Surviving T cells, surviving T cell populations, and drug compositions

Abstract

This disclosure provides a cell that does not starve or deplete even in glucose-depleted environments and is competitive with cancer cells. Specifically, this disclosure provides a surviving T cell with enhanced glucose uptake capacity, said T cell being modified to express glucose transporters and / or with enhanced expression of glucose transporters. Cell populations comprising such cells, pharmaceutical compositions, treatment methods, etc., are also provided.

Description

Technical Field

[0001] This disclosure relates to survivor T cells with enhanced glucose uptake capacity, populations of survivor T cells, and pharmaceutical compositions comprising such cells or populations. Background Technology

[0002] In order to give cancer cells an advantage in cell proliferation, they reprogram their metabolic system, actively utilizing the glycolysis system, which has low ATP production efficiency, even under aerobic conditions, thereby enhancing glucose uptake and lactic acid production (Warburg effect).

[0003] In the tumor microenvironment, many factors inhibit the metabolic activity and function of anti-tumor T cells. Besides immunosuppressive factors, metabolic competition between tumors and T cells also contributes to the formation of the immunosuppressive environment. When T cells are stimulated by the TCR, intracellular calcium levels... 2+ Increased concentration of phosphatase activates calmolysin. Due to the activation of calmolysin, the dephosphorylated transcription factor NFAT translocates to the nucleus, where it promotes and activates the transcription of genes such as IL-2 through interactions with other transcription factors.

[0004] On the other hand, because cancer cells consume large amounts of glucose, leading to their depletion, T cells require glucose as an energy source. eff After tumor-specific T cells infiltrate the tumor, even if the TCR receives the signal, intracellular Ca2+... 2+ The concentration also decreases, reducing the translocation of NFAT into the nucleus. As a result, T cells become dysfunctional, and cell proliferation and cytokine production are inhibited.

[0005] In addition to glucose, if nutrients such as amino acids and fatty acids are also consumed in large quantities by cancer cells, the competing T cells will become dysfunctional. Thus, in the tumor microenvironment, the metabolic mechanisms that support the active proliferation of cancer cells inhibit the anti-tumor effector function of tumor-specific T cells.

[0006] Therefore, it is desirable to have a cell that does not starve or deplete even in such an environment and is competitive with cancer cells. Summary of the Invention

[0007] [Methods used to solve problems]

[0008] This disclosure provides a glucose-enhanced T cell that can function without starvation and exhaustion even in a tumor environment; a cell population comprising the T cell; and a pharmaceutical composition comprising such cells.

[0009] Therefore, this disclosure provides the following content.

[0010] [Project 1] A T cell with enhanced glucose uptake capacity.

[0011] [Item 2] The T cell according to Item 1, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0012] [Item 2A] A T cell according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0013] [Item 3] The T cell according to any one of the above items, wherein the T cell has effector function.

[0014] [Item 4] The T cell according to any one of the preceding items, wherein the T cell is an effector T cell (T1). eff ) precursor cells.

[0015] [Item 5] A T cell according to any one of the preceding items, wherein the CAR is expressed in the T cell.

[0016] [Item 6] T cells according to any one of the preceding items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

[0017] [Item 7] T cells according to any one of the above items, wherein the glucose transporter is GLUT3.

[0018] [Item 8] The T cell according to any one of the above items is a human T cell.

[0019] [Item 9] A cell population comprising T cells with enhanced glucose uptake capacity, said T cells comprising cells that possess effector functional characteristics upon introduction into the body.

[0020] [Item 10] A cell population according to any one of the preceding items, wherein the T cells are modified to express glucose transporters and / or have enhanced expression of glucose transporters.

[0021] [Item 11] The cell population according to any one of the above items, wherein the T cells have effector functions.

[0022] [Item 12] A cell population according to any one of the preceding items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0023] [Item 12A] A cell population according to any one of the preceding items, wherein the T cells contain a chimeric antigen receptor (CAR).

[0024] [Item 13] A cell population according to any one of the preceding items, wherein the cell population comprises T eff .

[0025] [Item 14] A cell population according to any one of the preceding items, wherein the cell population comprises T eff The precursor cells.

[0026] [Item 14A] A cell population according to any one of the preceding items, wherein the cell population (or T) eff The precursor cells (Tnv, Tscm, Tcm and Temra) contain at least one cell selected from the group consisting of Tnv, Tscm, Tcm and Temra.

[0027] [Item 15] A cell population according to any one of the preceding items, wherein the CAR or TCR is expressed in the T cells.

[0028] [Item 16] A cell population according to any one of the preceding items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

[0029] [Item 17] A cell population according to any one of the preceding items, wherein the glucose transporter is GLUT3.

[0030] [Item 18] The cell population according to any one of the preceding items, wherein the T cells are human T cells.

[0031] [Item 19] A pharmaceutical composition comprising T cells as described in any one of the preceding items or a cell population as described in any one of the preceding items.

[0032] [Item 20] A pharmaceutical composition according to any one of the preceding items, used for the treatment or prevention of cancer, autoimmune diseases, allergies or infections.

[0033] [Item 20A] A pharmaceutical composition according to any one of the above items, used to cure cancer.

[0034] [Item 21] A pharmaceutical composition for preventing or treating a disease of a subject, the pharmaceutical composition being characterized in that:

[0035] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0036] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0037] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0038] [Item 21A] A pharmaceutical composition according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0039] [Item 21B] A pharmaceutical composition according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0040] [Item 21C] A pharmaceutical composition according to any one of the preceding items, wherein the disease includes cancer, autoimmune disease, allergy, or infection.

[0041] [Item 21D] A pharmaceutical composition according to any one of the preceding items, wherein the disease includes cancer.

[0042] [Item 22] The composition according to any one of the above items, wherein the glucose transporter is GLUT3.

[0043] [Item 23] The T cells or cell populations described in any of the above items, used as a drug.

[0044] [Item 24] Cells or cell populations according to any of the above items, used for the treatment or prevention of cancer, autoimmune diseases, allergies or infections.

[0045] [Item 24A] A cell or cell population according to any one of the preceding items, used to cure cancer.

[0046] [Item 25] A cell or cell population for preventing or treating a disease in a subject, characterized in that:

[0047] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0048] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0049] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0050] [Item 25A] A cell or cell population according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0051] [Item 25B] A cell or cell population according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0052] [Item 25C] Cells or cell populations according to any one of the preceding items, wherein the disease includes cancer, autoimmune diseases, allergies, or infectious diseases.

[0053] [Item 25D] A cell or cell population according to any one of the preceding items, wherein the disease includes cancer.

[0054] [Item 26] A cell or cell population according to any one of the preceding items, wherein the glucose transporter is GLUT3.

[0055] [Item 27] ​​A method of treating or preventing said object, comprising the step of administering an effective amount of any of the above items' T cells or any of the above items' cell populations to the object in need.

[0056] [Item 28] The method according to any one of the preceding items, wherein the treatment or prevention of the object is used to treat or prevent cancer, autoimmune diseases, allergies or infections.

[0057] [Item 28A] The method according to any one of the preceding items, wherein the treatment or prevention of said object is for the purpose of curing cancer.

[0058] [Item 29] A method for preventing or treating a disease in a subject, the method comprising:

[0059] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0060] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0061] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0062] [Item 29A] The method according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0063] [Item 29B] The method according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0064] [Item 29C] The method according to any one of the preceding items, wherein the disease includes cancer, autoimmune disease, allergy, or infection.

[0065] [Item 29D] The method according to any one of the preceding items, wherein the disease includes cancer.

[0066] [Item 30] The method according to any one of the above items, wherein the glucose transporter is GLUT3.

[0067] [Item 31] Use of any of the above items’ T cells or cell populations for manufacturing a medicament comprising said cells or cell populations.

[0068] [Item 32] The use according to any of the above items, wherein the drug is used to treat or prevent cancer, autoimmune diseases, allergies or infections.

[0069] [Item 32A] The use according to any of the above items, wherein the drug is used to cure cancer.

[0070] [Item 33] According to the use described in any of the above items, wherein the medicine is used to prevent or treat a disease of a subject, the medicine is characterized in that:

[0071] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0072] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0073] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0074] [Item 33A] The use according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0075] [Item 33B] The use according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0076] [Item 33C] The use according to any of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infection.

[0077] [Item 33D] The use as described in any of the above items, wherein the disease includes cancer.

[0078] [Item 34] According to the use described in any of the above items, wherein the glucose transporter is GLUT3.

[0079] This disclosure also provides the following content.

[0080] [Item B0] An effector T cell that is modified to express a glucose transporter and / or has enhanced expression of the glucose transporter, said T cell exhibiting at least the same effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM].

[0081] [Item B1] An effector T cell comprising a chimeric antigen receptor (CAR) is a modified effector T cell expressing a glucose transporter and / or with enhanced expression of the glucose transporter, said T cell exhibiting at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM].

[0082] [Item B2] The T cell according to the above item, wherein the CAR is expressed in the T cell.

[0083] [Item B3] T cells according to any one of the preceding items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

[0084] [Item B4] A T cell comprising a chimeric antigen receptor [CAR], which is a modified effector T cell expressing a glucose transporter and / or with enhanced expression of the glucose transporter, the T cell exhibiting at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM], and the glucose transporter being GLUT3.

[0085] [Project B5] An effector T cell with the GLUT3 gene introduced.

[0086] [Item B6] The T cell according to any one of the preceding items, wherein the T cell is a human T cell.

[0087] [Item B7A] A cell population comprising effector T cells modified to express glucose transporters and / or with enhanced expression of glucose transporters, said T cells exhibiting at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM].

[0088] [Item B8] A cell population according to any one of the preceding items, wherein the CAR is expressed in the T cells.

[0089] [Item B7] A cell population comprising effector T cells containing a chimeric antigen receptor [CAR] and modified to express glucose transporters and / or enhanced expression of glucose transporters, said T cells exhibiting at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM].

[0090] [Item B8] A cell population according to any one of the preceding items, wherein the CAR is expressed in the T cells.

[0091] [Item B9] A cell population according to any one of the preceding items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

[0092] [Item B10] A cell population comprising effector T cells containing a chimeric antigen receptor [CAR] and modified to express a glucose transporter and / or with enhanced expression of the glucose transporter, said T cells exhibiting at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM], wherein said glucose transporter is GLUT3.

[0093] [Item B11] A cell population comprising effector T cells with the GLUT3 gene introduced.

[0094] [Item B12] The cell population according to any one of the preceding items, wherein the T cells are human T cells.

[0095] [Item B13] A pharmaceutical composition comprising T cells or cell populations as described in any of the preceding items.

[0096] [Item B14] A pharmaceutical composition according to any one of the preceding items, used for the treatment or prevention of cancer, autoimmune diseases, allergies, or infections.

[0097] [Item B15] A pharmaceutical composition according to any one of the above items, used to cure cancer.

[0098] [Item B16] A pharmaceutical composition for the prevention or treatment of a disease in a subject, said pharmaceutical composition being characterized in that:

[0099] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0100] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0101] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0102] [Item B17] A pharmaceutical composition according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0103] [Item B18] A pharmaceutical composition according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0104] [Item B19] A pharmaceutical composition according to any one of the preceding items, wherein the disease includes cancer, autoimmune disease, allergy, or infection.

[0105] [Item B20] A pharmaceutical composition according to any one of the preceding items, wherein the disease includes cancer.

[0106] [Item B21] The composition according to any one of the above items, wherein the glucose transporter is GLUT3.

[0107] [Item B22] T cells according to any one of the preceding items or cell populations according to any one of the preceding items, used as a drug.

[0108] [Item B23] Cells or cell populations according to any one of the preceding items, used for the treatment or prevention of cancer, autoimmune diseases, allergies or infectious diseases.

[0109] [Item B24] A cell or cell population according to any one of the preceding items, used to cure cancer.

[0110] [Item B25] A cell or cell population for preventing or treating a disease in a subject, said cell or cell population being characterized by:

[0111] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0112] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0113] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0114] [Item B26] A cell or cell population according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0115] [Item B27] A cell or cell population according to any one of the preceding items, wherein the T cell contains a chimeric antigen receptor (CAR).

[0116] [Item B28] Cells or cell populations according to any one of the preceding items, wherein the disease includes cancer, autoimmune diseases, allergies, or infectious diseases.

[0117] [Item B29] The cell or cell population according to any one of the preceding items, wherein the disease includes cancer.

[0118] [Item B30] A cell or cell population according to any one of the preceding items, wherein the glucose transporter is GLUT3.

[0119] [Item B31] A method of treating or preventing said object, comprising the step of administering an effective amount of any of the above items' T cells or any of the above items' cell populations to the object in need.

[0120] [Item B32] The method according to any one of the preceding items, wherein the treatment or prevention of the object is used to treat or prevent cancer, autoimmune diseases, allergies or infections.

[0121] [Item B33] The method according to any one of the preceding items, wherein the treatment or prevention of said object is for the purpose of curing cancer.

[0122] [Item B34] A method for preventing or treating a disease in a subject, the method comprising:

[0123] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0124] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0125] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0126] [Item B35] The method according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0127] [Item B36] The method according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0128] [Item B37] The method according to any one of the preceding items, wherein the disease includes cancer, autoimmune disease, allergy, or infection.

[0129] [Item B38] The method according to any one of the preceding items, wherein the disease includes cancer.

[0130] [Item B39] The method according to any one of the above items, wherein the glucose transporter is GLUT3.

[0131] [Item B40] Use of any of the above items’ T cells or cell populations for manufacturing a medicament comprising said cells or cell populations.

[0132] [Item B41] The use according to any of the above items, wherein the drug is used to treat or prevent cancer, autoimmune diseases, allergies or infections.

[0133] [Item B42] The use according to any of the above items, wherein the drug is used to cure cancer.

