAR-T cells, rapid tumor removal was observed compared to 3C10 CAR-T cells. In addition, rejection was confirmed by tumor challenge.

[0483] (Example 2: Expression as needed)

[0484] This was performed on GLUT3-expressing CARs as needed. An overview of this is shown in Fig. 14.

[0485] Figure 14 illustrates an overview of a GLUT3-expressing CAR when needed. This was a system in which only the CAR is expressed and GLUT3 is not expressed under normal conditions, and GLUT3 is expressed when glucose demand increases, such as during CAR-T cell activation or in a tumor environment. To achieve GLUT3 expression when needed, a CAR construct was designed to express GLUT3 specifically in a tumor environment or specifically upon activation (Figure 15). GLUT3 expression is possible in response to increased NFAT upon activation (A.) and tumor hypoxia (B.).

[0486] Using the CAR-introduced cells of Fig. 15, the function and antitumor effect were analyzed.

[0487] (Example 3: Enhanced expression of glucose transporter)

[0488] To achieve enhanced expression of the glucose transporter, a CAR construct constitutively expressing GLUT3 was designed (stbl-GLUT3 = GLUT3 CAR in Fig. 15).

[0489] Using the CAR-introduced cells of Fig. 15, the function and antitumor effect were analyzed.

[0490] Figure 16 illustrates the structure of the NFATi-GLUT3 CAR plasmid. The structural details are as follows. In Figure 16, the CAR and inducible GLUT3 were introduced into a lentiviral vector. The CAR was encoded in this order of a single-strand antibody (3C10VH-linker-3C10VL) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM), CD8 hinge, CD28 transmembrane site, CD28 intracellular domain (CD28I), 4-1BB, and CD3z. The gene introduction marker Katushka was encoded downstream of the CAR via the P2A sequence. Additionally, human GLUT3 (hGLUT3) was encoded under the control of an 8-repeated NFAT binding site and a minimal promoter.

[0491] FIG. 17 illustrates the operation and basic functions of the inducible GLUT3 CAR system of the present disclosure. A system expressing GLUT3 in relation to NFAT nuclear potential (NFATi GLUT3 CAR-T) was constructed, and its operation was verified. The cytotoxic activity of the NFATi GLUT3 CAR-T cells (NFATi GLUT3 CAR-T) was equivalent to the cytotoxic activity of the GLUT3 CAR-T cells with constitutive expression (GLUT3 CAR-T), and cytokine production was at an intermediate level between 3C10 CAR-T (without GLUT3) and the GLUT3 CAR-T cells with constitutive expression (GLUT3 CAR-T).

[0492] Figure 18 illustrates the results of evaluating the antitumor effect of NFAT1GLUT3 CAR-T cells in a similar mouse model. CR was obtained in 4 mice treated with GLUT3 CAR-T and 2 mice treated with NFATi GLUT3 CAR-T.

[0493] Figure 19 illustrates the results of the tumor challenge and tumor re-challenge experiments. Specifically, the figure depicts the state of sudden death in mice in the GLUT3 CAR-T cell group. Tumors (U87d) were re-implanted into mice that had been cured in the experiment shown in the previous figure, and rejection ability was evaluated (“surrogate indicator of memory formation”). 1 × 10⁻⁶ 5 Tumor cells (four times the amount from the previous experiment) were re-implanted, and while tumor rejection was not achieved in 3C10 CAR-T, it was achieved in GLUT3 CAR-T and NFATiCAR-T. However, in the GLUT3 CAR-T group, three out of four mice died (deaths unrelated to tumor growth).

[0494] Figure 20 shows that apoptosis is inhibited after stimulation with NFATiGLUT3. CAR-T cells were repeatedly stimulated with U87d, and apoptosis of CAR-T cells was observed using Annexin V and 7-ADD.

[0495] Figure 21 illustrates that long-term survival is enhanced after various stimuli. Each CAR-T cell is observed over time under the above conditions. As a result, under low glucose conditions, the conventional CAR-T (3C10) did not exhibit effective cytokine production, whereas GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibited effective cytokine production ability. However, due to excessive glucose uptake, GLUT3 CAR-T cells differentiated into CCR7-negative Tem or Temra over time (day 3 or day 7), underwent terminal differentiation, lost stem cell characteristics, and underwent apoptosis. On the other hand, in the NFATi GLUT3 CAR-T cells, which are required cells, apoptosis is inhibited, and CCR7-positive Tcm and Tscm (memory cells, stem cells) are maintained.

[0496] In addition, when CAR-T cells (established under normal glucose conditions) were suddenly (Day 0) placed under low-glucose conditions of 0 mM or 0.5 mM, the glucose-dependent GLUT3 CAR-T cells were prone to apoptosis (conversely, NFATi GLUT-3 CAR-T cells exhibited resistance to low glucose).

[0497] Figure 22 illustrates that long-term survival is enhanced after stimulation. The antitumor effects, survival, and adverse events described above are investigated in a system similar to the mouse system described above. In Experiment 1, GLUT3 CAR-T and NFATi GLUT3 CAR-T exhibit equivalent antitumor activity. In Experiment 2, GLUT3 CAR-T hyperactivation induced by glucose exposure leads to apoptosis and hyperdifferentiation, resulting in the loss of antitumor activity. In contrast, excessive activation is suppressed in NFATi CAR-T cells, thereby exhibiting an effective antitumor effect. Upon re-challenge, NFATi CAR-T cells reject the tumor. In Experiment 4, dominant CAR-T cell engraftment and tumor T cell infiltration were observed in NFATi GLUT3 in proportion to the tumor effect. Additionally, Experiment 3 was conducted in a liver tumor model suggested to exhibit immunosuppression due to a low-glucose, high-lactic acid environment, and results similar to those of Experiment 2 were expected.

