Survivor T cells, survivor T cell populations, and pharmaceutical compositions

Abstract

The present disclosure provides cells that are competitive against cancer cells without starvation or exhaustion even in an environment where glucose is depleted. Specifically, the present disclosure provides survivor T cells in which the ability to take up glucose is enhanced, and the T cells are modified to express a glucose transporter and / or the expression of the glucose transporter is enhanced. Also provided are cell populations, pharmaceutical compositions, treatment methods, etc. containing such cells.

Description

Technical Field

[0001] The present disclosure relates to survivor T cells with enhanced glucose uptake ability, survivor T cell populations, and pharmaceutical compositions containing such cells or cell populations.

Background Art

[0002] Cancer cells reprogram their metabolic systems to be advantageous for their own cell proliferation, actively utilize the glycolytic system with low efficiency of ATP production even in the presence of oxygen, and enhance glucose uptake and lactate production (Warburg effect).

[0003] In the intratumoral microenvironment, there are many factors that suppress the metabolic activity and function of anti-tumor T cells. In addition to immunosuppressive factors, the competition between tumors and T cells in terms of metabolism is involved in the formation of an immunosuppressive environment. When T cells are stimulated by TCR, the intracellular concentration of Ca 2+ increases, and calcineurin is activated. The activation of calcineurin dephosphorylates the transcription factor NFAT, which translocates into the nucleus and promotes and activates the transcription of genes such as the IL-2 gene through interaction with other transcription factors.

[0004] On the other hand, since cancer cells consume a large amount of glucose and deplete it, type T eff tumor-specific T cells that require glucose as an energy source, even when they receive a signal from TCR when infiltrating into the tumor, the Ca 2+ concentration in the cells decreases, and the translocation of NFAT into the nucleus decreases. Therefore, T cells fall into dysfunction, and cell proliferation and cytokine production are suppressed.

[0005] In addition to glucose, when nutrients such as amino acids and fatty acids are also consumed in large amounts by cancer cells, competing T cells fall into dysfunction. Thus, in the intratumoral microenvironment, the metabolic mechanism that supports the active proliferation of cancer cells inhibits the anti-tumor effector action of tumor-specific T cells.

[0006] Therefore, even in such an environment, cells that are competitive against cancer cells without suffering from hunger or exhaustion are desired. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] The present disclosure provides T cells with enhanced glucose uptake ability, cells that function without suffering from hunger or exhaustion even in a tumor environment, a cell population containing the same, and a pharmaceutical composition containing such cells.