[0134] [Item B43] According to the use described in any of the above items, wherein the drug is used to prevent or treat a disease of a subject, the drug is characterized in that:

[0135] (A) Collect values ​​of the subject's glucose uptake capacity or related indicators;

[0136] (B) Based on the aforementioned indicators, select T cells with appropriate expression characteristics and enhanced glucose uptake capacity, or a cell population containing said T cells; and

[0137] (C) The glucose-enhanced T cells or a cell population containing the T cells are administered to the subject in an appropriate manner and dosage.

[0138] [Item B44] The use according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0139] [Item B45] The use according to any one of the preceding items, wherein the T cell comprises a chimeric antigen receptor (CAR).

[0140] [Item B46] The use according to any of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infection.

[0141] [Item B47] According to the use described in any of the above items, wherein the disease includes cancer.

[0142] [Item B48] According to the use described in any of the above items, wherein the glucose transporter is GLUT3.

[0143] In this disclosure, in addition to the explicit combinations, one or more of the above features may be intentionally combined to provide further embodiments. Furthermore, those skilled in the art will recognize further implementations and advantages of this disclosure upon reading and understanding the following detailed description.

[0144] In addition to the above, the features, significant effects and benefits of this disclosure will become clear to those skilled in the art by referring to the following sections and drawings of the embodiments of the invention.

[0145] [Invention Effects]

[0146] According to this disclosure, it is possible to provide T cells and / or related cell populations with enhanced glucose uptake capacity, and by using such cells and / or related cell populations, it is possible to provide chimeric antigen receptor (CAR) T cells and / or related cell populations that function without starvation and exhaustion even in a tumor setting.

[0147] The cells disclosed herein essentially apply the mechanism by which tumor cells survive in the tumor environment to metabolically enhanced T cells (surviving T cells) or related cell populations. Based on the cells and related cell populations disclosed herein, effective cytotoxic activity and cytokine production can be maintained in low-glucose environments such as the tumor microenvironment, which has not been achieved by previous immune checkpoint inhibitory therapies, resulting in excellent in vivo anti-tumor effects. Furthermore, by utilizing a transient glucose transporter expression system that expresses glucose transporters in response to increased glucose demand, T cell over-differentiation and exhaustion due to overactivation can be avoided, and memory phenotype T cells capable of maintaining efficacy long-term can be induced. Attached Figure Description

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[0173] The present disclosure will now be described while demonstrating the best implementation method. Throughout this specification, unless otherwise specified, singular expressions should be understood to include the concept of their plural forms as well. Therefore, unless otherwise specified, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the concept of their plural forms as well. Furthermore, unless otherwise specified, the terminology used in this specification should be understood to be used in the sense commonly understood in the art. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0174] The following provides appropriate definitions of terms and / or basic technical content used in this specification.

[0175] In this specification, "about" refers to ±10% of the following value. For example, "about 20" includes "18 to 22". The range of values ​​includes all values ​​between the two endpoints and the values ​​at the two endpoints. The "about" with respect to a range applies to both endpoints of the range. Therefore, for example, "about 20 to 30" includes "18 to 33".

[0176] In this specification, "T cell" is used in the broad sense as used in the art, referring to cells that are lymphocytes generated in the bone marrow, migrate to the thymus, and mature. T cells can be CD45- and CD3-positive cells from a normal subset of mononuclear cells derived from peripheral blood and bone marrow. The T cells used may be T cells isolated from a donor, particularly a human donor, but are not limited to this. Examples of T cells and cells derived from them include: T cells isolated without passage in culture; T cells passaged and maintained under cell culture conditions without immortalization; and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions. It is known that T cells exist in various types according to their function, including effector memory T cells (T cells). eff The types of T cells include: naive T cells (TnV), stem cell-like memory T cells (Tscm), central memory T cells (Tcm), and terminally differentiated RA-positive T cells (Temra). In this instruction manual, "peripheral T cells" refers to T cells located outside the thymus and can be obtained from peripheral blood, lymph nodes, and other tissues. When referring to "peripheral T cells" in this instruction manual, it is sufficient that the cell population contains peripheral T cells; it is not necessary to isolate the T cells. Cell subsets containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMCs), can also be used.

[0177] In this specification, "cell population" refers to a group containing two or more cells. For example, it can be a state in which cells are aggregated together on a plane, or a clump of cells adhering to each other in three-dimensional space. Furthermore, a "cell population" can be formed by a single type of cell or can contain multiple cell types. In this disclosure, when referring to a T cell population, it is sufficient that it contains at least one cell capable of effector function at the site where it is to perform its function (typically in vivo), such as Tnv, Tscm, Tcm, Temra, etc.

[0178] In this specification, "flow cytometry" refers to a technique for measuring the number of cells, individuals, and other biological particles suspended in a liquid, as well as the physical / chemical and biological properties of each particle. The apparatus using this technique is called a "flow cytometer." In this disclosure, the "positive" and "negative" status of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) is determined by flow cytometry as is commonly used in the art. More specifically, in flow cytometry, cells are arranged in a line and flow, and the number of cells is counted using spectroscopic methods. For example, cells labeled with fluorescence or luminescent enzymes are irradiated with a laser, and the fluorescence or luminescence signals emitted from the cells are detected by a detector such as a photodiode, thereby counting the number of target cells. Alternatively, the detector results can be imported into a computer to generate and display a two-dimensional graph. Thus, the presence and number of target cells can be easily determined.

[0179] In this specification, "effective function" in T cells refers to biological activities such as cytotoxicity, cytokine production, and cell division / proliferation mediated by TCR or CAR signaling. In other immune cells, it refers to biological activities induced by the Fc region of antibodies. Effector functions include, for example, 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 B cell activation. In this disclosure, whether a T cell possesses effector function can be determined by confirming its cytotoxicity, cytokine production, and cell division / proliferation using the methods illustrated in the examples.

[0180] In this manual, "effective cells" or "effective T cells" refer to T cells with effector functions, representing immune cells that exert cytotoxic effects on target cells, and are sometimes also called T cells. effIn this specification, whether a cell is considered an "effect cell" can be determined using methods such as cytotoxicity assays, surface antigen analysis via flow cytometry, and intracellular cytokine staining. Effector T cells are immune cells that perform effector functions, mediating antibody-dependent cytotoxicity (ADCC), etc. Effector T cells include, for example, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils, and can be isolated from natural tissues such as blood.

[0181] In this manual, "effect T cells (T cells)" are referred to as "effect T cells". eff"Precursor cells" refer to cells that, while not effector cells, acquire effector functions after migrating to their intended sites (e.g., within the body) or through other stimuli. Such cells include lymphocyte progenitor cells, TnV, Tscm, Tcm, and Temra. Precursor cells of effector T cells are activated in the initial stages of the immune response, proliferate, and differentiate into effector T cells, but some precursor cells have the ability to differentiate into memory T cells. These precursor cells possess specific gene expression patterns and metabolic states, which contribute to prolonging memory function. This prolongation of memory function involves several molecular and metabolic characteristics. First, precursor cells of effector T cells exhibit specific characteristics in metabolic programs. In these cells, oxidative phosphorylation (Ophosphorylation)... Phosphorylation, a metabolic pathway predominantly associated with effector T cells rather than glycolysis, is highly energy-efficient, enabling cells to survive longer. This metabolic characteristic is a crucial factor supporting their long-term function as memory T cells. Secondly, transcription factors play a vital role. Precursor cells of effector T cells express transcription factors such as T-bet or Eomes, which promote differentiation into memory T cells, enabling them to survive and function long-term. Furthermore, these precursor cells expressing surface markers such as CD62L or CCR7 have the ability to migrate to secondary lymphoid tissues. Based on this homing function, precursor cells receive necessary survival signals in secondary lymphoid tissues, contributing to the maintenance of memory function. Further, inhibition of antigen stimulation also helps prolong memory function. Some precursor cells are derived from… Released from antigen stimulation, the progenitor cells of effector T cells inhibit their complete differentiation into effector T cells, instead retaining their characteristics as memory T cells. Through this mechanism, excessive effector responses are suppressed while memory function is preserved. Furthermore, the cytokine environment surrounding the cell plays a crucial role. In particular, cytokines such as IL-7 and IL-15 provide the survival signals necessary to maintain memory T cells, which greatly contributes to the prolongation of memory function. Due to these factors, compared to typical effector T cells, the precursor cells of effector T cells can survive for a longer period and maintain the memory function necessary for rapid and potent secondary immune responses. This characteristic is believed to play a vital role not only in immune defense against infectious diseases but also in cancer immunotherapy and vaccine development. Therefore, understanding the characteristics of effector T cell precursor cells is of great significance in both basic immunological research and clinical applications.

[0182] In effector T cell precursor cells, maintaining the memory T cell phenotype refers to the phenomenon where, during differentiation into effector T cells, some precursor cells simultaneously retain the memory T cell-specific phenotypes (e.g., surface markers, metabolic state, transcription factor expression, etc.). These precursor cells can survive after the immune response ends and possess the ability to provide long-term immune memory. This phenomenon is considered an important mechanism that allows T cells to simultaneously perform their effector functions and maintain long-term survival while providing memory for rapid secondary immune responses. The characteristics of this phenomenon are explained below. First, effector T cell precursor cells maintain memory T cell-specific surface markers. These include homing molecules such as CD62L and CCR7, through which precursor cells maintain their ability to migrate to secondary lymphoid tissues. Second, these precursor cells are also characterized by maintaining a metabolic state. Specifically, oxidative phosphorylation is dominant compared to the glycolytic system, enabling long-term survival by maintaining an energy-efficient metabolic program. This metabolic characteristic is an important factor in maintaining the function of memory T cells. Furthermore, progenitor cells maintain a characteristic balance in the expression of transcription factors. Appropriate expression of transcription factors such as T-bet or Eomes plays a role in both differentiating into effector T cells and maintaining the phenotype of memory T cells. Further, progenitor cells are responsive to cytokine signals, capable of receiving survival signals such as IL-7 or IL-15. Based on this responsiveness, progenitor cells can achieve long-term survival while maintaining memory T cell characteristics. Finally, these progenitor cells can maintain antigen-independent immunity. That is, memory T cell progenitor cells have the ability to maintain their phenotype and remain ready for a rapid immune response even in the absence of antigens. Based on these characteristics, effector T cell progenitor cells maintain the memory T cell phenotype, playing a crucial role in the flexibility of the immune system and the establishment of long-term memory immunity. These characteristics are considered to have significant implications for infectious disease defense, cancer immunotherapy, and even vaccine design.

[0183] In this specification, "antigen receptor" refers to any molecule that specifically binds to a target antigen and transmits signals to the intracellular space of a cell expressing that receptor. In this disclosure, antigen receptors are molecules capable of being introduced into T cells; they can be natural or artificial molecules, including, in addition to T cell receptors (TCRs) and B cell receptors (BCRs), chimeric antigen receptors (CARs).

[0184] In this specification, "chimeric antigen receptor (CAR)" refers to a modified receptor capable of conferring antigen specificity to cells (e.g., immune cells). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors. They are modified receptors that specifically transplant antigens into immune system cells (e.g., naive T cells, central memory T cells, effector memory T cells, or combinations thereof, NK cells, macrophages, etc.). CARs may include, for example, an antigen-specific targeting region, an extracellular domain, a transmembrane domain, a co-stimulatory domain, and / or an intracellular signaling domain. Additionally, bispecific CARs may be included that utilize multiple (mostly two) antigen-specific targeting regions. Preferably, the CAR disclosed herein comprises at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0185] In this manual, "T cell receptor (TCR)" refers to receptors present on T cells. A TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains. Normal T cells express αβ-type TCRs, and γδ-type TCRs have specific functions. α- and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ, CD3ε, CD3γ, and CD3δ chains) to transmit intracellular signals after antigen recognition and initiate various immune responses. Endogenous antigens, such as viral antigens that proliferate intracellularly during viral infection or cancer antigens derived from cancer cells, are presented as antigenic peptides on MHC class I molecules. Additionally, antigens derived from exogenous microorganisms are taken up by antigen-presenting cells via endocytosis, processed, and then presented on MHC class II molecules. These antigens are then activated by CD8+. + T cells or CD4 + The TCR expressed by T cells is recognized. Co-stimulatory molecules such as CD28, ICOS, and OX40 are known to be important in the stimulation mediated by TCR molecules. Regarding αβ-type TCRs, the gene products of α and β are expressed specifically through unique combinations.

[0186] In this specification, "glucose uptake capacity" refers to the ability of cells to take up glucose per unit time. Cellular glucose uptake can be evaluated using methods such as the 2-NBDG uptake assay.

[0187] In this specification, "enhanced glucose uptake capacity" refers to improving glucose uptake capacity by any means, which can be achieved by modifying immune cells (e.g., T cells) to express one or more glucose transporters, and / or by modifying them to enhance the expression of glucose transporters. Such genetically modified immune cells are expected to exhibit high glucose uptake, for example, in a hypoglycemic environment (e.g., in a tumor microenvironment). Therefore, immune cells co-expressing one or more glucose transporters and chimeric receptor peptides can exhibit superior biological activities (e.g., optionally in the presence of therapeutic antibodies, in tumor microenvironments such as hypoglycemic conditions), such as cell proliferation, activation (e.g., increased cytokine production, such as IL-2 or IFNγ), cytotoxicity, and / or in vivo antitumor activity.

[0188] In this specification, "glucose transporter" (GLUT) refers to a transporter that performs facilitated diffusion transport of glucose (dextrose). Molecular types of GLUTs, GLUT1 through GLUT14, have been reported. For example, GLUT1 is expressed in a wide range of tissues and performs constitutive baseline glucose transport; GLUT2 operates at elevated glucose concentrations, enhancing glucose uptake; GLUT3 plays a central role in the central nervous system, performing transport functions even at low glucose concentrations; and GLUT4 operates upon insulin stimulation, each with its own characteristics.