[0498] Figure 23 illustrates data (in vitro data) showing the enhancement of long-term survival after stimulation. At each time point on the left, 7-AAD and Annexin V staining data reflecting apoptosis and cell death are shown. Apoptosis in each CAR-T cell was investigated using Annexin V and 7-AAD. Under unstimulated conditions, differences were observed according to glucose concentration, but no substantial differences were observed between each CAR-T cell.

[0499] Figure 24 presents in vitro data illustrating the enhancement of long-term survival after stimulation. As shown, the survival and apoptosis of each CAR-T cell after stimulation were evaluated in the same manner as in the previous figure. Compared to 3C10 CAR-T cells and GLUT3 CAR-T cells, survival was enhanced (apoptosis was inhibited) in NFATi GLUT3 CAR-T cells.

[0500] Figure 25 illustrates in vitro data showing the enhancement of long-term survival after stimulation. The proportion of CAR-positive cells in each CAR-T cell population was analyzed after stimulation. After stimulation, the proportion of CAR-positive cells decreased, particularly in GLUT3 CAR-T cells with constitutive expression, and this fraction was considered to have undergone apoptosis.

[0501] Figure 26 illustrates in vitro data showing the enhancement of long-term survival after stimulation. The number of T cells on day 7 is shown. As a model reflecting the kinetics following administration in the patient body (the dynamics of cells infiltrating from blood with sufficient glucose concentration into the tumor environment, which is a low-glucose to glucose-free environment after administration), cells were established and cultured under normal glucose conditions (10 mM glucose). After stimulation with EGFRvIII-binding beads, the cells were cultured under glucose-free (0 mM), low-glucose (0.5 mM), or normal glucose (10 mM) conditions, and the number of surviving cells after 7 days was evaluated. The graph shows the relative number of cells with the number of mock T cells set to 1. In GLUT3 CAR-T, almost all cells died under 0 mM conditions. In contrast, many cells were maintained in NFATi CAR-T. It has been suggested that apoptosis may have resulted from hyperactivation induced by constitutive GLUT3 expression and glucose-dependent metabolism.

[0502] Figure 27 illustrates the expression of the inhibitory molecule (PD-1). In NFATi GLUT3 CAR-T cells, the expression of the inhibitory molecule (PD-1) was lower than in GLUT3 CAR-T cells.

[0503] Figure 28 illustrates the suppressed expression of the inhibitory molecule (TIM3). In NFATi GLUT3 CAR-T cells, the expression of the inhibitory molecule (TIM3) was lower than in GLUT3 CAR-T cells.

[0504] (Example 4: Example using progenitor cells of other effector T cells)

[0505] Similar experiments were performed using other T cells. For example, unrefined peripheral T cells included Tnv, Tcm, Tem, Temra, etc., and a CAR (CAR, constitutive GLUT3 CAR, or GLUT3 CAR if necessary) could be introduced into any of these fractions.

[0506] In this embodiment, a CAR (CAR, constitutive GLUT3 CAR or GLUT3 CAR if necessary) was introduced into any purified and concentrated T cell memory fraction. In other experiments, CAR-T cells enriched with any T cell memory fraction were induced under culture conditions.

[0507] Experimental Procedure:

[0508] From T cell sources such as peripheral blood, bone marrow, or umbilical cord blood, T eff Tnv, Tscm, and Tcm, which are progenitor cells expected to survive longer and maintain stemness, or T which are already differentiated and primarily perform effector functions eff and Temra fractions, respectively, T cell memory-related surface antigen expression patterns, namely CD45RA + CCR7 + CD95 - , CD45RA + CCR7 + CD95 + , CD45RA - CCR7 + , CD45RA - CCR7 and CD45RA + CCR7 - Based on this, purification and concentration were performed using FACS classification or magnetic beads.

[0509] Each fraction was stimulated with CD3 / 28 beads, etc., and CAR was introduced using a lentivirus vector.

[0510] In addition, as another method, after delivering genes to bulk T cells, the type and amount of added cytokines are adjusted (by using IL-7 or IL-15 or platelet lysates) to T eff Progenitor cell-dominant CAR-T cells were obtained by inhibiting differentiation and inducing the dominant proliferation of Tnv, Tscm, and Tcm.

[0511] In vitro evaluation: These cells were co-cultured with U87d tumor cell lines or EGFRvIII beads, and after 1, 3, and 7 days, division and proliferation were evaluated by CFSE staining and cell counting, and activation and exhaustion status was evaluated by flow cytometry.

[0512] In vivo evaluation: These cells were administered to the U87d intracranial pharmaceutical model mice described above, and the antitumor effect was evaluated based on antitumor activity and survival induced by BLI. Additionally, tumors were re-administered to mice that had achieved a cure (re-challenge), and tumor rejection was evaluated.

[0513] result:

[0514] CARs are bulk T cells or more differentiated T cells eff Or compared to the case where it was introduced into Temra, CAR is T such as Tnv, Tscm, and Tcm eff CAR-T cells introduced into progenitor cells or CAR-T cells predominantly proliferated with Tnv, Tscm, and Tcm were expected to have high dividing and proliferative capacity, and long-term division was expected to be observed. In addition, the expression of PD1, TIM3, and LAG3 was low, and the cells were expected to have resistance to exhaustion.

[0515] In addition, these CAR-T cells were shown to have excellent antitumor activity and improved survival in mouse models. Furthermore, efficient tumor rejection was expected to be observed in tumor challenge (a surrogate indicator of long-term effects and prevention of recurrence).

[0516] (Example 5: Demonstration Example)

[0517] In this embodiment, an embodiment demonstrated on a larger scale was provided.

[0518] Materials and Methods

[0519] cell line

[0520] 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 Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. An EGFRvIII-packaged lentivirus vector was introduced into U-87 MG and U-251 MG cells, and the transduced cell lines were named U-87 MGΔ and U-251 MGΔ, respectively. SUP-T1, NALM6, and AsPC-1 cells were purchased from ATCC and cultured in RPMI1640 (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS and 1% penicillin / streptomycin. A CD19 cleavage vector was introduced into NALM6 cells, and a CD19-expressing AsPC1 cell line was obtained.