[0008] Accordingly, the present disclosure provides the following. [Item 1] T cells with enhanced glucose uptake ability. [Item 2] The cell according to Item 1, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 2A] The T cell according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item 3] The T cell according to any one of the above items, wherein the T cell has effector function. [Item 4] The T cell according to any one of the above items, wherein the T cell is a progenitor cell of effector T cell (T eff ). [Item 5] The T cell according to any one of the above items, wherein the CAR is expressed in the T cell. [Item 6] The T cell according to any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item 7] The T cell according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 8] The T cell according to any one of the above items, which is a human T cell. [Item 9] A cell population containing T cells with enhanced glucose uptake ability, wherein the T cells have the property of having effector function when introduced into the body. [Item 10] The cell population according to any one of the above items, wherein the T cells are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter. [Item 11] The cell population according to any one of the above items, wherein the T cells have effector functions. [Item 12] The cell population according to any one of the above items, wherein the T cells contain a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 12A] The cell population according to any one of the above items, wherein the T cells contain a chimeric antigen receptor (CAR). [Item 13] The cell population according to any one of the above items, which contains T eff . [Item 14] The cell population according to any one of the above items, which contains progenitor cells of T eff . [Item 14A] The cell population (or progenitor cells of T eff ) contains at least one cell selected from the group consisting of Tnv, Tscm, Tcm, and Temra. The cell population according to any one of the above items. [Item 15] The cell population according to any one of the above items, wherein the CAR or TCR is expressed in the T cells. [Item 16] The cell population according to any one of the above items, wherein the glucose transporter includes GLUT1, GLUT2, GLUT3, and GLUT4. [Item 17] The cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 18] The cell population according to any one of the above items, wherein the T cells are human T cells. [Item 19] A pharmaceutical composition containing the T cells according to any one of the above items or the cell population according to any one of the above items. [Item 20] The pharmaceutical composition according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune diseases, allergies, or infectious diseases. The pharmaceutical composition according to any one of the above items, which is for the cure of cancer. [Item 21] A pharmaceutical composition for preventing or treating a disease in a subject, wherein the pharmaceutical composition (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell, which has appropriate expression characteristics according to the index; (C) administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate usage and dosage. A pharmaceutical composition characterized by the above. [Item 21A] The pharmaceutical composition according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 21B] The pharmaceutical composition according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item 21C] The pharmaceutical composition according to any one of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infectious disease. [Item 21D] The pharmaceutical composition according to any one of the above items, wherein the disease includes cancer. [Item 22] The composition according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 23] The T cell according to any one of the above items for use as a medicine, or the cell population according to any one of the above items. [Item 24] The cell or cell population according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease. [Item 24A] The cell or cell population according to any one of the above items, which is for the cure of cancer. [Item 25] A cell or cell population for preventing or treating a disease in a subject, wherein the cell or cell population (A) Collecting the value of the glucose uptake ability or an index related thereto in the subject; (B) Selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell having appropriate expression characteristics according to the index; (C) Administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate usage and dosage; A cell or cell population, characterized by the above. [Item 25A] The cell or cell population according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 25B] The cell or cell population according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item 25C] The cell or cell population according to any one of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infectious disease. [Item 25D] The cell or cell population according to any one of the above items, wherein the disease includes cancer. [Item 26] The cell or cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 27] A method for treating or preventing a subject, including the step of administering an effective amount of the T cell according to any one of the above items or the cell population according to any one of the above items to a subject in need thereof. [Item 28] The method according to any one of the above items, wherein the treatment or prevention of the subject is for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease. [Item 28A] The method according to any one of the above items, wherein the treatment or prevention of the subject is for the cure of cancer. [Item 29] A method for preventing or treating a disease in a subject, the method comprising: (A) collecting the value of the glucose uptake ability or an index related thereto in the subject; (B) Selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell, which has appropriate expression characteristics according to the index; (C) Administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate usage and dosage; A method comprising the above steps. [Item 29A] The method according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 29B] The method according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item 29C] The method according to any one of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infectious disease. [Item 29D] The method according to any one of the above items, wherein the disease includes cancer. [Item 30] The method according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 31] Use for manufacturing a medicament containing the T cell according to any one of the above items or the cell population according to any one of the above items. [Item 32] The use according to any one of the above items, wherein the medicament is for treating or preventing cancer, autoimmune disease, allergy or infectious disease. [Item 32A] The use according to any one of the above items, wherein the medicament is for curing cancer. [Item 33] The use according to any one of the above items, wherein the medicament is for preventing or treating a disease in a subject, and the medicament (A) Collecting the value of the glucose uptake ability or an index related thereto in the subject; (B) Selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell, which has appropriate expression characteristics according to the index; (C) Administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate usage and dosage. Use, characterized by [Item 33A] Use according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 33B] Use according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item 33C] Use according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy or an infectious disease. [Item 33D] Use according to any one of the above items, wherein the disease comprises cancer. [Item 34] Use according to any one of the above items, wherein the glucose transporter is GLUT3. The present disclosure also provides the following. [Item B0] An effector T cell modified to express a glucose transporter and / or having enhanced expression of a glucose transporter, wherein the T cell exhibits at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM]. [Item B1] An effector T cell modified to express a glucose transporter and / or