[0189] In this specification, the terms "tumor microenvironment" and "tumor environment" are used interchangeably. The tumor microenvironment (TME) refers to the local biological environment surrounding tumor tissue, encompassing various factors involved in tumor progression, proliferation, metastasis, and drug resistance. This environment includes not only tumor cells but also non-tumor cells such as immune cells (e.g., T cells, 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. The tumor microenvironment is further influenced by physical factors such as oxygen concentration, pH, and nutritional status. Changes in cell-cell interactions and physical factors within the tumor microenvironment are considered to play a crucial role in tumor malignancy and treatment responsiveness. In particular, the immunosuppressive microenvironment has attracted considerable attention as a factor reducing the effectiveness of immunotherapy. In this invention, the "tumor microenvironment" refers to the aggregate of biological and physical factors including these elements and their interactions, and is an important object in tumor treatment and diagnosis.

[0190] In this specification, "tumor microenvironment conditions" refers to the tumor microenvironment or conditions equivalent to it. Therefore, "tumor microenvironment conditions" is a broad concept, encompassing not only the conditions constituting the tumor microenvironment but also similar or equivalent environmental conditions encountered by the tumor within the body. Specifically, it includes not only the local biological environment surrounding the tumor tissue but also the endogenous and exogenous factors affecting the tumor while it exists within the body. This environment includes: the interaction between tumor cells and non-tumor cells (e.g., immune cells, fibroblasts, vascular endothelial cells, etc.); extracellular matrix (ECM) or humoral factors such as cytokines, chemokines, and growth factors; and physical factors such as oxygen concentration, pH, nutritional status, and physical stress. Furthermore, since "tumor microenvironment conditions" also includes the interaction between the tumor and the broader physiological environment within the body, it is not limited to the local environment surrounding the tumor. In this invention, "tumor microenvironment conditions" comprehensively represents the extensive environmental conditions encountered by the tumor within the body, including all factors affecting tumor progression and treatment responsiveness.

[0191] In this specification, "modification" broadly refers to any structural, functional, or expression-related alteration of a gene (including nucleic acids and their corresponding proteins), including naturally occurring or artificial changes. Specifically, this includes: sequence changes resulting from base substitutions, deletions, insertions, additions, or combinations thereof; alterations to the amino acid sequence or properties of the encoded protein; or chemical modifications aimed at imparting or enhancing functionality (e.g., glycan addition, phosphorylation, acetylation, etc.). Furthermore, "modification" also includes: introducing genes from external sources (e.g., through transfection or viral vectors), transformation, or specific gene knockout or knock-in using genome editing technologies, and the suppression of gene expression (e.g., suppression through RNA interference or CRISPR technology). Further, epigenetic modifications (e.g., DNA methylation, histone modifications, etc.) and alterations to expression regulatory elements are also included. In this specification, "modification" comprehensively refers to any form of alteration 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.

[0192] In this specification, "disease" is interpreted in the broadest sense, referring to a state of discomfort or impairment in the physical or mental health of a person or animal. It includes any undefined state that cannot be described as health, such as disease, impairment, or various symptoms. Diseases to which this disclosure is applicable include, but are not limited to, diseases that may be related to immune responses, such as cancer, autoimmune diseases, allergies, and infectious diseases.

[0193] In this instruction manual, "immune response" in a subject to a certain component or substance means that the subject produces an immune reaction against that component or substance. This component or substance can be determined by observing changes in various immune cells or increases or decreases in immune-related substances (e.g., cytokines) in the subject or biological components derived from the subject (e.g., cells). This can be determined through objective indicators or by subjective judgment based on experience by physicians or others.

[0194] In this specification, whether a subject “has immune memory” regarding a certain component or substance can be evaluated by measuring, in the subject or biological components (e.g., cells, etc.) derived from the subject, whether the component or substance (i) induces antigen-dependent cytokine production or has a proliferative effect on memory CD4-positive T cells; (ii) alters the expression of surface antigens on memory regulatory T cells; (iii) alters the ratio of Tregs to Th1s; (iv) induces T-bet-positive Th1 cells to produce IFN-γ; (v) alters the ability to produce IFN-γ; (vi) alters the ability to produce IL-2; and (vii) alters the ability to produce TNF-α; and (viii) possesses a specific antibody against a certain component or substance in the blood; and confirming that at least one of these assays is positive.

[0195] In this instruction manual, "infectious disease" can refer to any infectious disease, including viral infections (including any viral form such as single-stranded or double-stranded DNA viruses and RNA viruses), bacterial infections, protozoan infections, mycoplasma infections, and any type of infectious disease. Examples include tuberculosis, coronavirus, malaria, yellow fever virus, smallpox virus, cowpox, measles / rubella, poliomyelitis, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonellosis, pathogenic Escherichia coli, toxoplasmosis, Zika virus, herpesvirus type 1, EBV / Epstein-Barr (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza, MARS, rabies, and diphtheria.

[0196] In this manual, "immune abnormality" refers to any disease, disorder, or condition that is at least partially caused by or suspected to be caused by an abnormality in the immune system. It refers to a state where the immune system becomes abnormal due to some cause, making one susceptible to infections or causing allergic reactions. Immune abnormalities may include allergies, autoimmune diseases, etc., but are not limited to these. The condition of self-antigens is usually called an autoimmune disease, while the condition of foreign antigens is called an allergy. When the immune response is strong, the condition is an autoimmune disease state for self-antigens and an allergic state for non-self antigens; conversely, when the immune response is weak, the condition is a cancerous state for self-antigens and an infectious disease for non-self antigens.

[0197] In this specification, "autoimmune disease" refers to an excessive immune response to a specific self-antigen. Autoimmune diseases can be described as diseases caused by the disruption of immune tolerance, where the immune system, normally responsible for recognizing and eliminating foreign substances, overreacts and attacks even its own normal cells and tissues, thus causing symptoms. Examples of autoimmune diseases or disorders include, but are not limited to, the following: inflammatory reactions 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 syndromes (including adult respiratory distress syndrome: ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic states such as eczema, asthma, and other conditions accompanied by T-cell infiltration and chronic inflammation; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE). lupus erythematosus: SLE (including but not limited to lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic myelitis; Sjögren's syndrome; juvenile-onset diabetes; and, typically, acute and delayed-type hypersensitivity reactions mediated by cytokines and T lymphocytes, as observed in tuberculosis, sarcoidosis, polymyositis, granulomatous diseases, and vasculitis; pernicious anemia (Addison's disease); diseases with leukocytic exudation; central nervous system disorders. Inflammatory disorders of the CNS (Central Nervous System); multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorders; Reiter's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP); autoimmune uveitis or autoimmune thrombocytopenia.

[0198] In this instruction manual, "allergy" refers to an excessive immune response against a specific non-self antigen, a disease characterized by an immune reaction against an "allergen." An "allergen" is an antigen that can react with the antibodies of a subject suffering from an allergic disease. Examples include: allergens derived from the pollen of woody plants (such as acacia, alder, velvet ash, European beech, birch, maple, pine, redwood, box willow, cypress, American elm, autumn elm, yellow fir, rubber tree, eucalyptus, hackberry, hickory, American linden, sugar maple, mulberry, paper mulberry, oak, olive, American hickory, pepper, pine, privet, oleaster, American sycamore, ailanthus, black walnut, black willow, etc.) and allergens derived from the pollen of herbaceous plants (such as cotton, bermudagrass, Kentucky bluegrass, brome, corn, tall fescue, false sorghum, oats, orchardgrass, creeping bentgrass, ryegrass, rice, yellow cogon grass, and timothy grass). Allergens include, but are not limited to, allergens derived from insects (silkworms, mites, bees, wasps, ants, cockleburs, etc.), allergens derived from fungi (Alternaria, Aspergillus, Clostridium bovis, Candida, Cephalosporium, Curvularia, Acanthopanax, Dermatophytes, Fusarium, Helicobacter, Cladosporium, Mucor, Penicillium, Stemdrops, Rhizopus, etc.), allergens derived from animal hair (dogs, cats, birds, etc.), allergens derived from house dust, proteins, and food allergens (OVA, etc.). Representative diseases associated with "allergies" include atopic dermatitis, allergic rhinitis (such as hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergies, drug allergies, and urticaria.

[0199] (Preferred Implementation)

[0200] The preferred embodiments of this disclosure are described below. The embodiments provided below are for a better understanding of this disclosure, and the scope of this disclosure should not be limited to the following description. Therefore, those skilled in the art, referring to the description in this specification, will obviously be able to make appropriate modifications within the scope of this disclosure. Furthermore, the following embodiments of this disclosure can be used individually or in combination.

[0201] In one aspect, this disclosure provides immune cells such as T cells with enhanced glucose uptake capacity, cell populations containing such cells, pharmaceutical compositions containing such cells or cell populations, and other related technologies.

[0202] In one aspect of this disclosure, a T cell with enhanced glucose uptake is provided. Cells modified to express glucose transporters and / or with enhanced expression of these transporters have shown unexpectedly significant effects against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc. (see reference). Figure 16 The advantage of the cells disclosed herein lies in their enhanced glucose uptake capacity. Furthermore, the cells of this disclosure are preferably effector cells that, upon introduction into the site where they are to function, particularly in vivo. T cells are an example of such cells. eff However, it is not limited to this; it can also be T. eff Precursor cells, such as lymphocyte lineage precursor cells, Tnv, Tscm, Temra, etc.

[0203] In one embodiment, this disclosure may be an effector T cell comprising a chimeric antigen receptor (CAR) and modified to express a glucose transporter and / or with enhanced glucose transporter expression. In another embodiment, this disclosure may be a cell population comprising effector T cells containing a chimeric antigen receptor (CAR) and modified to express a glucose transporter and / or with enhanced glucose transporter expression. This disclosure provides an "effector T cell" not available in the prior art. In this embodiment, the effector T cell refers to an immune cell (e.g., a T cell) that exerts cytotoxic effects on target cells (described in other parts of this specification). As described in other parts of this specification, effector functions include cytotoxic activity arising from signaling mediated by TCR or CAR, cytokine production, and division / proliferation, but one of the important functions of effector T cells is to exert cytotoxic effects on target cells.

[0204] On the other hand, existing technologies do not provide evidence of effector T cells exerting cytotoxic effects on target cells. Rather than a lack of guidance, there are no examples in existing technologies of confirmed cures through experiments such as re-challenge; for instance, there are instances of residual nodules, many of which show no cure. While various cancer treatments are known, most have only shrunk tumors, not eradicated them. This is because they lack the cytotoxic effect on target cells as described in this disclosure. In existing technologies, tumor suppression effects can sometimes be observed through the action of cytokines, but most anticancer agents are merely indirect effects. Unlike this disclosure, in in vivo models, even when tumors are suppressed, they do not exert cytotoxic effects on target cells; rather, the tumor remains, indicating a lack of cytotoxic function. This disclosure achieves a different effect from these existing technologies in this respect.

[0205] In detail, in the embodiments of this disclosure, chimeric antigen receptor T (CAR-T) cell therapy has previously failed to demonstrate effectiveness in solid cancers, but this disclosure also addresses this issue. Here, the inventors have discovered that in solid cancers, the dysfunction of CAR-T cells is caused by glucose deficiency in the tumor microenvironment (TME), and that supplementing this metabolism can significantly improve the anti-tumor effect of CAR-T cells. Cancer cells consume large amounts of glucose, reducing glucose levels in the solid cancer TME, resulting in CAR-T cell damage. In specific embodiments of this disclosure, it has been found that when GLUT-type glucose transporters are expressed in CAR-T cells, cytokine production and killing activity are restored, or effector function is improved. This provides a treatment method for various solid cancers that have failed to be cured even with combination therapies such as chemotherapy drugs and radiation. Some solid cancers have metabolically harsh TMEs, which impair CD8+. + The function of effector T cells, including T cells, is achieved by adoptively transferring CAR-T cells into the TME. This disclosure is particularly effective in cases where nervous system cells, such as neurons and glial cells, require glucose as an energy source. Furthermore, some aggressive cancer cells consume more glucose than normal cells due to their cancer cell-specific metabolism, namely aerobic glycolysis (known as the Warburg effect), leading to glucose depletion in the TME. On the other hand, naive T cells, to meet the increased energy demand, switch energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation. Therefore, intense metabolic competition via glucose occurs within the TME between activated T cells and cancer cells. Cancer cells can survive and proliferate by taking up sufficient glucose within the TME, while activated T cells suffer functional impairment due to failure in this metabolic competition. CAR-T cells that express GLUT externally or enhance internal expression to achieve equal or higher levels of GLUT can achieve sufficient competition with cancer cells.