[0521] Flow cytometry testing

[0522] Flow cytometry assays were performed as previously described [References 40 to 43]. Briefly, cells were washed twice with FACS buffer, and Fc blocking was performed using Fc receptor blocking solution (BioLegend, San Diego, CA) for 10 minutes under dark conditions at 4°C. Cells were washed twice with FACS buffer; antibodies targeting cell surface molecules were added; and cells were incubated for 20 minutes under dark conditions at 4°C. Subsequently, cells were washed twice with FACS buffer. For intracellular antigen staining, cells were incubated in fixation / permeability solution (BD Biosciences, San Jose, CA) at room temperature for 1 hour according to the manufacturer's instructions, followed by washing twice with wash buffer (BD Biosciences). Antibodies targeting intracellular antigens were added, and after incubation for 20 minutes under dark conditions at 4°C, cells were washed twice. For the apoptosis assay, cells were washed once with FACS buffer and once with Annexin binding buffer. Subsequently, cells were stained with Annexin V and 7-AAD at room temperature for 15 minutes. After washing, flow cytometry (FCM) was performed using an LSRFortessa X-20 cytometer (BD Biosciences), and analysis was performed using FlowJo ver. 10 software (BD Biosciences). The staining solution was prepared according to the manufacturer's instructions.

[0523] Cytokine staining

[0524] Intracellular cytokine staining was performed as previously described [References 40 to 43]. Monensin was added to the medium to maintain cytokines within the cells during the last 5 hours of 6-hour T-cell stimulation. After staining cell surface markers, Cytofix / Cytoperm reagent (BD Biosciences) was added, and the cells were incubated in the dark at 4°C for 20 minutes. Subsequently, the cells were washed twice with a washing solution, and antibodies targeting cytokines were added. The cells were incubated in the dark at 4°C for 20 minutes. After washing, FCM analysis was performed using an LSRFortessa X-20 cell analyzer (BD Biosciences), and analyzed using FlowJo ver. 10 software (BD Biosciences). The antibodies used for cell staining are summarized in the table. The staining antibodies were prepared according to the manufacturer's instructions.

[0525] [Table 2]

[0526]

[0527] Murder check

[0528] Total 1 × 10 5Canine luciferase-expressing cells (U-87 MGΔ, U-251 MGΔ, AsPC-1, and NALM6) were co-cultured with CAR-T cells in 200 μL of RPMI 1640 medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with low glucose (0.5 mM) or high glucose (10 mM) in flat-bottom 96-well white plates at the indicated ratios. After 24 hours of incubation, Bio-Glo (Promega, Madison, WI) was added to each well, and luminescence was measured using a Cytation5 multi-plate reader (Agilent Technologies, Santa Clara, CA). % specific lysis was calculated according to the following formula: % specific lysis = % specific lysis = [(Experimental lysis - Spontaneous lysis) / (Maximum lysis - Spontaneous lysis)] × 100.

[0529] Immunofluorescence staining

[0530] After washing, cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and permeated with cooling methanol at -20°C for 10 minutes. Cells were blocked with 3% BSA / PBS at room temperature for 60 minutes. After blocking, cells were incubated with the primary antibody at 4°C for 2 hours and with the secondary antibody at 4°C for 1 hour. Next, DAPI was added, and cells were incubated at room temperature for 5 minutes. After washing, stained cells were fixed to slides using Cytospin and analyzed using BZ-X710 (KEYENCE Corporation, Osaka, Japan).

[0531] Immunohistochemical (IHC) staining

[0532] IHC staining was performed on 5 μm-thick sections of formalin-fixed, paraffin-embedded specimens. Antigen removal was performed using citrate buffer (pH 6.0) in a steamer, followed by deparaffinization and rehydration. Slides were incubated with the primary antibody for 16 hours and with the HRP-labeled secondary antibody for 1 hour, after which they were developed with a diaminobenzidine substrate. Anti-CD3ε antibody (clone: ​​SP7, catalog number: ab16669) (Abcam, Waltham, MA) was used for primary staining. The stained slides were counterstained with hematoxylin. Hematoxylin and eosin (HE) staining were performed according to standard protocols. Deparaffinized and hydrated slides were placed in hematoxylin solution and incubated for 4 minutes. After washing, the slides were incubated with eosin solution for 2 minutes. Stained slides were scanned at 40× and 400× magnifications using a BZ-X710 (Keyence Corporation). Two pathologists independently evaluated the stained slides.

[0533] ELISA method

[0534] Total 2.0 × 10 5 10 U-87 MGΔ cells and 2.0 × 10 5 Canine CAR-T cells were co-cultured in 24-well plates. After 24 hours of culture, the supernatant was collected, and ELISA was performed to measure cytokine concentrations. ELISA kits for IFN-γ, IL-2, and TNFα (R&D Systems, Minneapolis, MN) were used according to the manufacturer's instructions. Cytokines in mouse serum were analyzed by the high-sensitivity LUMINEX assay according to the manufacturer's instructions (Merck Millipore, Burlington, MA).

[0535] Manufacturing of CAR-T cells

[0536] Peripheral blood was collected from healthy subjects, 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-to-bead ratio of 1:1 (Day 0). 24 hours after stimulation (Day 1), T cells were transfected with CAR lentivirus at MOI 3 and cultured in 30 U / ml IL-2. Half of the medium was replaced after 24 hours. After 5 days, the CD3 / 28 beads were removed. During culture, half of the medium was replaced on Days 4 and 10, and the T cell concentration was set to 0.7 × 10⁶. 6 The cell count was adjusted to 10 cells / mL. On day 10, established CAR-T cells were collected and used for subsequent analysis.