having enhanced expression of a glucose transporter, comprising a chimeric antigen receptor (CAR), wherein the T cell exhibits at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM]. [Item B2] The T cell according to the above item, wherein the CAR is expressed in the T cell. [Item B3] The T cell according to any one of the above items, wherein the glucose transporter comprises GLUT1, GLUT2, GLUT3, and GLUT4. [Item B4] An effector T cell that expresses a glucose transporter and / or has enhanced expression of a glucose transporter, and is modified to express a chimeric antigen receptor [CAR], wherein the T cell exhibits at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM], and the glucose transporter is GLUT3. [Item B5] An effector T cell into which the GLUT3 gene has been introduced. [Item B6] The T cell according to any one of the above items, which is a human T cell. [Item B7A] A cell population comprising effector T cells that express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM]. [Item B8] The cell population according to any one of the above items, wherein the CAR is expressed in the T cells. [Item B7] A cell population comprising effector T cells that express a glucose transporter and / or have enhanced expression of a glucose transporter, and are modified to express a chimeric antigen receptor [CAR], wherein the T cells exhibit at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM]. [Item B8] The cell population according to any one of the above items, wherein the CAR is expressed in the T cells. [Item B9] The cell population according to any one of the above items, wherein the glucose transporter includes GLUT1, GLUT2, GLUT3, and GLUT4. [Item B10] A cell population comprising effector T cells that are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, and that contain a chimeric antigen receptor [CAR], wherein the T cells exhibit at least equivalent effector function under low glucose conditions [0.5 mM] as under normal glucose conditions [10 mM], and wherein the glucose transporter is GLUT3. [Item B11] A cell population comprising effector T cells into which the GLUT3 gene has been introduced. [Item B12] The cell population according to any one of the above items, wherein the T cells are human T cells. [Item B13] A pharmaceutical composition comprising the T cells according to any one of the above items or the cell population according to any one of the above items. [Item B14] The pharmaceutical composition according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune diseases, allergies or infectious diseases. [Item B15] The pharmaceutical composition according to any one of the above items, which is for the cure of cancer. [Item B16] A pharmaceutical composition for the prevention or treatment of a disease in a subject, the pharmaceutical composition comprising: (A) collecting the value of the glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell or a cell population containing the T cell with enhanced glucose uptake ability and having appropriate expression characteristics according to the index; (C) administering the T cell or the cell population containing the T cell with enhanced glucose uptake ability to the subject at an appropriate dosage form and dosage. A pharmaceutical composition characterized by the above. [Item B17] The pharmaceutical composition according to any one of the above items, wherein the T cells contain a chimeric antigen receptor [CAR] or a T cell receptor [TCR]. [Item B18] The pharmaceutical composition according to any one of the above items, wherein the T cells contain a chimeric antigen receptor [CAR]. [Item B19] The disease is the pharmaceutical composition according to any one of the above items, including cancer, autoimmune disease, allergy or infectious disease. [Item B20] The disease is the pharmaceutical composition according to any one of the above items, including cancer. [Item B21] The composition according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B22] The T cell according to any one of the above items for use as a medicament or the cell population according to any one of the above items. [Item B23] The cell or cell population according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease. [Item B24] The cell or cell population according to any one of the above items, which is for the cure of cancer. [Item B25] A cell or cell population for the prevention or treatment of a disease in a subject, wherein the cell or cell population (A) collecting the value of the glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell having appropriate expression characteristics according to the index; (C) administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate dosage and regimen Characterized by a cell or cell population. [Item B26] The cell or cell population according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B27] The cell or cell population according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item B28] The cell or cell population according to any one of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infectious disease. [Item B29] The cell or cell population according to any one of the above items, wherein the disease includes cancer. [Item B30] The cell or cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B31] A method for treating or preventing a subject, comprising the step of administering to the subject in need thereof an effective amount of the T cell according to any one of the above items or the cell population according to any one of the above items. [Item B32] The method according to any one of the above items, wherein the treatment or prevention of the subject is for the treatment or prevention of cancer, autoimmune disease, allergy or infectious disease. [Item B33] The method according to any one of the above items, wherein the treatment or prevention of the subject is for the cure of cancer. [Item B34] A method for the prevention or treatment of a disease in a subject, the method comprising: (A) obtaining a value of the glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell having appropriate expression characteristics according to the index; (C) administering to the subject the T cell with enhanced glucose uptake ability or the cell population containing the T cell at an appropriate dosage and usage. comprising. [Item B35] The method according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B36] The method according to any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item B37] The method according to any one of the above items, wherein the disease comprises cancer, autoimmune disease, allergy or infectious disease. [Item B38] The method according to any one of the above items, wherein the disease comprises cancer. [Item B39] The method according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B40] Use of the T cell according to any one of the above items or the cell population according to any one of the above items for manufacturing a medicament containing the cell or cell population. [Item B41] The use according to any one of the above items, wherein the medicament is for treating or preventing cancer, autoimmune disease, allergy or infectious disease. [Item B42] The use according to any one of the above items, wherein the medicament is for curing cancer. [Item B43] The use according to any one of the above items, wherein the medicament is for preventing or treating a disease in a subject, and the medicament (A) collecting the glucose uptake ability or the value of an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population containing the T cell having appropriate expression characteristics according to the index; (C) administering the T cell with enhanced glucose uptake ability or the cell population containing the T cell to the subject at an appropriate dosage form and dosage. Characterized by the use. [Item B44] The use according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B45] The use according to any one of the above items, wherein the T cell contains a chimeric antigen receptor (CAR). [Item B46] The use according to any one of the above items, wherein the disease includes cancer, autoimmune disease, allergy or infectious disease. [Item B47] The use according to any one of the above items, wherein the disease includes cancer. [Item B48] The use according to any one of the above items, wherein the glucose transporter is GLUT3.