[0206] In detail, as a preferred and non-limiting embodiment of this disclosure, the following observations have been made. That is, although chimeric antigen receptor T (CAR-T) cell therapy has achieved clinical success in hematologic malignancies, its application in refractory solid tumors, including glioblastoma (GBM), has not been successful. Clinical trials of CAR-T cells targeting solid tumors have so far failed to demonstrate effectiveness, and this disclosure addresses this issue. Here, the inventors have discovered that the dysfunction of CAR-T cells in GBM is caused by glucose deficiency in the tumor microenvironment (TME), and that supplementing this metabolism can significantly improve the anti-tumor effect of CAR-T cells. Cancer cells consume large amounts of glucose, reducing glucose levels in the GBM TME, resulting in CAR-T cell damage. In a preferred embodiment of this disclosure, when CAR-T cells stably express the high-affinity glucose transporter GLUT3, cytokine production and killing activity are restored. Glioblastoma (GBM) is a devastating malignant brain tumor with an annual incidence of 3.19 cases per 100,000 people. In 2005, with the advent of combination therapies consisting of temozolomide and radiation, Novo Therapeutic Fields (NTF) therapy for primary GBM was developed, improving the prognosis of GBM patients in clinical practice. However, the five-year overall survival rate for GBM remains below 20%. Given the clinical success of chimeric antigen receptor T-cell (CAR-T cell) therapy in hematologic malignancies, several preclinical studies and clinical trials of CAR-T cell therapy for GBM are being actively investigated. Although some patients treated in clinical trials have shown promising results such as CAR-T cell infiltration into the tumor microenvironment (TME) and reduced tumor burden, most patients do not respond to CAR-T cell therapy. GBM has a metabolically harsh TME, which impairs CD8+. +The function of effector T cells, including T cells, is restored, while CAR-T cells are adopted into the TME. In the brain, nervous system cells such as neurons and glial cells require glucose as an energy source; the brain consumes 25% of the body's energy from glucose. Furthermore, invasive GBM cells, due to cancer cell-specific metabolism—aerobic glycolysis (known as the Warburg effect)—consume three times more glucose than normal neurons. This leads to glucose depletion in the GBM TME. On the other hand, naive T cells, to meet the increased energy demand, switch energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation. Therefore, intense metabolic competition via glucose occurs between activated T cells and cancer cells within the GBM TME. In a preferred embodiment, GLUT3, encoded by SLC2A3 and physiologically expressed in neurons or glial cells, has a glucose affinity five times higher than GLUT1, the major glucose transporter expressed in T cells. As a result, GBM cells expressing GLUT3 are able to survive and proliferate continuously by taking up sufficient glucose within the TME, while activated T cells suffer functional impairment due to failure in metabolic competition. In this preferred embodiment, considering the significant glucose uptake mediated by high-affinity GLUT3 in GBM cells, CAR-T cells exhibit equal or higher expression of GLUT3, enabling them to compete effectively with GBM cells. In a preferred embodiment of this disclosure, a construct is provided that links SLC2A3 to a CAR to induce GLUT3 expression in CAR-T cells.

[0207] (cell population)

[0208] In one aspect of this disclosure, a cell population comprising immune cells such as T cells with enhanced glucose uptake is provided. Among at least a portion of the T cells included in this cell population, cells that express and / or enhance glucose transporters through modification have shown unexpectedly significant effects on diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc. (see reference). Figure 16 (etc.). The advantage of the cell population disclosed herein lies in its enhanced glucose uptake capacity. Furthermore, the cell population of this disclosure preferably comprises cells that possess effector functional characteristics after introduction into the site where they are to perform their function, particularly in vivo. Examples of such cells include T cells. eff However, it is not limited to this; it can also be T. eff Precursor cells, such as lymphocyte lineage precursor cells, Tnv, Tscm, Temra, etc.

[0209] In one embodiment of this disclosure, a cell population comprising the aforementioned T cells is involved, wherein it is advantageous for T cells possessing the characteristics of this disclosure (e.g., enhanced or expressed GLUT, effector function, or both) to constitute more than 10% of the cell population. In one embodiment, the proportion of T cells possessing the characteristics of this disclosure in the cell population may be more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 97%, or more than 99%.

[0210] In one embodiment, the cell population disclosed herein comprises T cells that have effector functional properties when introduced into the body.

[0211] In one embodiment, the T cells in the cell population disclosed herein comprise chimeric antigen receptors (CARs) or T cell receptors (TCRs), preferably chimeric antigen receptors (CARs).

[0212] In one embodiment, the cell population of this disclosure includes T cells that are modified to express glucose transporters and / or have enhanced expression of glucose transporters.

[0213] In one embodiment, the cell population disclosed herein includes T cells that have effector functions.

[0214] In one embodiment, the cell population of this disclosure comprises T eff and / or T eff Precursor cells. Preferably, the cell population (or T) eff Precursor cells include at least one of the following: lymphocyte lineage precursor cells, Tnv, Tscm, Tcm, Temr, etc.

[0215] In one embodiment, in the cell population of this disclosure, the CAR is expressed in the T cells.

[0216] In one embodiment, the glucose transporters in the cell population disclosed herein include GLUT1, GLUT2, GLUT3, and GLUT4, preferably GLUT3.

[0217] In one embodiment, the T cells in the cell population disclosed herein are human T cells.

[0218] In one embodiment, the cell populations of this disclosure are provided as a medicine or pharmaceutical composition. Their uses may include prevention or treatment of cancer, cure of cancer, prevention of cancer metastasis, or prevention of cancer recurrence. In specific embodiments, the cell populations of this disclosure may be used to prevent or treat cancer to prevent its recurrence.

[0219] <Glucose transporters>

[0220] To favor their own cell proliferation, cancer cells reprogram their metabolic systems, actively utilizing the inefficient glycolysis system, even under aerobic conditions, to enhance glucose uptake and lactate production (the Warburg effect). Therefore, due to the massive glucose consumption and subsequent depletion by cancer cells, T cells require glucose as an energy source. eff Tumor-specific T cells, after infiltrating a tumor, even if they receive TCR signals, will still exhibit increased calcium levels in their cells. 2+ The concentration will also decrease, leading to starvation and depletion.

[0221] Therefore, in one embodiment of this disclosure, a genetically engineered T cell and cell population expressing a glucose transporter are provided to promote aerobic glycolysis and enhance the competitiveness and / or adaptability of immune cells in a glucose-depleted tumor microenvironment (TME).

[0222] In one embodiment, GLUT1, GLUT2, GLUT3, and GLUT4 can be listed as glucose transporters, among which GLUT3 is a glucose transporter with a high affinity for glucose. Therefore, in a preferred embodiment, GLUT3 is the glucose transporter used in the cells and cell populations of this disclosure.

[0223] GLUT3 was initially identified in the mouse brain and was initially defined as a neuroglucose transporter. Subsequent studies have shown that GLUT3 is also expressed in other glucose-dependent cells, such as mouse sperm, which ensures energy for movement, and the blastocyst, which is crucial for post-implantation development. Furthermore, GLUT3 is expressed in immune cells such as lymphocytes, monocytes, macrophages, and platelets, and is typically stored in intracellular vesicles, migrating to the cell surface upon activation to maintain metabolic switches. More recent studies have shown that T cells, particularly CD8 cells, are also involved in this process. + T cells highly express GLUT3 during differentiation and activation, therefore their glucose uptake depends not only on GLUT1 but also on GLUT3.

[0224] In T eff In this study, when glucose transporters such as GLUT3 are activated, enhanced effector functions such as cytotoxic activity and cytokine production can be observed through glucose uptake.

[0225] In one embodiment of the present disclosure, the T cells of the present disclosure or the T cells included in a cell population may include effector cells.

[0226] In one embodiment of the present disclosure, it may be advantageous when the T cells included in the T cells and cell populations of the present disclosure are human T cells.

[0227] <Chimeric antigen receptor (CAR)>

[0228] In other aspects of the present disclosure, there is provided a T cell with enhanced glucose uptake ability or a cell population containing such cells, wherein the T cell includes a chimeric antigen receptor (CAR), and the T cell is modified to express a glucose transporter and / or the expression of the glucose transporter is enhanced. The CAR included in the T cells or cell population of the present disclosure, as long as it can function as a CAR, can be included either as a protein or as a nucleic acid molecule expressing the CAR. In a representative embodiment, the CAR of the present disclosure may be expressed in T cells.

[0229] The CAR disclosed in this specification includes at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0230] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that includes an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain through a transmembrane domain. The characteristics of CAR include, for example, that it does not rely on MHC, but uses the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to a selected target. Antigen recognition independent of MHC can endow T cells expressing CAR or T cells included in a cell population with the ability to recognize antigens independently of the antigen processing process, thereby avoiding immune escape of tumors.

[0231] In the intracellular T cell signaling domain of CAR, for example, it may include a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a part of CAR that includes the intracellular domain of the T cell receptor, such as the intracellular part of the CD3ζ (zeta) protein. The co-stimulatory signaling domain refers to a part of CAR that includes the intracellular domain of a co-stimulatory molecule, and the co-stimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand that is necessary for lymphocytes to produce an effective response to an antigen.

[0232] <T cell receptor (TCR)>

[0233] In other aspects of this disclosure, a T cell or cell population containing enhanced glucose uptake is provided, wherein the T cell contains a T cell receptor (TCR) and is modified to express a glucose transporter and / or with enhanced glucose transporter expression. The TCR contained in the T cell or cell population of this disclosure may be contained as a protein or as a nucleic acid molecule expressing a TCR, provided it can function as a TCR. In a representative embodiment, the TCR of this disclosure may be expressed in the T cell.

[0234] In this specification, TCRs are heterodimeric receptor molecules composed of two TCR polypeptide chains. There are αβ-type TCRs expressed by normal T cells and γδ-type TCRs with specific functions. α-chain and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, CD3δ chain) to transmit intracellular signals after antigen recognition, initiating various immune responses. Endogenous antigens, such as viral antigens that proliferate intracellularly during viral infection and cancer antigens derived from cancer cells, are presented as antigenic peptides on MHC class I molecules. Additionally, antigens derived from exogenous microorganisms are taken up by antigen-presenting cells via endocytosis, processed, and then presented on MHC class II molecules. These antigens are respectively activated by CD8+. + T cells or CD4 + T-cell recognition of TCRs. Among the known TCR-mediated stimuli, co-stimulatory molecules such as CD28, ICOS, and OX40 are also important. Regarding αβ-type TCRs, the gene products of α and β can express specificity through unique combinations. TCRs can be appropriately modified to include T-cell receptor signaling domains, T-cell co-stimulatory signaling domains, or both. T-cell receptor signaling domains include the intracellular domains of T-cell receptors, such as the intracellular portion of the CD3ζ protein. Co-stimulatory signaling domains contain the intracellular domains of co-stimulatory molecules, which are cell surface molecules other than antigen receptors or their ligands, essential for an effective lymphocyte response to antigens.

[0235] (Extracellular domain)

[0236] In one embodiment, the CAR used in T cells comprising the T cells or cell populations disclosed herein includes an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof may be appropriately selected based 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 ligands that function as cell surface markers associated with a specific disease state on the target cell. Therefore, examples of cell surface markers capable of acting as ligands targeting the antigen-binding domain in the CAR of this disclosure include tissue-specific markers, tumor-specific markers, viral, bacterial, and parasitic infections, autoimmune diseases, and markers associated with cancer cells.

[0237] The extracellular binding domain of a CAR can be composed of a single-chain variable fragment (scFv) obtained by fusing the variable heavy chain region and the light chain region of a mouse or humanized monoclonal antibody. Alternatively, scFv derived from Fab (not from the antibody, but for example, from a Fab library) can be used. The scFv can fuse to the transmembrane domain and then to the intracellular signal transduction domain.

[0238] In one embodiment, the antigen-binding domain portion of the CAR disclosed herein may target antigens including: (1) allogeneic antigens such as MHC class I and MHC class II; (2) extracellular autoantigens such as TSHR (thyroid stimulating hormone receptor), DSG3 (desmoglein 3) and Cytokeratin 8; (3) foreign antigens such as Gliadin and Ara h2; (4) targeting molecules such as CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin) and MadCam1 (mucosal vascular addressin cell adhesion molecule 1), but the antigens that the antigen-binding domain portion of the CAR disclosed herein may target are not limited to these.

[0239] In one embodiment, the CAR of this disclosure may be modified to include an antigen-binding domain specific to the desired target antigen, depending on the antigen to be targeted. For example, when CD19 is the target antigen, an antibody against a cancer antigen such as CD19 may be used as the antigen-binding domain in the CAR. Non-limiting examples of cancer antigens include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folate receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGEA-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, IL13Rα, IL13Rα2, AIM-2, AIM-3, NY-ESO-1, and 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 protein, LivinP protein, MRP-3, Nestin, OLIG2, ART1, ART4, Cyclin B, Grill, Cav-1, Cathepsin B, CD74, E-Cadherin, EphA2 / Eck, Fra-1 / Fosl 1. GAGE-1, gangliosides / GD2, GnT-V, p1, 6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOX10, SOX11, Survivin, phCG, WT1, mesothelin, Melan A, NY-BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2, etc. In several embodiments, the effector T cells disclosed in this specification are engineered to express more than one CAR, thereby recognizing more than one antigen.

[0240] (Transmembrane domain)

[0241] The CAR used in T cells or cell populations disclosed in this specification may include one or more transmembrane domains fused with extracellular domains.

[0242] In one embodiment, a linker domain derived from an extracellular domain can be linked to a transmembrane domain. The transmembrane domain can be natural or synthetic. As a natural transmembrane domain, it can be derived from any membrane-binding or transmembrane protein. Transmembrane regions particularly used in the present disclosure can be derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0243] In one embodiment, for the CAR used in the T cells contained in the T cells or cell populations disclosed in this specification, a spacer domain can also be configured between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain preferably can have a sequence that promotes the binding of the CAR to the antigen and enhances signal transduction into the cell.

[0244] (Intracellular domain)

[0245] The cytoplasmic signaling domain (or intracellular signaling domain) of the CAR is involved in the activation of at least one normal effector function of the immune cells expressing the CAR. The intracellular signaling domain refers to the protein portion that transmits the effector function signal and instructs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any intact, mutant, or truncated portion of the intracellular signaling domain of a specific protein that is sufficient to transmit a signal that initiates or inhibits the effector function of the immune cell.

[0246] In one embodiment, as examples of the intracellular signaling domain used in the CAR, the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction after antigen receptor binding can be cited.

[0247] (Alloantigens, allergens, and haptens related to rejection reactions)

[0248] For the CAR used in the T cells contained in the T cells or cell populations disclosed in this specification, receptors related to alloantigens, allergens, and haptens related to rejection reactions can be cited.