[0537] animal models

[0538] Female NSG mice (6 weeks old) were purchased from Jackson Laboratory. Prior to the painful procedure, an anesthetic was injected intraperitoneally to prevent pain. Using a stereotactic frame, tumor cells were inoculated at positions 2 mm to the right of the bregma, 3 mm posterior to the bregma, and 3 mm deep from the brain surface. A total of 2.0 × 10⁶ in 5 mL of PBS 4 After injecting the cells over a period of 1 minute, the needle was withdrawn 1 mm and left in place for an additional 1 minute before removal. Four days after tumor transplantation, tumor growth was confirmed by bioluminescence imaging (BLI), and patients were randomized to their respective treatment groups. On the 5th day, 1.0 × 10⁶ 6Canine CAR-T cells were injected via the tail vein. Tumor growth was monitored twice a week using BLI after CAR-T cell injection. Animal care and experiments were conducted in accordance with the guidelines of the Animal Committee of the National Cancer Center after obtaining approval from the Institutional Animal Care and Use Committee of the National Cancer Center.

[0539] Glucose concentration measurement

[0540] Tissue samples (5 × 5 mm) were washed to remove attached blood. The samples were chopped and centrifuged at 4°C for 10 minutes, and tumor interstitial fluid was collected. Glucose concentration in the interstitial fluid was measured using a Multiskan GO (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0541] Metabolic analysis

[0542] OCR (in pmol / min) and ECAR (in mpH / min) were evaluated using a Seahorse XF-24 metabolic extracellular flux analyzer (Agilent Technologies). CAR-T cells (conventional EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and GLUT3 EGFRvIII CAR-T cells as needed) stimulated with EGFRvIII beads (ACRO Biosystems, Beijing, China) for 24 hours were resuspended in glucose-free, unbuffered RPMI-1640 medium (Agilent Technologies) and plated on Seahorse cell plates coated with poly-L-lysine (BD Bioscience) (2.0 × 10⁶ per well). 5Canine cells). 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 manufactured by Agilent Technologies). Experiments using the Seahorse system were performed under the following assay conditions: mixing for 3 minutes, waiting for 2 minutes, and measurement for 3 minutes. Afterward, the values ​​of the metabolic parameters were calculated.

[0543] RNA sequencing and subsequent analysis

[0544] CAR-T cells classified using BD FACSymphony S6 (BD Biosciences) (conventional EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and GLUT3 EGFRvIII CAR-T cells as needed) 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 collected on days 3 and 7, and RNA was extracted using the 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). Next-generation sequencing of 150-bp paired-end reads from both ends was performed on an RNA-seq library using Novaseq X plus (Illumina, San Diego, CA). For expression profiling using RNA-seq data, paired-end reads were aligned to the hg38 human genome assembly by STAR [Ref. 44]. RNAseqChef was a web-based platform for systematic transcriptome analysis, gene expression analysis, principal component analysis, and pathway analysis [Ref. 45].

[0545] Statistical analysis

[0546] GraphPad Prism 9 (GraphPad Software, San Diego, CA) was used for statistical analysis. Relationships between groups were compared using t-tests or one-way ANOVA. Survival was analyzed using the Kaplan-Meier method and statistically compared using the log-rank test. A P-value of <0.05 was considered statistically significant.

[0547] result

[0548] GBM had low glucose levels in TME, which impaired the function of CAR-T cells.

[0549] Although accumulating evidence indicates that low glucose levels in the TME are a potential barrier to CAR-T cell therapy in solid tumors, the actual glucose concentrations in the TME and the effects of low-glucose conditions on CAR-T cell function have not yet been elucidated [Ref. 21]. The inventors investigated glucose concentrations in interstitial fluid and serum derived from surgical specimens (Fig. 1a). Glucose concentrations in the interstitial fluid of GBM specimens were approximately 10 times lower than those in serum [less than 0.5 mM (average: 0.217 mM)] (Fig. 1b). While glucose concentrations were generally low in various types of cancer, such as non-small cell lung cancer and colorectal cancer, GBM exhibited the lowest glucose concentrations in the TME. This indicated that low glucose concentrations in the TME are characteristic of GBM (Fig. 1c).

[0550] Next, the inventors investigated the effects of a low-glucose environment on CAR-T cell functions, such as cytokine production. CAR-T cells targeting the GBM-associated antigen EGFRvIII (conventional EGFRvIII CAR-T cells) were co-cultured with a human GBM cell line (U-87 MGΔ) expressing EGFRvIII under low-glucose conditions (0.5 mM) that replicate the TME of GBM. Upon stimulation by U-87 MGΔ, cytokine production (IFN-γ, IL-2, and TNFα) was significantly reduced under the low-glucose environment even after short-term (16 hours) exposure (Figs. 29d and 29e). To further investigate functional changes in CAR-T cells under low-glucose conditions, CAR-T cells were stimulated for 16 hours under low-glucose (0.5 mM) or high-glucose (10 mM) conditions, and gene expression profiles were analyzed. Gene expression profiles were significantly altered by exposure to low-glucose conditions (Fig. 35a). The set of genes related to the cell cycle, differentiation, and cytokine production was significantly reduced, while the set of genes related to cellular responses to glucose starvation and endogenous apoptosis was increased under low-glucose conditions (Figs. 29f and 29g). As described above, the effector function of CAR-T cells was immediately impaired when exposed to low-glucose conditions such as the TME of GBM.

[0551] Overexpression of GLUT3 enhanced the metabolic fitness of CAR-T cells.