[0009] In the present disclosure, it is intended that one or more of the above features may be provided in combination in addition to the explicitly stated combinations. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading the following detailed description as necessary.

[0010] In addition to the above, the features and remarkable functions and effects of the present disclosure will become clear to those skilled in the art by referring to the sections and drawings of the following embodiments of the invention.

Effects of the Invention

[0011] The present disclosure can provide T cells and / or related cell populations with enhanced glucose uptake ability. By using such cells and / or related cell populations, chimeric antigen receptor (CAR) T cells and / or related cell populations that can function without starvation and exhaustion even in the tumor environment can be provided.

[0012] The cells of the present disclosure can be referred to as metabolism-enhanced T cells (survivor T cells) or related cell populations that apply the mechanism by which tumor cells survive in the tumor microenvironment to T cells. According to the cells and related cell populations of the present disclosure, it is possible to maintain effective cytotoxic activity and cytokine production ability in a low-glucose environment such as the tumor microenvironment, which could not be achieved by conventional immune checkpoint inhibition therapies, and a high antitumor effect in the body can be exerted. In addition, by using a transient glucose transporter expression system in which glucose transporters are expressed in accordance with the increased demand for glucose, over-differentiation and exhaustion of T cells due to excessive activation can be avoided, and memory phenotype T cells capable of maintaining the effect for a long time can be induced.

Brief Description of the Drawings

[0013]

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Best Mode for Carrying Out the Invention

[0014] Hereinafter, the present disclosure will be described while showing the best mode. Throughout this specification, it should be understood that the singular expressions include the concepts of their plural forms unless otherwise specified. Therefore, the singular articles (e.g., "a", "an", "the" in English, etc.) should be understood to include the concepts of their plural forms unless otherwise specified. Also, the terms used in this specification should be understood to be used in the meanings commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meanings as those generally understood by those skilled in the art to which the present disclosure pertains. In case of contradiction, this specification (including the definitions) shall prevail.

[0015] Hereinafter, the definitions of the terms particularly used in this specification and / or the basic technical contents will be appropriately explained.

[0016] In this specification, "about" means ±10% of the numerical value that follows. For example, "about 20" shall include "18 to 22". The numerical range includes all numerical values between the two endpoints and the numerical values of the two endpoints. "About" regarding the range is applied to both endpoints of the range. Therefore, for example, "about 20 to 30" shall include "18 to 33".

[0017] As used herein, "T cell" is used in the broad sense as used in the art, and refers to lymphocytes generated in the bone marrow that migrate to the thymus and mature. T cells can be CD45-positive and CD3-positive cells among the normal fractions of peripheral blood and bone marrow-derived monocytes. The T cells used can be, but are not limited to, T cells isolated from donors, particularly human donors. Examples of T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells passaged and maintained under cell culture conditions without immortalization, and T cells that can be immortalized and maintained indefinitely under cell culture conditions. T cells are known to exist in multiple types according to their functions, including effector memory T cells (T eff ), naive T cells (Tnv), stem cell memory T cells (Tscm), central memory T cells (Tcm), terminally differentiated RA-positive T cells (Temra), and the like. As used herein, "peripheral T cells" refer to T cells present outside the thymus and can be obtained from peripheral blood, lymph nodes, and other tissues. When referring to "peripheral T cells" herein, it is sufficient that the cell population contains peripheral T cells, and the T cells do not need to be isolated. Cell fractions containing various lymphocytes in addition to T cells, such as peripheral blood mononuclear cells (PBMC), may also be used.

[0018] As used herein, a "cell population" is a population containing two or more cells. For example, it may be a state in which cells are gathered flatly, or it may be a cell mass formed by cells adhering to each other three-dimensionally. Also, a "cell population" may be formed by a single type of cell or may contain multiple types of cells. When referring to the cell population of T cells of the present disclosure, it is sufficient that at least one cell capable of having an effector function is included at the location where it should function (typically in the body), such as Tnv, Tscm, Tcm, Temra, etc.

[0019] As used herein, "flow cytometry" refers to a technique for measuring the number of cells, individuals, and other biological particles suspended in a liquid, as well as their individual physical, chemical, and biological properties. An apparatus using this technique is called a "flow cytometer". In the present disclosure, the "positive" and "negative" of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are defined by flow cytometry as commonly used in the art. More specifically, in flow cytometry, cells are lined up and flowed, and the number of cells is counted by spectroscopic methods. For example, laser light is irradiated onto cells labeled with fluorescence or a luminescent enzyme, and the fluorescence or luminescence signal emitted from the cells is detected by a detector such as a photodiode, thereby counting the number of target cells. Also, the detection results by the detector can be captured by a computer to generate and display a two-dimensional plot. Thereby, the presence or absence of target cells, their number, etc. can be easily grasped.

[0020] As used herein, "effector function" in T cells refers to biological activities such as cytotoxic activity, cytokine production, and division / proliferation caused by signals via TCR or CAR, and in other immune cells, refers to biological activities caused by the Fc region of an antibody. Examples of effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and activation of B cells. In the present disclosure, whether a T cell has an effector function can be determined by confirming the presence of cytotoxic activity, cytokine production, and division / proliferation using the methods exemplified in the examples and the like.

[0021] As used herein, "effector cell" or "effector T cell" means a T cell having an effector function, which is an immune cell that exerts a cytotoxic effect on a target cell, and T effIt may also be referred to as such. Whether it is an "effector" (cell) in this specification can be determined by judgment methods such as a cytotoxicity assay, surface antigen analysis by flow cytometry, and intracellular cytokine staining. An effector T cell is an immune cell that executes an effector function and mediates antibody-dependent cell 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, etc., and can be isolated from natural tissues such as blood, for example.