[0249] (Medicinal uses of T cells contained in a T cell or cell population)

[0250] In one aspect of this disclosure, a pharmaceutical composition is provided comprising T cells with enhanced glucose uptake or a cell population containing such T cells, wherein the T cells are modified to express a glucose transporter and / or have enhanced glucose transporter expression. In another aspect of this disclosure, a pharmaceutical composition is provided comprising T cells with enhanced glucose uptake or a cell population containing such T cells, wherein the T cells contain a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the T cells are modified to express a glucose transporter and / or have enhanced glucose transporter expression. In one embodiment of this disclosure, the T cells or cell population contained in the T cells of this disclosure may possess one or more of the characteristics of the aforementioned T cells.

[0251] The cells, cell populations, etc. disclosed herein can be used in immunotherapy. Immunotherapy is considered effective for diseases that produce antigenic lesions (e.g., cancer, autoimmune diseases, allergies, infectious diseases, etc.), as well as diseases in which abnormal immune responses to specific antigens are involved in the pathogenesis or progression of the disease. For example, the cells, etc., disclosed herein can be used for the management, treatment, or prevention of autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD), rejection, or transplant failure during transplantation. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), Sjögren's syndrome, systemic lupus erythematosus (SLE), antiphospholipid syndrome, polymyositis / dermatomyositis, systemic sclerosis, mixed connective tissue disease, vasculitis syndrome, type I diabetes, Graves' disease, Hashimoto's disease, idiopathic Addison's disease, autoimmune hepatitis, Goodpassuia 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. Furthermore, the cells and cell populations disclosed herein can also be used for treatment or prevention of diseases in which abnormal immune responses to specific antigens are involved in the pathogenesis or progression of the disease.

[0252] In certain embodiments, this disclosure provides immunotherapies including TCR-T therapy or CAR-T therapy. In this specification, "TCR-T therapy" refers to cell therapy utilizing a modified T-cell receptor (TCR), such as a therapy for cancer treatment. In this specification, "CAR-T therapy" refers to a gene / cell therapy method that involves introducing a chimeric antigen receptor (CAR) (e.g., a genetically manipulated receptor to overcome tumor immune evasion mechanisms) into a patient's T cells, expanding and culturing these T cells in vitro, and then infusing them into the patient.

[0253] The cells, cell populations, etc. disclosed herein can be appropriately combined with other cancer treatments and used as combination therapies. Typically, they can be administered in combination with one or more additional agents. Alternatively, the combination therapy can also be a combination with radiotherapy. The one or more additional agents can be any chemotherapy drug or may contain immune checkpoint inhibitors. Alternatively, other cancer therapies used in the combination therapy include, but are not limited to, other cancer immunotherapies (e.g., immune checkpoint inhibitors), hyperthermia, surgery, etc.

[0254] Furthermore, in one aspect, a therapeutic agent comprising the T cells or cell populations disclosed herein is provided, which diagnoses a disease in a subject and selects, based on the diagnosis, a suitable CAR or TCR, etc., contained in the T cells or cell population.

[0255] In another embodiment, this disclosure provides a pharmaceutical composition comprising any of the immune cells described herein (e.g., T cells such as effector T cells) or a cell population containing such cells, and a pharmaceutically acceptable carrier. When the immune cells express peptides such as CARs or TCRs, 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 is capable of binding to target antigens such as those specific to tumor antigens, pathogen antigens, or self-antigens on immune cells. Pathogen antigens may be bacterial antigens, viral antigens, or fungal antigens.

[0256] In one embodiment, the Fc-containing therapeutic agent may be a therapeutic antibody including, but not limited to, the following: adalimumab, trastuzumab-metazine conjugate, alenzumab, baribizumab, bevacizumab, belimumab, brentuximab, cananulumab, cetuximab, cetrus, dacrolimus, denosumab, denutoxicum, eculizumab, efazolinumab, epazolizumab, gemetuzumab, and gorlinumab. Immorumab, hu14.18K322A, teimomab, infliximab, ipilimumab, labetizumab, moromona, natazumab, ozoglucomutizumab, ofatolimumab, omalizumab, pallizumab, panitumumab, pertuzumab, ramosimumab, ranitizumab, rituximab, tocilizumab, trastuzumab, tosimomumab, ustekinumab, moglizumab, and vedelizumab.

[0257] Furthermore, this disclosure provides a kit comprising: (i) a first pharmaceutical composition containing any of the T cells described herein or a cell population containing such T cells and a pharmaceutically acceptable carrier; and (ii) a therapeutic drug containing Fc as described herein and a pharmaceutically acceptable carrier.

[0258] In other aspects of this disclosure, a method is provided 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), comprising administering the cells, cell populations, and / or pharmaceutical compositions of this disclosure to the subject. In one embodiment, at least a portion of the cells expressing the target antigen may be in a low-glucose environment.

[0259] In one embodiment, the object of treatment by the method of this disclosure can be a patient suffering from cancer, such as carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Examples of exemplary target cancers include, but are not limited to, B-cell-derived cancers such as breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B-cell-derived cancers include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin lymphoma.

[0260] In addition to treating target diseases or disorders such as cancer or infectious disorders, the use of the T cells or cell populations disclosed herein for manufacturing targeted medical therapeutics is also within the scope of this disclosure.

[0261] In one embodiment, a pharmaceutical composition comprising effector T cells containing a chimeric antigen receptor (CAR) of this disclosure can be used for cell therapy. In cell therapy, T cells comprising a chimeric antigen receptor (CAR) or a T-cell receptor (TCR) of this disclosure, or a cell population containing such T cells, can be injected into a recipient as a pharmaceutical composition or as a formulation of a cell population expressing a therapeutically effective CAR or T-cell receptor (TCR) of this disclosure. The T cells or cell population containing such T cells injected into the recipient can treat the recipient's target disease or disorder, such as cancer or an infectious disorder. The recipient can be the same recipient from whom the cells were obtained (autologous cell therapy), or the cells can be derived from different recipients of the same species (allogeneic cell therapy).

[0262] In one embodiment, T cells comprising the CAR or (TCR) of this disclosure, or cell populations comprising such T cells, can be formulated into a preparation for administration to a subject using techniques known to those skilled in the art. In one embodiment, a preparation comprising therapeutically effective T cells, including the CAR or (TCR) of this disclosure, or cell populations comprising such T cells, may contain pharmaceutically acceptable excipients (carriers or diluents). The excipients included in the preparation may have different purposes, for example, depending on the characteristics of the antigen-binding domain of the CAR of this disclosure. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonic agents, expanders, and lubricants.

[0263] In this specification, when gene names and their products are described using all capital letters, as is the usual practice, it may refer to both the gene and the protein. For example, sometimes the terms FOXP3 gene and FOXP3 protein may be used interchangeably, and when referred to as FoxP3, it refers to both the concept and the physical entity of the gene or protein (as a whole).

[0264] Preparations containing therapeutically effective T cells, including the CAR of this disclosure, or cell populations containing such T cells, can be administered to subjects using methods and techniques known to those skilled in the art. Intravenous injection is exemplified, but is not limited to, for example. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid region), intracranial, intraspinal, and intrathecal (spinal fluid) administration.

[0265] (General Technology)

[0266] The molecular biological, biochemical, and microbiological methods used in this manual are well-known and commonly used in the field, as described in, for example, Sambrook J. et al. (1989). *Molecular Cloning: A Laboratory Manual*, *Cold Spring Harbor* and its 3rd 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: Methods and Applications*. *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, FM (1995).*Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology*, Greene Pub. Associates; Innis, MA et al. (1995). *PCR Strategies*, Academic Press; Ausubel, FM (1999). *Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology*, Wiley and annual updates; Sninsky, JJ et al. (1999). *PCR Applications: Protocols for Functional Genomics*, Academic Press. Press); Experimental Medicine Supplement "Experimental Methods for Gene Introduction and Expression Analysis" (Gene Introduction & Expression Analysis Experimental Methods), Yotsu Publishing House, 1997, etc. Relevant portions (or all portions) of these documents are cited in this instruction manual as references.

[0267] Regarding DNA synthesis techniques and nucleic acid chemistry for artificially synthesized genes, gene synthesis or fragment synthesis services provided by companies such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Additionally, references are made to, for example, 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 (1996). Bioconjugate Techniques, Academic Press, etc. Relevant portions of these documents are cited in this manual as references.

[0268] In this specification, "or" is used when "at least one or more" of the items listed herein are applicable. The same applies to "or". When this specification explicitly states a "range" of "two values", that range also includes the two values ​​themselves.

[0269] All scientific literature, patents, patent applications, and other references cited in this specification are used here for reference in their entirety to the extent that they are identical to their respective specific contents.

[0270] For ease of understanding, preferred embodiments have been shown and the present disclosure has been described above. The present disclosure will now be described based on embodiments; however, the above description and the following embodiments are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments and examples specifically described in this specification, but is limited only by the claims.

[0271]

Example

[0272] In this embodiment, various functions of T cells with enhanced glucose uptake were investigated. The reagents specifically used were the products described in the embodiment, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nacalai, R&D Systems, USCN Life Science INC, etc.) can be used as substitutes.

[0273] (Example 1)

[0274] Methods and Materials

[0275] Fabrication of GLUT3 3C10-CAR construct and viral vector preparation

[0276] A lentiviral vector, 3C10 CAR-Katushka2S, was constructed by inserting an SLC2A3 sequence (GLUT3) with a P2A sequence immediately upstream of an anti-EGFRvIII CAR backbone plasmid (3C10 CAR-Katushka2S) containing a CD8 leader sequence, an anti-EGFRvIII single-stranded variable region (scFv), a CD8 hinge region (CD8 hinge), a CD8 transmembrane region (TM), a CD28 intracellular domain (ICD), and a 4-1BB ICD-CD3z-P2A-Katushka2S structure. Figure 1 Lentiviral vectors were transfected into 293T cells along with packaging vectors psPAX2 (Addgene#12260) and pMD2.G (Addgene#12259), and lentivirus was obtained from the supernatant.

[0277] Gene importation

[0278] Peripheral blood mononuclear cells from healthy individuals were isolated from heparinized whole blood using the Ficoll method (Ficoll-Paque PLUS, GE Healthcare), and then stimulated with anti-CD3 / 28 magnetic beads (Dynabeads T-Activator CD3 / CD28, Veritas). CAR gene delivery was performed on day 1 at an MOI of 4. Cells were cultured in the presence of IL-2 at 30 U / ml and cryopreserved from day 8 to 12, or used directly for various evaluations.

[0279] Immunostaining

[0280] 1×10 6 GLUT3 3C10 CAR-T cells were fixed with 4% PFA and permeabilized with methanol. They were blocked with 3% BSA for 1 hour and incubated with anti-GLUT3 antibody (ab15311) for 2 hours. After washing with 0.05% PBST, they were incubated with anti-rabbit IgG antibody (Alexa flour 488) for 1 hour. After washing with 0.05% PBST, nuclear staining was performed with DAPI. The stained cells were then attached to slides using Cytospin (Thermo Fisher Scientific), mounted with VECTASHIELD (VECTOR LABORATORIES), and observed using a Keyence XZ-800 microscope.

[0281] Phenotypic analysis (FCM)

[0282] The antibodies used for FCM analysis are shown in Table 1.

[0283] Table 1

[0284]

[0285] Cells were washed using 4% FBS-PBS. Dead cell staining and surface staining were performed. After washing, data were acquired using FACSymphonyA3 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0286] In vitro CAR-T cell survival and functional analysis Figure 17 )

[0287] The long-term survival, memory formation, and functional maintenance of CAR-T cells were evaluated. CAR-T cells were stimulated 1:1 with stimulating factors (medium medium as a negative control, EGFRvIII stimulation magnetic beads, and U87d cell line). After 24 hours, the stimulating factors were removed by magnetic beads or sorting, and CAR-T cells were divided into 0mM, 0.5mM, and 10mM groups for further culture. Before stimulation and on days 1, 3, and 7 after stimulation, in addition to cell counting, cell survival / cell death markers such as Annexin V / 7AAD, CD27, CCR4, and CD45RA, as well as inhibitory factors such as PD-1, TIM3, and LAG3, were analyzed and evaluated. Figure 19 Other indicators (transcription factors, activation / senescence biomarkers) were also analyzed. Additionally, RNA was extracted from each sample and subjected to detailed analysis including RNA sequencing. After a series of analyses, some cells from day 7 were stimulated again in the same group, and the survival / functionality of the repeatedly stimulated cells was analyzed.

[0288] Cytokine analysis (FCM)

[0289] 1×10 5 CAR-T cells and 5×10 5 EGFRvIII-expressing U87 cell line (U87Δ) was co-cultured in 1 ml of medium for 12 hours, followed by 6 hours of culture in the presence of 5 μg / ml monensin (BD 554724 BD Bioscience) to stimulate CAR T cells. The medium consisted of glucose-free RPMI 1640 (Wako) supplemented with 10% dialyzed FBS (Cytiva), and the glucose concentration was adjusted to 10 mM and 0.5 mM with glucose solution (Gibco). Cells were fixed using fixation / permeabilization dilution (Invitrogen), and intracellular cytokines were stained with the antibodies shown in Table 1. Data were acquired using LSR Tortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0290] Cytotoxic activity analysis

[0291] In a 96-well plate, with 2×10⁻⁶ holes per well... 5Cells were seeded into target cells (luciferase-expressing U87Δ cells) 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, the live cells were fluoresced with VivoGlo luciferin (Promega), and luciferase activity was measured using Cytation (Promega) to calculate cytotoxic activity.