[0552] Among the SLC2 family of glucose transporters (GLUTs), GLUT3 encoded by SLC2A3 is characterized by having the highest affinity and being predominantly expressed in neurons and glial cells with high glucose requirements [Reference 16]. Accordingly, the inventors investigated whether the expression of GLUT3 by CAR-T cells could promote glucose uptake in the low-glucose TME of the GBM, thereby promoting the activity of CAR-T cells. By fusing SLC2A3 to a conventional anti-EGFRvIII CAR construct via a self-cleaved P2A sequence, the simultaneous stable expression of CAR and GLUT3 was enabled (GLUT3 EGFRvIII CAR) (Fig. 30a). GLUT3 was expressed on the membranes of T cells introduced with GLUT3 EGFRvIII CAR (GLUT3 EGFRvIII CAR-T cells) (Fig. 30b), and its expression level was approximately twice that of parent anti-EGFRvIII CAR-T cells (conventional EGFRvIII CAR-T cells) and similar to that of the GBM cell line U-87 MGΔ, whereas no change was observed in the expression level of GLUT1 (Figs. 30c and 36a). Glucose uptake measured by a glucose uptake assay using the glucose analog 2-NBDG was higher in GLUT3 EGFRvIII CAR-T cells than in conventional EGFRvIII CAR-T cells (Fig. 30d). Therefore, glycolysis and glycolytic capacity of GLUT3 EGFRvIII CAR-T cells were significantly improved (Figs. 30e and 36b). Metabolic analysis showed that GLUT3 EGFRvIII CAR-T cells had a higher oxygen consumption rate (OCR) compared to conventional EGFRvIII CAR-T cells. high and high extracellular acidification rate (ECAR) highIt was further confirmed that they transitioned to a more energetic state (Fig. 30f). In a glucose competition assay in which GLUT3 EGFRvIII CAR-T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were co-cultured with conventional EGFRvIII CAR-T cells at a 1:1 ratio, the number of GLUT3 EGFRvIII CAR-T cells relative to conventional 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 in terms of glucose uptake, particularly in low glucose environments (Figs. 36c and 36d). Thus, stable GLUT3 expression by CAR-T cells promotes glucose uptake, enables CAR-T cells to be competitive in terms of glucose uptake, and enhances the metabolic fitness of CAR-T cells in a limited-glucose environment.

[0553] Since the expression of GLUT3 improved the metabolic fitness of CAR-T cells, its effector function and safety profile were investigated. Accordingly, the inventors investigated the effector function of GLUT3 EGFRvIII CAR-T cells in both in vitro and in vivo assays. Compared to conventional EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells significantly increased cytokine production (IFN-γ, IL-2, and TNFα) at ​​both low glucose levels (0.5 mM) and high glucose levels (10 mM) (Figs. 30g and 30h, and Fig. 37a). CD8 + T cell subset and CD4 +Cytokine production was enhanced in both T cell subsets (Figs. 37b and 37c). GLUT3 EGFRvIII CAR-T cells exhibited increased cytotoxicity against U-87 MGΔ cells and another EGFRvIII-expressing GBM cell line, U-251 MGΔ cells, under both low-glucose and high-glucose conditions (Figs. 30i, 37d, and 37e). After antigen stimulation, the expression of exhaustion-related molecules, including PD-1 and Tim-3, was higher in GLUT3 EGFRvIII CAR-T cells than in conventional EGFRvIII CAR-T cells (Figs. 30j and 30k, and Figs. 37f and 37g). Furthermore, enhancement of effector function by stable GLUT3 expression was similarly observed in CAR-T cells targeting a different antigen, CD19 (GLUT3 CD19 CAR-T cells). This indicated that the enhancement of metabolic fitness by GLUT3 expression is universal in all CARs regardless of the single-strand variable fragment (scFv) or target antigen used (Figs. 38a to 38c). Thus, stable GLUT3 expression allowed CAR-T cells to be activated in a TME with low glucose levels, even though an exhaustion phenotype was present.

[0554] Stable GLUT3 expression overactivated CAR-T cells and caused severe adverse effects.

[0555] Using an intracranial U-87 MGΔ xenograft model, the effects of stable GLUT3 expression in CAR-T cells on antitumor efficacy and safety profiles were further investigated (Fig. 31a). GLUT3 EGFRvIII CAR-T cells were administered intravenously 5 days after intracranial tumor inoculation. As expected, GLUT3 EGFRvIII CAR-T cell therapy demonstrated enhanced antitumor efficacy (CR: 4 / 6) compared to conventional EGFRvIII CAR-T cell therapy (CR: 2 / 6) (Fig. 31b). Nevertheless, GLUT3 EGFRvIII CAR-T cell therapy did not improve survival due to a high incidence of treatment-related death (TRM) associated with deterioration of general condition and severe weight loss (Figs. 31c to 31e). Consistent with these findings, pathological analysis revealed T cell hyperactivation in the brain tissue of mice treated with GLUT3 EGFRvIII CAR-T cells, specifically accompanied by diffuse infiltration from the ventricular walls into the brain parenchyma (Fig. 31f). Additionally, diffuse alveolar damage manifested as thickening of the alveolar walls accompanied by T cell infiltration was detected in the lung tissue (Fig. 31g). Indeed, effector memory T cells (CD45RA CCR7 -- The frequency of ) was significantly higher in mice administered GLUT3 EGFRvIII CAR-T cells than in mice administered conventional EGFRvIII CAR-T cells (Fig. 31h). In addition, gene expression assays indicated that a set of genes related to cytokine production, T cell differentiation, and T cell activation was significantly upregulated in GLUT3 EGFRvIII CAR-T cells even on day 7 after stimulation (Fig. 31i). Therefore, stable GLUT3 expression by CAR-T cells caused dysregulation of the T cell activation state due to excessive glucose uptake, indicating the need to finely regulate metabolic compatibility.

[0556] As-needed expression of GLUT3 by antigen stimulation was achieved by the CAR-NFAT-GLUT3 construct.