[0022] In this specification, "effector T cell (T" effThe term "precursor cells" refers to cells that are not effector cells but can be transferred to the location where they should function (e.g., in the body) or acquire effector functions upon other stimuli. Such cells include lymphocyte precursor cells, Tnv, Tscm, Tcm, Temra, etc. Precursor cells of effector T cells are activated in the initial stage of the immune response, mature into effector T cells through proliferation and differentiation, and some precursor cells have the ability to differentiate into memory T cells. These precursor cells have specific gene expression patterns and metabolic states, which contribute to the extension of memory function. The extension of this memory function involves several molecular and metabolic characteristics. First, precursor cells of effector T cells have specific characteristics in their metabolic programs. In these cells, oxidative phosphorylation, rather than glycolysis (a characteristic of effector T cells), is dominant. This metabolic pathway is highly energy-efficient and enables long-term cell survival. This metabolic characteristic is an important factor supporting the long-term maintenance of memory T cells. Second, the role of transcription factors is also important. In precursor cells of effector T cells, transcription factors such as T-bet and Eomes are expressed, which promote differentiation into memory T cells and enable their long-term survival and function. These precursor cells expressing surface markers such as CD62L and CCR7 have the ability to migrate to secondary lymphoid tissues. Through this homing function, precursor cells receive the necessary survival signals in secondary lymphoid tissues, contributing to the maintenance of memory function. Furthermore, the suppression of antigen stimulation is also involved in the extension of memory function. Some precursor cells are released from antigen stimulation, which suppresses their complete differentiation into effector T cells and instead retains the properties of memory T cells. This mechanism suppresses excessive effector responses while maintaining memory function. In addition, the cytokine environment around the cells also plays an important role. In particular, cytokines such as IL-7 and IL-15 provide the necessary survival signals for the maintenance of memory T cells, which greatly contributes to the extension of memory function.Due to these factors, the precursor cells of effector T cells survive for a longer period compared to normal effector T cells and possess a memory function that enables a rapid and strong secondary immune response. This property is thought to play an important role not only in immune defense against infections but also in cancer immunotherapy and vaccine development. Thus, understanding the characteristics of precursor cells of effector T cells has great significance in both basic research and clinical applications of immunology.

[0023] In progenitor cells of effector T cells, the maintenance of the memory T cell phenotype refers to the phenomenon that during the process of differentiating into effector T cells, some progenitor cells exist while retaining the phenotype specific to memory T cells (e.g., surface markers, metabolic state, expression of transcription factors, etc.). Such progenitor cells survive even after the end of the immune response and have the ability to provide immune memory over a long period. This phenomenon is considered an important mechanism for reconciling both the process by which T cells exert effector functions and the memory function that enables long-term survival and rapid secondary immune responses. Explain the characteristics of this phenomenon. First, progenitor cells of effector T cells maintain surface markers specific to memory T cells. This includes homing molecules such as CD62L and CCR7, and the expression of these molecules enables progenitor cells to retain the ability to migrate to secondary lymphoid tissues. Next, the maintenance of their metabolic state is also characteristic. Specifically, oxidative phosphorylation is more dominant than glycolysis, and by maintaining a metabolic program with high energy efficiency, long-term survival becomes possible. This metabolic characteristic is an important factor for maintaining the function as memory T cells. Also, progenitor cells maintain a characteristic balance in the expression of transcription factors. Appropriate expression of transcription factors such as T-bet and Eomes plays a role in reconciling both the differentiation into effector T cells and the maintenance of the memory T cell phenotype. Furthermore, progenitor cells are responsive to cytokine signals and can receive survival signals such as IL-7 and IL-15. Due to this responsiveness, progenitor cells can survive for a long time while retaining the characteristics of memory T cells. Finally, these progenitor cells can be maintained in an antigen-independent manner. That is, progenitor cells of memory T cells have the ability to retain their phenotype even in the absence of antigen and maintain a state of readiness for a rapid immune response. Due to the above characteristics, the maintenance of the memory T cell phenotype by progenitor cells of effector T cells plays an extremely important role in the flexibility of the immune system and the establishment of long-term memory immunity. These characteristics are considered to have great significance in infectious disease defense, cancer immunotherapy, and even vaccine design.

[0024] As used herein, the term "antigen receptor" refers to any molecule that specifically binds to a target antigen and transmits a signal into the cell expressing the receptor. In the present disclosure, the antigen receptor can be introduced into T cells and can be either a natural or artificial molecule. For example, in addition to T cell receptors (TCRs) and B cell receptors (BCRs), chimeric antigen receptors (CARs) are also included.