[0292] Metabolic analysis

[0293] Cellular metabolic capacity was measured using an XFe24 cell throughput analyzer (Bioscience) and an XF glycolysis stress assay kit (Agilent Technologies) according to the product protocol. The day before analysis, 24-well plates were coated with poly-D-lysine (0.1 mg / ml) in a CO2-free incubator, and the sensor cartridges were hydrated. On the day of analysis, cells were washed with analytical medium (Seahorse XF RPMI medium, L-glutamine 2 mM) and then subjected to a 2×10⁻⁶ ion exchange rate. 5 Cells were seeded on an analytical plate coated with poly-D-lysine and incubated at 37°C in CO2-free conditions for 60 minutes. To achieve a final concentration of 20 μM for oligomycin and 10 μM for FCCP, glucose was added to each port of the sensor housing to a final concentration of 10 mM or 0.5 mM, oligomycin to a final concentration of 1 μM, and 2-DG to a final concentration of 50 mM. The cells were then analyzed using a throughput analyzer, and metabolic function was evaluated by OCR and ECAR.

[0294] Glucose uptake capacity assessment

[0295] 2×10 4 GLUT3 CAR-T cells were seeded in 96-well plates and cultured for 5 hours with 200 μl of glucose-free RPMI 1640 (Wako). Five minutes before analysis, 1 μl of 2-NDBG (abcam) was added to each well. After washing, data were acquired using an LSR Tortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0296] Intracranial tumor xenotransplantation model

[0297] 2.5 × 10⁻⁶ cells were injected into the intracranial cavity (base of the right brain) of NSG mice. 4After successful transplantation, a U87Δ tumor cell line expressing luciferase (U87Δ-luc) was administered via tail vein with 2×10 Mock, 3C10 CAR-T cells, and GLUT3 CAR-T cells respectively. 6 Individual cells. Tumor burden was monitored over time using in vivo imaging, and survival was observed.

[0298] (method)

[0299] Figure 1 The structure of the metabolically altered CAR (EGFRvIII_CAR_GLUT3) is shown. A single-chain antibody (asFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) was constructed and linked to the CD8 hinge region, CD28 transmembrane region (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BB ICD, and CD3z to construct the CAR. Downstream of the CAR, a high-affinity glucose transporter (GLUT3) is encoded via a P2A sequence.

[0300] (result)

[0301] The results are shown in Figure 2 And then thereafter. For example... Figure 2 As shown, enhanced GLUT3 expression and glucose uptake on the T cell surface are evident. CAR-T cells were obtained by introducing conventional CAR (3C10 CAR) and metabolically modified CAR (GLUT3 CAR) genes into T cells derived from healthy donors. Uniform GLUT3 expression on the GLUT3 CAR-T cell membrane was confirmed by fluorescence microscopy. Furthermore, compared to T cells and conventional CAR-T cells (3C10 CAR-T cells), GLUT3 CAR-T cells exhibited enhanced glucose uptake.

[0302] Then, Figure 3 This demonstrates T cell expansion efficiency comparable to conventional CAR-T cells. Figure 3 As shown, the T cell proliferation efficiency was analyzed after CAR gene induction following stimulation with anti-CD3 / 28 magnetic beads. GLUT3 CAR-T cells showed a similar establishment efficiency to conventional CAR-T cells (3C10 CAR-T cells).

[0303] Then, Figure 4This study demonstrates how GLUT3 expression enhances the glycolytic capacity of T cells. As shown in the figure, the metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Flax analyzer. GLUT3 CAR enhanced glycolytic capacity under both low-glucose and normal-glucose conditions.

[0304] Then it was shown that GLUT3 CAR-T cells had a competitive advantage under hypoglycemic conditions. Figure 5 ).like Figure 5 As shown, 3C10 CAR-T cells and GLUT3 CAR-T cells were co-cultured under normal glucose and low glucose conditions. Under normal glucose conditions, the survival rates of both types were found to be comparable, but under low glucose conditions, GLUT3 CAR-T cells showed significant survival, indicating their ability to efficiently take up glucose.

[0305] Then, Figure 6 This indicates that GLUT3 CAR-T cells have enhanced cytokine production capabilities. For example... Figure 6 As shown, after antigen stimulation of 3C10 CAR-T cells and GLUT3 CAR-T cells, the proportion of cells producing cytokines was analyzed by FCM. Significantly increased expression of cytokine groups (IFN-γ, IL-2, TNF-α), which are important for T cell maintenance / activation and anti-tumor activity, was confirmed.

[0306] Then, Figure 7 This study demonstrates that GLUT3 CAR-T cells can maintain function under hypoglycemic conditions. As shown in the figure, cytokine production capacity was compared between normal and hypoglycemic conditions. In conventional CAR-T cells (3C10 CAR-T cells), extreme functional reduction was confirmed under hypoglycemic conditions. On the other hand, GLUT3 CAR-T cells exhibited high cytokine production capacity under normal glucose conditions and performed comparable to 3C10 CAR-T cells under normal glucose conditions, even under hypoglycemic conditions.

[0307] Then, Figure 8 The enhanced cytotoxic activity of GLUT3 CAR-T cells is shown. As shown in the figure, cytotoxic activity was analyzed using cell lines expressing EGFRvIII as targets. Cytotoxic activity was analyzed under both low-glucose and high-glucose conditions. Under both conditions, GLUT3 CAR-T cells showed higher cytotoxic activity than 3C10 CAR-T cells.

[0308] Then, Figure 9 The characteristics of memory phenotype produced by GLUT3 CAR are shown. As shown in the figure, the memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. In GLUT3 CAR-T cells, a tendency to differentiate into effector T cells was confirmed.

[0309] Then, Figure 10 This study demonstrates the inhibition of inhibitory molecule expression in GLUT3 CAR-T cells. The expression of inhibitory molecules under unstimulated conditions and under EGFRvIII antigen-positive cell stimulation conditions was compared. Inhibition of PD-1, LAG3, and Tim3 expression was confirmed in GLUT3 CAR-T cells.

[0310] Then, Figure 11 This presentation shows an mRNA analysis of GLUT3 CAR-T cells (evaluation of metabolism, exhaustion, activation, and differentiation). Detailed analysis of 3C10 CART and GLUT3 CAR-T cells was performed using mRNA expression. In GLUT3 CAR-T cells, similar to FCM, a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors were observed. Overall, this data indicates enhanced T cell effector functions, including activation and cytokine production.

[0311] Then, Figure 12 This demonstrates that the effect is not limited to 3C10 CARs; it can also be achieved with CD19 CARs, proving its versatility. GLUT3 was also incorporated into a CD19 CAR, confirming the same effect as with 3C10 CARs. Furthermore, by inducing CD19 expression in pancreatic cancer cell lines, its effectiveness against pancreatic cancer was also demonstrated.

[0312] like Figure 13 The results demonstrate rapid tumor eradication in an intracranial xenograft model. Antitumor activity was observed in the U87Δ intracranial xenograft model. Faster tumor eradication was confirmed in GLUT3 CAR-T cells compared to 3C10 CAR-T cells. Furthermore, tumor rejection was confirmed in tumor re-challenge.

[0313] (Example 2: Enhanced expression of glucose transporters)

[0314] To achieve enhanced expression of glucose transporters, a constitutive CAR construct for expressing GLUT3 was designed (and... Figure 12 (The stbl-GLUT3=GLUT3 CAR is the same).

[0315] Use import to have Figure 12 We analyzed the function and anti-tumor effects of CAR cells.

[0316] Figure 15 The results show the evaluation of the antitumor effect of GLUT3 CAR-T cells in the same mouse model. No complete remission (CR) was achieved in the Mock T cell and 3C10 CAR-T cell groups, while 4 mice in the GLUT3 CAR-T group achieved complete remission (CR).

[0317] Figure 16 The results of tumor rechallenge are shown. Specifically, the sudden death of mice in the GLUT3 CAR-T cell group is shown. The tumor (U87d) was re-transplanted into the cured mice from the previous experiment to evaluate their rejection ability ("a surrogate indicator of memory formation").

[0318] Figure 17 Enhanced long-term survival was observed after various stimuli. CAR-T cells were observed over time under different conditions. Results showed that under hypoglycemic conditions, conventional CAR-T (3C10) did not show effective cytokine production, while GLUT3CAR-T demonstrated effective cytokine production. However, GLUT3 CAR-T cells, activated by excessive glucose uptake, differentiated over time (day 3 or day 7) into CCR7-negative Tem or Temra cells (terminal differentiation, loss of stemness), and entered apoptosis.

[0319] In addition, when CAR-T cells (established under normal glucose) are abruptly (day 0) placed under low glucose conditions of 0 mM or 0.5 mM, GLUT3 CAR-T cells with high activation and high glucose dependence are prone to apoptosis.

[0320] Figure 18 Enhanced long-term survival after stimulation was observed. The antitumor effects, survival, and adverse events were investigated in the same system as the aforementioned mice. In Experiment 1, GLUT3 CAR-T cells showed equivalent antitumor activity. In Experiment 2, due to glucose exposure leading to overactivation of GLUT3 CAR-T cells, apoptosis and overdifferentiation occurred, resulting in the loss of antitumor activity. In the rechallenge, GLUT3 CAR-T cells rejected the tumor. In Experiment 4, CAR-T cell transplantation and tumor T cell infiltration proportionally superior to the tumor response were observed in GLUT3 CAR-T cells. Furthermore, Experiment 3 was conducted in a liver tumor model inducing immunosuppression due to a hypoglycemic, hyperlactatic environment, with the expectation of obtaining similar results to Experiment 2.

[0321] Figure 19Data showing enhanced long-term survival after stimulation (in vitro data) are presented. Annexin V and 7-AAD staining data reflecting cell death and apoptosis are shown at the time points on the left. Cell death of CAR-T cells was investigated using Annexin V and 7-AAD. While differences due to glucose concentration were observed in the unstimulated state, no significant differences were observed among the individual CAR-T cells.

[0322] (Example 3: CAR-T Example)

[0323] The following are further empirical examples of CAR-T.

[0324] (Materials and Methods)

[0325] (cell line)

[0326] 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 and Kazumitsu Pure Chemicals Co., Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. EGFRvIII-packaged lentiviral vectors were introduced into U-87 MG and U-251 MG cells, and the transduced cell lines were 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 and Kazumitsu Pure Chemicals Co., Ltd.) supplemented with 10% FBS and 1% penicillin / streptomycin. A truncated CD19 vector was introduced into NALM6 cells to obtain the CD19-expressing AsPC1 cell line.

[0327] (Flow cytometry analysis)

[0328] Flow cytometry analysis was performed according to previously described methods (40. Kumagai S, Togashi Y, Kamada T, et al (2020) "The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies", Nat Immunol;21(11):1346-58.; 41. Kumagai S, Koyama S, Itahashi K, et al (2022) "Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments", CancerCell;40(2):201-18.e9.; 42. Tada Y, Togashi Y, Kotani D, et al (2018) "Targeting VEGFR2 with Ramucirumab strongly impacts effector / activated regulatory T cells and CD8+ T cells in the tumor microenvironment", J Immunother Cancer;6(1):106.1 and 43. Tanegashima T, Togashi Y, Azuma K, et al (2019) "Immune Suppression by PD-L2 against Spontaneous and Treatment-Related Antitumor Immunity", ClinCancer Res;25(15):4808-19.In brief, cells were washed twice with FACS buffer and blocked with Fc receptor blocking solution (BioLegend, San Diego, California) for 10 minutes in the dark at 4°C. Cells were then washed twice with FACS buffer, and antibodies targeting cell surface molecules were added, followed by incubation in the dark at 4°C for 20 minutes. Cells were then washed twice with FACS buffer. For intracellular antigen staining, cells were incubated in fixation / permeabilization solution (BD Biosciences, San Jose, California) at room temperature for 1 hour, followed by washing twice with wash buffer (BD Biosciences) according to the manufacturer's instructions. Antibodies targeting intracellular antigens were added, and cells were incubated in the dark at 4°C for 20 minutes, followed by washing twice. For apoptosis analysis, cells were washed once with FACS buffer and once with Annexin binding buffer. Cells were then stained with Annexin V and 7-AAD at room temperature for 15 minutes. After washing, flow cytometry (FCM) analysis was performed using an LSR Tortessa X-20 cytometer (BD Biosciences) and analyzed using FlowJover.10 software (BD Biosciences). The staining solutions were prepared according to the manufacturer's instructions.

[0329] (Cytokine staining)

[0330] Intracellular cytokine staining was performed according to previously described methods (40. Kumagai S, et al (2020) atImmunol;21(11):1346-58.; 41. Kumagai S, et al (2022) Cancer Cell;40(2):201-18.e9.; 42. Tada Y, et al (2018) J Immunother Cancer;6(1):106.l and 43. Tanegashima T, et al (2019) Clin Cancer Res;25(15):4808-19.). Monensin was added to the culture medium during the last 5 hours of 6-hour T cell stimulation to retain cytokines within the cells. After staining for 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 washing buffer and then treated with antibodies targeting the cytokines. Incubate in the dark at 4°C for 20 minutes. After washing, perform FCM analysis using an LSR Tortessa X-20 cytometer (BD Biosciences) and analyze with FlowJo ver.10 software (BD Biosciences). The antibodies used for cell staining are summarized in Table 2. Antibodies were prepared according to the manufacturer's instructions.

[0331] Table 2

[0332]

[0333] (Killing Experiment)

[0334] Total 1×10 5Cells expressing luciferase (U-87 MGΔ, U-251 MGΔ, AsPC-1, and NALM6) were co-cultured with CAR-T cells at a specified ratio in 200 μL of RPMI 1640 medium (manufactured by Fujifilm and Kodenki Chemical Co., Ltd.) supplemented with low glucose (0.5 mM) or high glucose (10 mM) on flat-bottomed 96-well white plates. After 24 hours of culture, Bio-Glo (Promega, Madison, WI, WA) was added to each well, and luminescence was measured using a Cytation 5 microplate reader (Agilent Technologies, Santa Clara, CA, WA). Specific lysis (% specific lysis) was calculated using the following formula: % specific lysis = [(experimental lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis)] × 100.