[0557] Considering the severe toxicity caused by the overactivation of GLUT3 EGFRvIII CAR-T cells, the inventors investigated whether the benefits of GLUT3 expression could be maximized and toxicity minimized by expressing GLUT3 in response to glucose requirements, specifically at the time of T-cell stimulation upon target antigen recognition in the TME. A construct in which SLC2A3 is positioned downstream of the NFAT-binding domain was developed to enable GLUT3 expression when needed (GLUT3 CAR when needed) (Fig. 32a). T-cell activation by antigen recognition induced nuclear translocation of NFAT and promoted GLUT3 expression. Recognition of the target antigen by T-cells carrying the GLUT3 CAR when needed construct (GLUT3 EGFRvIII CAR-T cells when needed) induced GLUT3 expression. GLUT3 expression returned to initial levels 7 days after the last antigen stimulation (Figs. 32b and 32c). In fact, glucose uptake and glycolysis were higher in required GLUT3 EGFRvIII CAR-T cells than in conventional EGFRvIII CAR-T cells (Figs. 32d, 32e, and 39a). In required GLUT3 EGFRvIII CAR-T cells, metabolism was in a more energetic state on day 3 (OCR high ECAR highIt was converted to and recovered to a level similar to conventional EGFRvIII CAR-T cells on day 7 after the last antigen stimulation (Fig. 33f). Cytokine production was significantly higher in GLUT3 EGFRvIII CAR-T cells than in conventional EGFRvIII CAR-T cells at both low-glucose (0.5 mM) and high-glucose (10 mM) levels (Figs. 33g, 33h, and 39b). GLUT3 EGFRvIII CAR-T cells also exhibited increased cytotoxicity against U-87 MGΔ cells and U-251 MGΔ cells under both low-glucose and high-glucose conditions (Figs. 33i and 39c). In summary, the GLUT3 CAR construct successfully improved metabolic compatibility as needed, and the GLUT3 EGFRvIII CAR-T cells enhanced cytokine production and cytotoxicity under both low-glucose and high-glucose conditions.

[0558] When necessary, GLUT3 expression enabled the sustained effector function of CAR-T cells.

[0559] Since we have successfully developed GLUT3 EGFRvIII CAR-T cells for as-needed use possessing enhanced cytokine production and cytotoxicity, the inventors decided to investigate a comprehensive immunological profile focusing on persistence, function, and molecular expression. First, cell proliferation after antigen stimulation was investigated. While GLUT3 EGFRvIII CAR-T cells for as-needed use continued to proliferate after antigen stimulation, GLUT3 EGFRvIII CAR-T cells did not proliferate (Figs. 40a and 40b). This failure of proliferation in GLUT3 EGFRvIII CAR-T cells was primarily attributed to the induction of apoptosis, which was caused by hyperactivation known as activation-induced apoptosis (AICD), a phenomenon critical to T cell homeostasis [Reference 22] (Fig. 34a). Through gene expression analysis, it was further confirmed that under low-glucose conditions, the expression of genes related to apoptosis increased in GLUT3 EGFRvIII CAR-T cells compared to GLUT3 EGFRvIII CAR-T cells and conventional EGFRvIII CAR-T cells (Fig. 34b). Additionally, the expression of apoptosis-related genes was closely correlated with the expression of TNFα signaling genes, suggesting a role of TNFα in apoptosis in GLUT3 EGFRvIII CAR-T cells (Fig. 34c). Meanwhile, the expression of the T cell activation-related gene family increased in GLUT3 EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells compared to conventional EGFRvIII CAR-T cells (Fig. 34b). Therefore, GLUT3 EGFRvIII CAR-T cells [develop] a central memory (CD62L CCR7 ++ CD45RA - ) and naive phenotype (CD62L CCR7 ++ CD45RA + While ) maintained, GLUT3 EGFRvIII CAR-T cells exhibited the final differentiated phenotype (CD62L CCR7 -- CD45RA+ ... (Figs. 34d and 40c). In addition, the expression of exhaustion-related molecules, including PD-1 and Tim-3, was lower in GLUT3 EGFRvIII CAR-T cells when needed compared to GLUT3 EGFRvIII CAR-T cells and conventional EGFRvIII CAR-T cells (Figs. 34e and 40d). Furthermore, the composite gene expression profiles of GLUT3 EGFRvIII CAR-T cells and GLUT3 EGFRvIII CAR-T cells when needed were similar on day 3 after stimulation, but significantly different on day 7 after stimulation (Fig. 34f). RNA-seq analysis of each CAR-T cell population on day 7 after stimulation further confirmed that while GLUT3 EGFRvIII CAR-T cells relied on glycolysis, when needed, GLUT3 EGFRvIII CAR-T cells maintained oxidative phosphorylation and fatty acid oxidation reflecting an activated state, namely naive / stem cell memory and central memory phenotypes (Fig. 34g). These findings indicated that when needed, GLUT3 expression prevents the overactivation of CAR-T cells and maintains functional CAR-T cells for a long period. From the above, particularly from the results in Fig. 34, it can be said that constitutively expressed GLUT3 enhances the antitumor effect (i.e., effector function) of EGFRvIII CAR-T cells. It can also be said that when needed, 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. In addition, it can be concluded that adverse events such as weight loss and treatment-related death induced by constitutively expressed GLUT3 were prevented, and as a result, significant improvement in survival, including cure, was achieved.

[0560] When necessary, GLUT3 EGFRvIII CAR-T cells demonstrated an enhanced antitumor effect without severe toxicity in an intracranial human GBM cell xenograft model.