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

[0026] As used herein, the "T cell receptor (TCR)" refers to the receptor present on T cells. TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains, and there are an αβ type TCR expressed by normal T cells and a γδ type TCR with special functions. The α and β chain TCR molecules form a complex with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, CD3δ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens such as viral antigens that have proliferated intracellularly following viral infection and cancer antigens derived from cancer cells are presented as antigenic peptides on MHC class I molecules. In addition, antigens derived from foreign microorganisms are taken up by antigen-presenting cells through endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by the TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via the TCR molecule. Regarding the αβ type TCR, the gene products of α and β are each thought to express specificity through unique combinations.

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

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

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

[0030] In this specification, "tumor microenvironment" can be used interchangeably with "tumor environment". The tumor microenvironment (TME) refers to the local biological environment surrounding tumor tissue and is a concept that includes various factors involved in tumor progression, proliferation, metastasis, drug resistance, etc. This environment includes not only tumor cells but also non-tumor cells such as immune cells (e.g., T cells and macrophages), fibroblasts, vascular endothelial cells, and adipocytes. Additionally, humoral factors such as the extracellular matrix (ECM), cytokines, chemokines, and growth factors are also important components. Furthermore, the tumor microenvironment is also affected by physical factors such as oxygen concentration, pH, and nutritional status. Changes in cell-cell interactions and physical factors in the tumor microenvironment are thought to play an important role in tumor malignancy and treatment responsiveness. In particular, the immunosuppressive microenvironment has attracted attention as a factor that reduces the effectiveness of immunotherapy. In the present invention, "tumor microenvironment" refers to an aggregate of biological and physical factors including these elements and their interactions, and is an important target in tumor treatment and diagnosis.

[0031] In this specification, "tumor microenvironment conditions" means the tumor microenvironment or conditions equivalent thereto. Therefore, "tumor microenvironment conditions" means a broad concept that includes, in addition to the conditions constituting the tumor microenvironment, similar or equivalent environmental conditions in which the tumor is placed in the body. Specifically, it includes not only the local biological environment surrounding the tumor tissue but also endogenous and exogenous factors that affect the tumor when it exists in the body. This environment includes interactions between tumor cells and non-tumor cells (e.g., immune cells, fibroblasts, vascular endothelial cells, etc.), humoral factors such as the extracellular matrix (ECM), cytokines, chemokines, and growth factors, and physical factors such as oxygen concentration, pH, nutritional status, and physical pressure. Also, "tumor microenvironment conditions" includes the situation where the tumor interacts with the extensive physiological environment in the body, and thus is not limited to the local environment around the tumor. In the present invention, "tumor microenvironment conditions" comprehensively represents the broad environmental conditions encountered by the tumor in the body, and this includes all factors that affect tumor progression and treatment responsiveness.

[0032] As used herein, "modification" broadly refers to any structural, functional, or expression-related change made to a gene (including nucleic acids and their corresponding proteins), and includes naturally occurring or artificial changes. Specifically, it includes changes in the sequence due to substitution, deletion, insertion, addition of bases, or combinations thereof, changes in the amino acid sequence and properties of the encoded protein, or chemical modifications (e.g., glycosylation, phosphorylation, acetylation, etc.) for the purpose of adding or improving functionality. Also, "modification" includes introduction of external genes (e.g., transfection or gene introduction via viral vectors), transformation, knockout or knock-in of specific genes using genome editing techniques, suppression of gene expression (e.g., suppression by RNA interference or CRISPR technology), etc. Furthermore, epigenetic modifications (e.g., DNA methylation, histone modification, etc.) and modification of expression regulatory elements are also included. "Modification" as used herein comprehensively refers to any form of change that affects the structure, expression, function, or biological behavior of a gene, and includes not only internal changes but also changes due to external introduction or manipulation.

[0033] As used herein, "disease" is construed in a broad sense to refer to a state in which discomfort or inconvenience occurs in the mind or body of a human or animal, and refers to any state that cannot be said to be a healthy state that is not specifically defined, such as illness, disorder, various symptoms, etc. Diseases that may be the subject of the present disclosure include, but are not limited to, diseases in which an immune response may be involved, such as cancer, autoimmune diseases, allergies, infectious diseases, etc.

[0034] As used herein, for a certain component or substance, when it is said that a certain subject "has an immune response", it means that some immune reaction occurs against the said component or substance. In the subject or biological components derived from the subject (e.g., cells, etc.), the component or substance can be identified by observing changes in various immune cells or increases or decreases in substances related to immunity (e.g., cytokines, etc.), and can be judged by objective indicators, or subjective judgment based on the experience of a doctor, etc. can also be made.