[0335] (Immunofluorescence staining)

[0336] After washing, cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then permeabilized with cold methanol at -20°C for 10 minutes. Cells were then blocked with 3% BSA / PBS at room temperature for 60 minutes. After blocking, cells were incubated with primary antibody at 4°C for 2 hours, and with secondary antibody at 4°C for 1 hour. DAPI was then added, and cells were incubated at room temperature for 5 minutes. After washing, stained cells were fixed onto slides using Cytospin and analyzed using a BZ-X710 (KEYENCE, Osaka, Japan).

[0337] (Immunohistochemical (IHC) staining)

[0338] IHC staining was performed on 5 μm thick sections of formalin-fixed paraffin-embedded specimens. Antigen retrieval was performed in a steam oven using citrate buffer (pH 6.0), followed by dewaxing and rehydration. Slides were incubated with primary antibody for 16 hours, then with HRP-labeled secondary antibody for 1 hour, followed by staining with diaminobenzidine substrate. Primary antibody staining was performed using anti-CD3ε antibody (clone: ​​SP7, catalog number: ab16669) (Abcam, Waltham, MA). The stained slides were counterstained with hematoxylin.

[0339] Hematoxylin-eosin (HE) staining was performed according to standard procedures. Dewaxed and hydrated slides were placed in hematoxylin solution and incubated for 4 minutes. After rinsing, the slides were incubated in eosin solution for 2 minutes. Stained slides were scanned using a BZ-X710 (Keyence) at 40x and 400x magnification. The stained slides were independently evaluated by two pathologists.

[0340] (ELISA method)

[0341] Total 2.0 × 10 5 10 U-87 MGΔ cells and 2.0 × 10 5 CAR-T cells were co-cultured in 24-well plates. After 24 hours of culture, the supernatant was recovered, and ELISA was performed to determine cytokine concentrations. ELISA kits for IFN-γ, IL-2, and TNFα were used according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, Minnesota). Cytokine levels in mouse serum were analyzed using a high-sensitivity LUMINEX assay (Merck Millipore, Burlington, MA, Massachusetts) according to the manufacturer's instructions.

[0342] (CAR-T cell production)

[0343] Peripheral blood was collected from healthy individuals, and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-Paque (GE Healthcare, Chicago, IL, Illinois). T cells were isolated from the PBMCs using negative selection via MojoSort (BioLegend). The isolated T cells were stimulated with anti-CD3 / 28 magnetic beads (ThermoFisher Scientific, Waltham, MA) at a T cell:bead ratio of 1:1 (day 0). Twenty-four hours after stimulation (day 1), the T cells were inoculated with CAR lentivirus at MOI 3 and cultured in 30 U / ml IL-2. Half of the culture medium was replaced after 24 hours. The CD3 / 28 beads were removed after 5 days. During culture, half of the culture medium was replaced on days 4 and 10, and the T cell concentration was adjusted to 0.7 × 10⁻⁶. 6 CAR-T cells / mL. The prepared CAR-T cells were harvested on day 10 for subsequent analysis.

[0344] (Animal model)

[0345] Female NSG mice (6 weeks old) were purchased from Jackson Laboratory. Anesthesia was administered intraperitoneally to prevent pain prior to analgesia. Using a positioning frame, tumor cells were seeded from the surface of the brain to a position 2 mm to the right, 3 mm to the back, and 3 mm deep of the anterior fontanelle. A total of 2.0 × 10⁻⁶ cells were injected into 5 mL of PBS over 1 minute. 4 One cell was extracted, then the 1mm needle was withdrawn, and the cell was left to stand for 1 minute before being withdrawn again. Four days after tumor transplantation, tumor growth was confirmed by bioluminescence imaging (BLI), and patients were randomly assigned to different treatment groups. On day 5, 1.0 × 10⁻⁶ cells were injected via the tail vein. 6 One CAR-T cell was injected. Tumor proliferation was monitored twice weekly via BLI after CAR-T cell infusion. Animal husbandry and experiments were conducted in accordance with the guidelines of the Animal Committee of the National Cancer Center of Japan, following approval from the Animal Ethics Review Committee.

[0346] (Glucose concentration determination)

[0347] Tissue samples (5×5 mm) were cleaned to remove adhering blood. The samples were chopped and centrifuged at 4°C for 10 minutes to recover the tumor interstitial fluid. The glucose concentration of the interstitial fluid was determined using a Multiskan GO (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0348] (Metabolic analysis)

[0349] OCR (pmol / min) and ECAR (mpH / min) were evaluated using a Seahorse XF-24 metabolic extracellular flux analyzer (Agilent Technologies). CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and on-demand GLUT3 EGFRvIII CAR-T cells) stimulated for 24 hours with EGFRvIII magnetic beads (ACRO Biosystems, Beijing, China) were resuspended in glucose-free unbuffered RPMI-1640 medium (Agilent Technologies) and seeded on poly-L-lysine-coated Seahorse cell culture plates (2.0 × 10⁶ cells per well). 5(Cells). Perturbation analysis of CAR-T cell metabolic pathway utilization was performed by adding glucose (10 mM or 0.5 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (50 mM) (all from Agilent Technologies). Experiments using the Seahorse system were performed under the following analytical conditions: mixing for 3 minutes, waiting for 2 minutes, and measuring for 3 minutes. Subsequently, the values ​​of metabolic parameters were calculated.

[0350] (RNA sequencing and subsequent analysis)

[0351] CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and on-demand GLUT3 EGFRvIII CAR-T cells) sorted with BDFACSymphony S6 (BD Biosciences) at a 1:1 ratio were stimulated with EGFRvIII magnetic beads under low glucose (0.5 mM) or high glucose (10 mM) conditions and supplemented with 30 U / ml IL-2. CAR-T cells were recovered on days 3 and 7, and RNA was extracted using the RNeasy kit (RNeasykit, QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from the isolated RNA using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA). RNA-seq libraries were sequenced using Novaseq X plus (Illumina, San Diego, CA) with 150 bp at each end (paired-end reads). To utilize RNA-seq data for expression profiling, paired end reads were aligned with the hg38 human genome assembly using STAR (Dobin A, Gingeras TR (2015), Mapping RNA-seq Reads with STAR. Curr Protoc Bioinformatics, 51:11.4.1-4.9). RNAseqChef is a web-based platform for systematic transcriptome analysis, gene expression analysis, principal component analysis, and pathway analysis (Etoh K, Nakao M (2023), A web-based integrative transcriptome analysis, RNAseqChef, reveals the cell / tissue type-dependent action of sulforaphane (J Biol Chem;299(6):104810).

[0352] (Statistical Analysis)

[0353] Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA). Relationships between groups were compared using t-tests or one-way ANOVA. Survival rates were analyzed using the Kaplan-Meier method and statistically compared using the log-rank test. A p-value <0.05 was considered statistically significant.

[0354] (result)

[0355] (GBM has low glucose concentration within the TME, which impairs the function of CAR-T cells.)

[0356] Although accumulated evidence suggests that low glucose levels within the TME are a potential obstacle to CAR-T cell therapy in solid tumors, the actual glucose concentration within the TME and the impact of low glucose status on CAR-T cell function remain unclear [Peng JJ, Wang L, Li Z, et al (2023) Metabolic challenges and interventions in CAR T cell therapy, Sci Immunol;8(82):eabq3016]. The inventors measured the glucose concentration in surgical samples and serum interstitial fluid (...). Figure 20 a). The glucose concentration in the interstitial fluid of GBM samples was approximately 10 times lower than that in serum [less than 0.5 mM (average 0.217 mM)] ( Figure 20 b). Although glucose concentrations are generally low in many types of cancer, such as non-small cell lung cancer and colon cancer, GBM exhibits the lowest glucose concentration in the TME. This suggests that low glucose concentration in the TME is a characteristic of GBM. Figure 20 c).

[0357] Then, the effects of a hypoglycemic environment on CAR-T cell function, including cytokine production, were investigated. CAR-T cells targeting the GBM-associated antigen EGFRvIII (conv EGFRvIII CAR-T cells) were co-cultured with a human GBM cell line expressing EGFRvIII (U-87 MGΔ) under hypoglycemic conditions (0.5 mM) that replicated the TME of GBM. Stimulation with U-87 MGΔ significantly reduced the production of cytokines (IFN-γ, IL-2, TNFα) even with short-term (16 hours) exposure under hypoglycemic conditions. Figure 20(d, e). To further investigate the functional changes of CAR-T cells under hypoglycemic conditions, CAR-T cells were stimulated for 16 hours under either hypoglycemic (0.5 mM) or hyperglycemic (10 mM) conditions, and their gene expression profiles were analyzed. Gene expression profiles changed significantly due to exposure to hypoglycemic conditions. Figure 22 a). Gene sets related to cell cycle, differentiation, and cytokine production were significantly reduced, while gene sets related to cellular responses to glucose starvation and endogenous apoptosis increased under low glucose conditions. Figure 20 (f, g). Thus, when exposed to low glucose conditions such as TME (tumor methyl phospholipids) like GBM, the effector function of CAR-T cells is immediately impaired.

[0358] (Overexpression of GLUT3 enhances the metabolic adaptability of CAR-T cells.)

[0359] Among the SLC2 family of glucose transporters (GLUTs), GLUT3, encoded by SLC2A3, has the highest affinity and is characterized by preferential expression in neurons and glial cells with high glucose demand [Flavahan WA, Wu Q, Hitomi M, et al (2013) Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake Nat Neurosci;16(10):1373-82.]. Therefore, the inventors investigated whether GLUT3 expressed by CAR-T cells could promote CAR-T cell activity by promoting glucose uptake in the low-glucose TME of the GBM. SLC2A3 was fused into a conventional anti-EGFRvIII CAR construct via a self-cleaving P2A sequence, enabling stable simultaneous expression of CAR and GLUT3 (GLUT3 EGFRvIII CAR). Figure 21 a). In the membrane of T cells infused with GLUT3 EGFRvIII CAR (GLUT3EGFRvIII CAR-T cells) ( Figure 21 b) GLUT3 was expressed at a level approximately twice that of parental anti-EGFRvIII CAR-T cells (conv EGFRvIII CAR-T cells), comparable to that of GBM cell line U-87 MGΔ, but no change in GLUT1 expression was observed. Figure 21 c and Figure 23a). Glucose uptake, as determined by a glucose uptake assay using the glucose analog 2-NBDG, was greater in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells. Figure 21 d). Therefore, the glycolysis and glycolytic capacity of GLUT3 EGFRvIII CAR-T cells were significantly improved. Figure 21 e and Figure 23 b). Metabolic analysis further confirmed that GLUT3 EGFRvIII CAR-T cells, compared to conv EGFRvIII CAR-T cells, transitioned to a more energy-intensive state under conditions of higher oxygen consumption rate (OCR) and higher extracellular acidification rate (ECAR). Figure 21 f). In a glucose competition experiment in which GLUT3 EGFRvIII CAR-T cells labeled with carboxyfluorescein succinimide (CFSE) were co-cultured with convEGFRvIII CAR-T cells at a 1:1 ratio, the number of GLUT3 EGFRvIII CAR-T cells increased relative to conventional cells at low glucose levels (0.5 mM), while the number was comparable under high glucose conditions (10 mM). This suggests that GLUT3 EGFRvIII CAR-T cells are substantially competitive in terms of glucose uptake, especially in low glucose environments. Figure 23 (c, d). Therefore, stable GLUT3 expression in CAR-T cells can promote glucose uptake, making CAR-T cells competitive in glucose uptake and thus improving their metabolic adaptation in glucose-limited environments.

[0360] Since GLUT3 expression enhances the metabolic adaptability of CAR-T cells, its effector function and safety profile were investigated. Therefore, the inventors studied the effector function of GLUT3EGFRvIII CAR-T cells using both in vitro and in vivo experiments. Compared to conv EGFRvIII CAR-T cells, GLUT3EGFRvIII CAR-T cells showed significantly increased production of cytokines (IFN-γ, IL-2, TNFα) under both low glucose levels (0.5 mM) and high glucose levels (10 mM). Figure 21 g, h and Figure 24 a). Cytokine production occurs in CD8. + T cell subsets and CD4 + Enhancement was observed in all T cell subsets. Figure 24b, c). GLUT3 EGFRvIII CAR-T cells also showed enhanced 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. Figure 21 i、 Figure 24 d, e). Following antigen stimulation, among exhaustion-related molecules including PD-1 and Tim-3, GLUT3 EGFRvIII CAR-T cells showed higher expression than conv EGFRvIII CAR-T cells (d, e). Figure 21 j, k and Figure 24 f, g). Furthermore, enhanced effector function based on stable GLUT3 expression was also observed in CAR-T cells targeting different CD19 antigens (GLUT3 CD19 CAR-T cells). This indicates that the enhanced metabolic adaptation resulting from GLUT3 expression is universal across all CARs, regardless of the single-chain variable region fragment (scFv) used or the antigen targeted. Figure 25 Therefore, through stable expression of GLUT3, CAR-T cells, although exhibiting a depleted phenotype, can still be activated in a TME with low glucose levels.