[0561] Based on the superior phenotype of GLUT3 EGFRvIII CAR-T cells as needed, the antitumor efficacy and safety profile were investigated in an intracranial human GBM cell xenograft model (Fig. 35a). Compared to conventional EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells as needed and GLUT3 EGFRvIII CAR-T cells demonstrated superior antitumor effects, and most mice achieved CR (5 / 10 CR for conventional EGFRvIII CAR-T cells, 9 / 10 CR for GLUT3 EGFRvIII cells, and 9 / 10 CR for GLUT3 EGFRvIII CAR-T cells as needed) (Figs. 35b and 41a). However, as observed in previous experiments (Figs. 32a to 32e), GLUT3 EGFRvIII CAR-T cells induced severe toxicity accompanied by significant body weight loss and failed to improve survival. In contrast, when needed, GLUT3 EGFRvIII CAR-T cells demonstrated a significant improvement in survival without causing severe toxicity or body weight loss (Figs. 35c to 35e). Consequently, tumor rechallenge was suppressed only in mice administered with GLUT3 EGFRvIII CAR-T cells when needed (Fig. 35b). Thus, on day 14 after CAR-T cell injection, high frequencies of the naive / stem cell memory subset and the central memory subset were detected in the peripheral blood of mice treated with GLUT3 EGFRvIII CAR-T cells when needed, whereas the effector memory subset was more abundant in mice treated with GLUT3 EGFRvIII CAR-T cells (Fig. 35f). This increase in antitumor effect induced by GLUT3 EGFRvIII CAR-T cells as needed was similarly observed in another xenograft model (U-251 MGΔ); compared to mice in other treatment groups, mice treated with GLUT3 EGFRvIII CAR-T cells as needed showed superior survival without severe toxicity (Fig. 35g and Figs. 41b and 41c).Cytokine levels (TNFα and GM-CSF) remained high in the serum of mice administered GLUT3 EGFRvIII CAR-T cells even on day 14, when the tumor had already been rejected, and the cytokine reduction rate was significantly lower in mice administered GLUT3 EGFRvIII CAR-T cells than in mice administered conventional EGFRvIII CAR-T cells or as needed EGFRvIII CAR-T cells (Figs. 35h and 35i). Additionally, levels of cytokines (IL-5, IP-10, and MDC) reported to be involved in the development of cytokine release syndrome (CRS) were higher in the serum of mice treated with GLUT3 EGFRvIII CAR-T cells than in the serum of mice treated with GLUT3 EGFRvIII CAR-T cells as needed (Fig. 41d). Overall, on-demand GLUT3 expression by CAR-T cells is a promising strategy for achieving sustained antitumor effects in GBM without severe toxicity.

[0562] Discussion

[0563] Enhancing the antitumor effect by improving the metabolic compatibility of CAR-T cells in the TME is a novel concept aimed at accelerating the clinical application of CAR-T cell therapy for refractory solid tumors. Although some clinical trials of CAR-T cells in the GBM have failed to demonstrate clinical utility, immunological monitoring of thes...