[0035] In this specification, whether a subject has "immune memory" with respect to a certain component or substance is determined by measuring, in the subject or a biological component (such as cells, etc.) derived from the subject, whether the component or substance (i) enhances cytokine production in an antigen-dependent manner with respect to memory CD4-positive T cells or has a growth-promoting effect, (ii) alters the expression of surface antigens of memory regulatory T cells, (iii) changes the ratio of Treg to Th1, (iv) induces IFN-γ production from T-bet-positive Th1 cells, (v) varies the IFN-γ-producing ability, (vi) varies the IL-2-producing ability, and (vii) varies the TNF-α-producing ability, and (viii) whether it has antibodies specific to the component or substance present in the blood, and it can be evaluated by confirming that at least one of these results in a positive outcome.

[0036] As used herein, "infectious disease" can be any infectious disease, including any type of infectious disease such as viral infectious diseases (including any viral form such as single-stranded or double-stranded DNA viruses, RNA viruses, etc.), bacterial infectious diseases, protozoal infectious diseases, mycoplasma infectious diseases, etc. For example, it can be tuberculosis, coronavirus, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, whooping cough, Japanese encephalitis, meningococcal infection, Salmonella infection, pathogenic Escherichia coli, Toxoplasma, Zika virus, herpes simplex virus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza, MARS, rabies, and diphtheria, etc.

[0037] As used herein, "immune abnormality" refers to any disease, disorder or condition that is at least partially caused by or suspected of being caused by an abnormality of the immune system. It refers to a state in which the immune system is abnormal for some reason, making a person more susceptible to infectious diseases or causing allergic reactions. Immune abnormalities include, but are not limited to, allergies, autoimmune diseases, etc. When it is against one's own antigen, it is generally called an autoimmune disease, and when it is against an external antigen, it is called an allergy. When the immune response is strong, it will result in an autoimmune disease state against one's own antigen and an allergic state against a non-self antigen. On the other hand, when the immune response is weak, it can be said that it will result in a cancer state against one's own antigen and an infectious disease state against a non-self antigen.

[0038] As used herein, the term "autoimmune disease" refers to an excessive immune response against specific self-antigens. An autoimmune disease can be said to be a disease caused by a breakdown of immune tolerance, in which the immune system, which has the role of recognizing and eliminating foreign substances, overreacts and attacks even normal cells and tissues of one's own body, resulting in symptoms.Examples of autoimmune diseases or disorders include, but are not limited to, the following: inflammatory reactions such as psoriasis and dermatitis (e.g., atopic dermatitis) including inflammatory skin diseases; systemic sclerosis and scleroderma; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type I diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune reactions associated with acute and delayed hypersensitivity typically mediated by cytokines and T lymphocytes seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases with leukocyte extravasation; central nervous system (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disorders; Reiter's disease; stiff-man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP), autoimmune uveitis or autoimmune thrombocytopenia.

[0039] As used herein, "allergy" refers to an excessive immune response to specific non-self antigens, and is a disease in which an immune reaction occurs against an "allergen". An "allergen" refers to an antigen that can react with the antibodies of a subject having an allergic disease, and examples include allergens derived from tree pollens (acacia, Japanese cypress, Cryptomeria japonica, European beech, silver birch, maple, Japanese hemlock, red pine, Japanese spindle tree, Japanese cypress, American sweetgum, Liquidambar acalycina, Chinese sweetgum, mesquite, cashew, Quercus, olive, pecan, pepper, pine, Taxus cuspidata, Russian olive, American bladdernut, Japanese sumac, black walnut, black willow, etc.), allergens derived from herbaceous plant pollens (cotton, wildrye, reed, plantain, corn, Ambrosia artemisiifolia, sorghum, rye, Phragmites australis, Echinochloa crus-galli, rice, barnyard grass, Rana nigromaculata, Amaranthus retroflexus, thistle, Salsola collina, Solidago virgaurea, Isodon japonicus, white mustard, pansy, mallow, Malva verticillata, Malva neglecta, Malva rotundifolia, Chenopodium album, Artemisia princeps, Pteridium aquilinum, Pteridium revolutum, etc.), allergens derived from insects (silkworm, mite, honeybee, wasp, ant, cockroach, etc.), allergens derived from fungi (Alternaria, Aspergillus, Clostridium botulinum, Candida, Cephalosporium, Colletotrichum, Epicoccum nigrum, dermatophyte, Fusarium, Helminthosporium, Cladosporium cladosporioides, Penicillium chrysogenum, Phoma, Pullularia pullulans, Sarcoptes scabiei, etc.), allergens derived from animal hair (dog, cat, bird, etc.), allergen proteins derived from house dust, allergens derived from food (OVA, etc.), etc., but are not limited thereto. Representative diseases of "allergy" include atopic dermatitis, allergic rhinitis (such as hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, pediatric asthma, food allergy, drug allergy, or urticaria, etc.

[0040] (Preferred Embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are for better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Thus, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present disclosure with reference to the description in this specification. Also, the following embodiments of the present disclosure can be used alone or in combination with each other.