[0361] (discuss)

[0362] Enhancing anti-tumor effects by improving the metabolic adaptation of CAR-T cells in the tumor microenvironment (TME) is a novel concept proposed to accelerate the clinical application of CAR-T cell therapy in refractory solid tumors. Although some CAR-T cell clinical trials targeting GBM have failed to demonstrate clinical benefit, immunological monitoring of these clinical trials has shown that abundant CAR-T cells can be detected in the TME. [O'Rourke DM, Nasrallah MP, Desai A, et al (2017) A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma Sci Transl Med;9(399).; Bagley SJ, Logun M, Fraietta JA, et al (2024) Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results Nat Med;30(5):1320-9.; Brown CE, Hibbard JC, Alizadeh D, et al (2024) "Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial" NatMed;30(4):1001-12. Given the presence of CAR-T cells in the TME, antigen loss or migration impairment can be ruled out as the cause of failure, and it is speculated that specific conditions in the TME containing GBM are involved in inducing CAR-T cell dysfunction.Since effector T cells rely on glycolysis for survival and activation, glucose is an indispensable nutrient [Ho PC, Bihuniak JD, Macintyre AN, et al (2015) Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses Cell;162(6):1217-28.]. Therefore, considering the significant low glucose environment detected in the TME of GBM, the low glucose level in the TME of GBM must be a major metabolic stress for CAR-T cells. In fact, conv EGFRvIII CAR-T cells are in a state of dysfunction (reduced cytokine production and increased expression of exhaustion markers). Furthermore, PD-1 signaling disrupts PI3K / Akt / mTOR signaling and further inhibits glycolysis, thus effector T cells may fall into a negative metabolic cycle [Chang CH, Qiu J, O'Sullivan D, et al (2015) "Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression" Cell;162(6):1229-41].

[0363] Considering that nervous system cells such as neurons and glial cells, as well as GBM, especially GBM expressing EGFRvIII, utilize the high-affinity glucose transporter GLUT3 to meet their high glucose requirements [Vannucci SJ, Maher F, Simpson IA (1997) Glucosetransporter proteins in brain: delivery of glucose to neurons and glia Glia;21(1):2-21.; Chen S, Yang L, Li Z, et al (2021) EGFR / EGFRvIII partly regulates the tumorigenesis of glioblastoma through the SOX9-GLUT3 axis Am J Transl Res;13(6):6055-65.; Schmidt S, Hommel A, Gawlik V, et al (2009) [Essential role of glucose transporter GLUT3 for post-implantation embryonic development] J Endocrinol;200(1):23-33. If CAR-T cells express high levels of GLUT3, it is highly likely to overwhelm nutrient competition. In fact, the GLUT3EGFRvIII CAR construct developed by the inventors has been shown to enhance glucose uptake, as well as effector functions such as cytokine production and cytotoxicity in GLUT3 EGFRvIII CAR-T cells.

[0364] (Example 3: Precursor cells of other effector T cells)

[0365] Perform the same experiment using other T cells. For example, unpurified peripheral T cells containing Tnv, Tcm, Tem, Temra, etc., any subset can be introduced with CAR (e.g., constitutively expressing GLUT3 CAR).

[0366] In this embodiment, CAR (CAR constitutively expressed GLUT3 CAR) was introduced into a purified and concentrated random T cell memory subset. In another experiment, concentrated CAR-T cells were obtained by inducing random T cell memory subsets through culture conditions.

[0367] Experimental steps:

[0368] • T cells derived from peripheral blood, bone marrow, umbilical cord blood, etc., are analyzed based on the expression pattern of T cell memory-related surface antigens, namely CD45RA. + CCR7 + CD95 - CD45RA + CCR7 + CD95 + CD45RA - CCR7 + CD45RA - CCR7 and CD45RA + CCR7 - T cells, which are expected to have longer-term survival and stemness maintenance, were sorted using FACS or magnetic beads, respectively. eff Precursor cells Tnv and Tscm, Tcm, or differentiated T cells that primarily function as effectors eff The Temra subpopulation was purified and concentrated.

[0369] • Stimulate each subpopulation with CD3 / 28 magnetic beads, etc., and introduce CAR using lentiviral vectors.

[0370] • Alternatively, as another method, after introducing the gene into Bulk T cells, the type and amount of cytokines can be adjusted (using IL-7 and IL-15 or platelet lysate) to inhibit T cells. eff The differentiation and induction of Tnv, Tscm, and Tcm dominant proliferation were achieved to obtain CAR-T cells with precursor cells predominant.

[0371] • In vitro assessment: These cells were co-cultured with U87d tumor cell line or EGFRvIII magnetic beads. After 1, 3 and 7 days, cell division and proliferation were evaluated by CFSE staining and cell counting, and activation and exhaustion status were evaluated by flow cytometry.

[0372] • In vivo evaluation: These cells were administered to the above-mentioned U87d intracranial tumor-bearing mice, and the antitumor effect was evaluated by BLI-based antitumor activity and survival.

[0373] result:

[0374] • Introduced into Bulk T cells or into T cells undergoing differentiation. eff Compared to Temra, when introduced into Tnv, Tscm, Tcm, etc., which are used as precursor cells, eff In its precursor cells or in CAR-T cells that are predominantly expanded in Tnv, Tscm, and Tcm, it is expected to have high division / proliferation capacity and long-term cell division can be observed. In addition, it is expected to have low expression of PD1, TIM3, and LAG3 and be resistant to exhaustion.

[0375] (Example 4: TCR example)

[0376] The same experiment can be performed using the example of importing TCR instead of CAR. For example, TCR can be introduced into the TCR. eff TCR was introduced into subpopulations such as Tnv, Tcm, Tem, and Temra.

[0377] In this embodiment, TCRs (constitutively expressing GLUT3TCRs) were introduced into purified, concentrated random T cell memory subsets. In another experiment, TCR-T cells, which were concentrated from random T cell memory subsets, were induced under culture conditions.

[0378] Various in vitro and in vivo evaluations as shown in Examples 1-3 were performed.

[0379] (Example 5: Prevention, reinfection prevention and treatment of bacterial infections)

[0380] In this embodiment, the prevention of bacterial infections, prevention of reinfection, and / or treatment are verified.

[0381] (method)

[0382] In the prevention of bacterial infections (such as bacterial conjunctivitis), T cells of this disclosure modified with the antigens of the infectious pathogens of bacterial infections are administered to subjects who do not have bacterial infections.

[0383] In the prevention and / or treatment of recurrent bacterial infections (such as bacterial conjunctivitis), the T cells disclosed herein are administered to subjects who have had and / or are currently suffering from bacterial infections.

[0384] After administration, the subjects were monitored for prognostic effects of bacterial infection using conventional techniques in this field.

[0385] (Example 6: Prevention, reinfection prevention and treatment of parasitic infections)

[0386] In this embodiment, the prevention, reinfection prevention, and / or treatment of parasitic infections are verified.

[0387] (method)

[0388] In the prevention of parasitic infections (such as amoebic keratitis), T cells of this disclosure modified with antigens of the pathogen causing the parasitic infection are administered to subjects who do not have a parasitic infection.

[0389] In the prevention and / or treatment of recurrent parasitic infections (such as amoebic keratitis), the T cells disclosed herein are administered to subjects who have had and / or are currently suffering from parasitic infections.

[0390] After administration, the subjects were observed for prognostic effects of parasitic infection using conventional techniques in this field.

[0391] (Example 7: Prevention of infection, prevention of onset, prevention of reinfection, and treatment of viral infections)

[0392] In this embodiment, infection prevention, disease prevention, reinfection prevention and / or treatment of viral infections are verified.

[0393] (method)

[0394] In the prevention of viral infections (including but not limited to: tuberculosis, malaria, yellow fever virus, smallpox virus, cowpox, measles / rubella, poliomyelitis, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonellosis, pathogenic Escherichia coli, toxoplasmosis, Zika virus, herpes simplex virus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza, MARS, rabies, diphtheria, etc.), T cells of this disclosure modified with the antigen of the infectious pathogen of the viral infection are administered to subjects who do not have a viral infection.

[0395] In the prevention and / or treatment of relapse of the viral infection, the T cells of this disclosure are administered to subjects who have had and / or are currently suffering from a viral infection.

[0396] After administration, the subjects were monitored for prognostic effects of viral infection using conventional techniques in this field.

[0397] (Example 8: Prevention, recurrence prevention and treatment of allergies)

[0398] In this embodiment, the prevention, recurrence prevention, and / or treatment of allergies are verified.

[0399] (method)

[0400] In the prevention of allergies (e.g., allergic conjunctivitis), T cells of this disclosure modified with allergen antigens are administered to subjects who do not possess allergen antigens.

[0401] In the prevention and / or treatment of recurrent allergies (e.g., allergic conjunctivitis), the T cells disclosed herein are administered to subjects who have previously had allergy causative antigens and / or currently have allergy causative antigens.

[0402] After administration, the subjects were observed for allergic reactions using conventional techniques in this field.

[0403] (Example 9: Prevention, relapse prevention and treatment of autoimmune diseases)

[0404] In this embodiment, the prevention, relapse prevention, and / or treatment of autoimmune diseases are verified.

[0405] (method)

[0406] In the prevention of autoimmune diseases (such as autoimmune uveitis), T cells of this disclosure modified with antigens of the cause of autoimmune disease infection are administered to subjects who do not have allergic pathogenic factor antigens.

[0407] In the prevention and / or treatment of relapse of autoimmune diseases (e.g., autoimmune uveitis), the T cells disclosed herein are administered to subjects who have had pathogenic factor antigens of autoimmune diseases and / or currently have pathogenic factor antigens of autoimmune diseases.

[0408] After administration, the subjects were observed for the prognosis of autoimmune diseases using conventional techniques in this field.

[0409] (Note)

[0410] As described above, this disclosure has been exemplarily illustrated using preferred embodiments, but it should be understood that the scope of this disclosure should be interpreted solely by the claims. It should be understood that the contents of any patents, patent applications, and other documents referenced in this specification should be incorporated herein by reference in the same manner as specifically described herein. This application claims priority to Japanese Patent Application No. 2024-001532, filed with the Japanese Patent Office on January 9, 2024, the contents of which are incorporated herein by reference in their entirety.

[0411] [Industry Applicability]

[0412] According to this disclosure, even in glucose-depleted environments such as tumor microenvironment (TME), cells that do not starve or deplete and are competitive with cancer cells can be provided. Therefore, strategies to improve the efficacy of cell-based immunotherapies can be developed, which is expected to be applied in the medical field.

Claims

1. A type of T cell with enhanced glucose uptake capacity.

2. The T cell according to claim 1, wherein, The T cells contain chimeric antigen receptors, or CARs.

3. The T cell according to claim 1 or 2, wherein, The T cells possess effector functions.

4. The T cell according to claim 1 or 2, wherein, The T cells mentioned are precursor cells of effector T cells (Teff).

5. The T cell according to any one of claims 2 to 4, wherein, The CAR is expressed in the T cells.

6. The T cell according to any one of claims 1 to 5, wherein, The glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

7. The T cell according to any one of claims 1 to 6, wherein, The glucose transporter is GLUT3.

8. The T cell according to any one of claims 1 to 7, wherein, The T cells mentioned are human T cells.

9. An effector T cell comprising a chimeric antigen receptor (CAR), which is a modified effector T cell expressing a glucose transporter and / or with enhanced expression of the glucose transporter, said T cell exhibiting at least equivalent effector function under 0.5 mM low glucose conditions as under 10 mM normal glucose conditions.

10. The T cell according to claim 9, wherein, The CAR is expressed in the T cells.

11. The T cell according to claim 9 or 10, wherein, The glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

12. An effector T cell with the GLUT3 gene introduced.

13. The T cell according to any one of claims 9 to 12, wherein, The T cells mentioned are human T cells.

14. A cell population comprising T cells with enhanced glucose uptake capacity, said T cells comprising cells that possess effector functions when introduced into the body.

15. The cell population according to claim 14, wherein, The T cells are modified cells that express glucose transporters and / or cells with enhanced expression of glucose transporters.

16. The cell population according to claim 14 or 15, wherein, The T cells possess effector functions.

17. The cell population according to any one of claims 14 to 16, wherein, The T cells contain chimeric antigen receptors, or CARs.

18. The cell population according to any one of claims 14 to 17, wherein, The cell population contains T eff .

19. The cell population according to any one of claims 14 to 18, wherein, The cell population contains T eff The precursor cells.

20. The cell population according to any one of claims 17 to 19, wherein, The CAR is expressed in the T cells.

21. The cell population according to any one of claims 14 to 20, wherein, The glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

22. The cell population according to any one of claims 14 to 21, wherein, The glucose transporter is GLUT3.

23. The cell population according to any one of claims 14 to 22, wherein, The T cells mentioned are human T cells.

24. A cell population comprising effector T cells containing a chimeric antigen receptor (CAR) and modified to express glucose transporters and / or enhanced expression of glucose transporters, said T cells exhibiting at least equivalent effector function under 0.5 mM low glucose conditions as under 10 mM normal glucose conditions.

25. The cell population according to claim 24, wherein, The CAR is expressed in the T cells.

26. The cell population according to claim 24 or 25, wherein, The glucose transporters include GLUT1, GLUT2, GLUT3 and GLUT4.

27. A cell population comprising effector T cells with the GLUT3 gene introduced into them.

28. The cell population according to any one of claims 24 to 27, wherein, The T cells mentioned are human T cells.

29. A pharmaceutical composition comprising T cells according to any one of claims 1 to 13 or a cell population according to any one of claims 14 to 28.

30. The pharmaceutical composition according to claim 29, used for the treatment or prevention of cancer.

31. The pharmaceutical composition according to claim 29 or 30, for the treatment of cancer.

32. The pharmaceutical composition according to any one of claims 29 to 31, wherein, The cure for the cancer is confirmed by the eradication of the tumor.

33. The pharmaceutical composition according to any one of claims 29 to 32, for producing a long-term effect against cancer.

34. The pharmaceutical composition according to any one of claims 29 to 33, for preventing cancer metastasis or cancer recurrence.

35. The pharmaceutical composition according to any one of claims 29 to 34, for the prevention of cancer recurrence.

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