Claims

Claim 1 T cells having enhanced glucose uptake capacity in which the expression of glucose transporters in T cells is regulated in response to changes in tumor microenvironment conditions. Claim 2 In claim 1, the tumor microenvironment conditions include T cells including the degree of TCR-related signal transmission. Claim 3 In paragraph 2, the TCR-related signal is a T cell mediated by a molecule selected from the group consisting of a TCR signal or a part thereof, and a chimeric signal of an antigen different from the TCR signal (also called a CAR signal). Claim 4 In paragraph 2 or 3, the TCR-related signal is a T cell mediated by a molecule containing a TCR signaling domain. Claim 5 In any one of claims 1 to 4, the change in tumor microenvironment conditions comprises the presentation of tumor antigens and / or costimulatory ligands and / or changes in cytokine conditions of T cells. Claim 6 In any one of paragraphs 2 to 5, the degree of transmission of TCR-related signals is a T cell including the degree of TCR stimulation. Claim 7 In any one of paragraphs 2 to 6, the degree of transmission of TCR-related signals comprises at least one selected from the group consisting of the degree of TCR stimulation, TCR co-stimulation, and humoral factor. Claim 8 In any one of paragraphs 2 to 7, the degree of transmission of TCR-related signals comprises at least one selected from the group consisting of the degree of TCR stimulation and TCR co-stimulation and humoral factors. Claim 9 In any one of paragraphs 2 to 8, the degree of transmission of TCR-related signals comprises T cells including the degree of TCR stimulation, TCR co-stimulation, and humoral factors. Claim 10 In any one of claims 6 to 9, the TCR stimulation is a T cell selected from the group consisting of the NFAT, NF-κB, and STAT transcription factor families that translocate into the nucleus in connection with T cell activation. Claim 11 In any one of claims 1 to 10, the tumor microenvironment conditions include T cells with an oxygen concentration level. Claim 12 A T cell comprising a nucleic acid sequence encoding a glucose transporter and operably linked to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. Claim 13 In claim 12, the tumor microenvironment sensing element comprises: (i) an inducible activation motif that is activated in response to a change in tumor microenvironment conditions, and (ii) a T cell comprising a nucleic acid sequence encoding a glucose transporter degradation factor that is inactivated in response to a change in tumor microenvironment conditions. Claim 14 In claim 12 or 13, the tumor microenvironment sensing element comprises: (i) an inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to activation of the T cell binds; (ii) a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to a decrease in oxygen concentration binds; and (iii) a T cell comprising a nucleic acid sequence encoding a glucose transporter degrading enzyme whose activity of degrading a glucose transporter is inactivated in response to a decrease in oxygen concentration. Claim 15 A T cell according to any one of claims 1 to 14, wherein the nucleic acid sequence encoding a glucose transporter comprises a construct operably linked to a nucleic acid sequence encoding an inducible activation motif that is activated in response to a change in tumor microenvironment conditions and / or a degradation factor for the glucose transporter that is inactivated in response to a change in tumor microenvironment conditions. Claim 16 A T cell according to any one of claims 1 to 15, wherein the nucleic acid sequence encoding the glucose transporter comprises a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to activation of the T cell binds. Claim 17 In any one of claims 1 to 16, the nuclear translocation factor is a T cell selected from the group consisting of NFAT, NF-κB, and STAT transcription factor families. Claim 18 In claim 16 or 17, the nuclear translocation factor-binding inducible activation motif is a T cell selected from the group consisting of an NFAT-binding domain, a κB motif (GGGACTTTCC) (SEQ No. 1), a STAT-binding sequence (TTCNNNGAA), and sequences modified thereof. Claim 19 In any one of claims 16 to 18, the combination of a nuclear translocation factor and a nuclear translocation factor-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 T cell comprising a combination of a STAT transcription factor family and a STAT-binding sequence (TTCNNNGAA). Claim 20 A T cell according to any one of claims 1 to 19, wherein the nucleic acid sequence encoding the glucose transporter comprises a structure operably linked to a nuclear translocation factor-binding inducible activation motif to which a nuclear translocation factor translocating into the nucleus in response to a decrease in oxygen concentration binds. Claim 21 In any one of paragraphs 16 to 20, the nuclear translocation factor is a T cell selected from the group consisting of HIF-1. Claim 22 In any one of paragraphs 13 to 21, the nuclear translocation factor-binding inducible activation motif is a T cell selected from the group consisting of HIF-RE. Claim 23 In any one of claims 13 to 22, the combination of nuclear translocation factor and nuclear translocation factor-binding inducible activation motif is: (1) a T cell selected from the group consisting of combinations of HIF-1 and HIF-RE. Claim 24 A T cell according to any one of claims 1 to 23, wherein the nucleic acid sequence encoding a glucose transporter comprises a structure operably linked to a nucleic acid sequence encoding a factor whose activity of degrading the glucose transporter is inactivated in response to a decrease in oxygen concentration. Claim 25 In any one of paragraphs 15 to 24, the factor to be inactivated is a T cell selected from the group consisting of oxygen-dependent degradation factors (ODD). Claim 26 In any one of claims 1 to 25, the T cell is a T cell comprising a chimeric antigen receptor (CAR). Claim 27 In any one of paragraphs 1 to 26, the T cell is a T cell having an effector function. Claim 28 In any one of paragraphs 1 to 26, the T cell is a T cell that is a precursor cell of an effector T cell (Teff). Claim 29 In paragraph 28, a T cell in which the memory function of the precursor cell of the effector T cell is extended. Claim 30 In paragraph 28 or 29, a T cell in which the memory T cell phenotype of the precursor cell of the effector T cell is maintained. Claim 31 A T cell according to any one of claims 28 to 30, wherein the effector T cell is activated or the effector T cell activity is maintained, and the memory function of the effector T cell precursor cell is extended or the memory T cell phenotype of the effector T cell precursor cell is maintained. Claim 32 In any one of claim 1 or claims 26 to 31, expression and / or enhancement are T cells specifically achieved in a tumor environment. Claim 33 In any one of paragraphs 26 to 32, CAR is a T cell expressed in a T cell. Claim 34 In any one of claims 1 to 33, the glucose transporter is a T cell comprising GLUT1, GLUT2, GLUT3 and GLUT4. Claim 35 In any one of claims 1 to 34, the glucose transporter is a T cell in which GLUT3 is the glucose transporter. Claim 36 In any one of paragraphs 1 to 35, the T cell is a human T cell. Claim 37 A cell population comprising T cells having enhanced glucose uptake capacity in response to need (on demand). Claim 38 A cell population comprising T cells according to any one of paragraphs 1 to 36. Claim 39 In paragraph 37 or 38, T cells are a cell population comprising cells having the characteristic of exhibiting effector function when introduced into the body. Claim 40 In any one of paragraphs 37 to 39, the T cells are a cell population comprising a chimeric antigen receptor (CAR). Claim 41 A cell population according to any one of claims 37 to 40, wherein the T cells are modified so that the expression of a glucose transporter in the T cells is regulated in response to changes in tumor microenvironment conditions and / or comprise a nucleic acid sequence encoding a glucose transporter, wherein the nucleic acid sequence is operably connected to a tumor microenvironment sensing element that detects changes in tumor microenvironment conditions. Claim 42 In any one of paragraphs 37 to 41, T cells are a group of cells having effector functions. Claim 43 In any one of paragraphs 37 to 42, the cell population is a cell population including Teff. Claim 44 In any one of paragraphs 37 to 43, the cell population is a cell population comprising Teff's precursor cells. Claim 45 In any one of paragraphs 38 to 44, CAR is a population of cells expressed in T cells. Claim 46 In any one of claims 37 to 45, the glucose transporter is a cell population comprising GLUT1, GLUT2, GLUT3 and GLUT4. Claim 47 In any one of paragraphs 37 to 46, a cell population in which the glucose transporter is GLUT3. Claim 48 In any one of paragraphs 37 to 47, the T cells are a population of cells that are human T cells. Claim 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. Claim 50 In paragraph 49, the pharmaceutical composition is a pharmaceutical composition intended for the prevention or treatment of cancer. Claim 51 In paragraph 49 or 50, the pharmaceutical composition is a pharmaceutical composition intended to cure cancer. Claim 52 A pharmaceutical composition according to any one of claims 49 to 51, wherein the pharmaceutical composition is intended for the prevention of cancer metastasis or the prevention of cancer recurrence. Claim 53 A pharmaceutical composition according to any one of paragraphs 49 to 52, wherein the pharmaceutical composition is intended to prevent or treat cancer so as not to cause cancer to recur. Claim 54 In any one of paragraphs 1 through 36 or any one of paragraphs 37 through 48, the T cell or cell population is a T cell or cell population intended for use as a medicament. Claim 55 A method for preventing or treating a disease in a subject, comprising the step of administering to the subject an effective amount of T cells according to any one of claims 1 to 36 or a cell population according to any one of claims 37 to 48. Claim 56 Use of T cells according to any one of claims 1 to 36 or cell populations according to any one of claims 37 to 48 for the production of drugs.

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