[0041] In one aspect, the present disclosure provides immune cells such as T cells with enhanced glucose uptake ability, cell populations containing such cells, pharmaceutical compositions containing such cells or cell populations, and other related technologies.

[0042] In one aspect of the present disclosure, T cells with enhanced glucose uptake ability are provided. In T cells, those modified and / or enhanced to express glucose transporters unexpectedly show significant efficacy against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc. (see FIG. 16). The cells of the present disclosure advantageously have enhanced glucose uptake ability. Also, the cells of the present disclosure preferably have effector function after being introduced into the place where they should function, particularly in the body. Such cells include, but are not limited to, T eff and the like, and may also be precursor cells of T eff , for example, lymphoid precursor cells, Tnv, Tscm, Temra, etc.

[0043] In one embodiment, the present disclosure can be effector T cells that are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter and that include a chimeric antigen receptor (CAR). In another embodiment, the present disclosure can be a cell population that includes effector T cells that are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter and that include a chimeric antigen receptor (CAR). The present disclosure provides "effector T cells" that were not available in the prior art. The effector T cells referred to in this embodiment mean immune cells (e.g., T cells) that exert a cytotoxic effect on target cells (described elsewhere in this specification). As described elsewhere in this specification, effector function includes cytotoxic activity, cytokine production, and division and proliferation resulting from signals via TCR or CAR, but one of the important functions as effector T cells is to exert a cytotoxic effect on target cells.

[0044] On the other hand, it has not been provided in the prior art that effector T cells can exert a cytotoxic effect on target cells. Rather than being suggested, there are in fact no examples in the prior art where cure has been confirmed by experiments such as rechallenge, and for example, there are cases where nodules remain, indicating that many cases are not cured. Here, although various cancer therapeutics are known, most conventional therapeutics have only been able to reduce tumors and not eradicate them. This is because they do not have the function of exerting a cytotoxic effect on target cells as in the present disclosure. In the prior art, the tumor-suppressing effect may be observed due to the effects of cytokines and the like, and most anticancer agents are merely such indirect effects. In the prior art, in an in vivo model, unlike the present disclosure, even if a tumor is suppressed, it does not exert a cytotoxic effect on target cells. Rather, the tumor remains, and it can be said that it does not have the function of exerting a cytotoxic effect on target cells. The present disclosure exhibits different effects from these prior arts in this regard.

[0045] Specifically, in the embodiments of the present disclosure, in chimeric antigen receptor T (CAR-T) cell therapy, it has not been able to show effectiveness against solid cancers, but the present disclosure also solves this problem. Here, the inventors have found that the dysfunction of CAR-T cells in solid cancers is caused by glucose deficiency in the tumor microenvironment (TME), and that its metabolic supplementation significantly improves the anti-tumor effect of CAR-T cells. The massive consumption of glucose by cancer cells reduces the glucose level in the TME of solid cancers, and as a result, impairs CAR-T cells. In certain embodiments of the present disclosure, it has been found that when GLUTs, which are glucose transporters, are expressed in CAR-T cells, cytokine production and killing activity are restored, or effector function is improved. It provides a treatment method for various solid cancers that could not be cured even by combination therapies using chemotherapeutic agents and radiation. Some solid cancers have a metabolically harsh TME, which impairs the function of effector T cells including CD8 + T cells, and adoptively transfers CAR-T cells into the TME. The present disclosure is particularly effective in examples where nervous system cells such as neurons and glial cells require glucose as an energy source. Furthermore, certain aggressive cancer cells consume more glucose than normal cells due to aerobic glycolysis, which is a characteristic metabolism of cancer cells (known as the Warburg effect), leading to depletion of glucose in the TME. On the other hand, naive T cells shift energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to ensure an increased energy demand. Therefore, intense metabolic competition via glucose occurs in the TME between activated T cells and cancer cells. Cancer cells can survive and continue to proliferate while taking in sufficient glucose within the TME, but activated T cells lose their function due to the failure of metabolic competition. Expression of GLUT equivalent to or higher than that by CAR-T cells that externally express GLUT or enhance its internal expression may enable complete competition with cancer cells.

[0046] Specifically, the following are observed as preferred non-limiting embodiments of the present disclosure. That is, despite the clinical success of chimeric antigen receptor T (CAR-T) cell therapy in hematological malignancies, its application to refractory solid tumors including glioblastoma (GBM) has not yet been successful, and clinical trials of CAR-T cells against solid cancers have not been able to demonstrate efficacy so far, but the present disclosure also solves this problem. Here, the inventors have found that the dysfunction of CAR-T cells in GBM is caused by glucose deficiency in the tumor microenvironment (TME), and that its metabolic supplementation significantly improves the anti-tumor effect of CAR-T cells. The massive consumption of glucose by cancer cells reduces the glucose level in the TME of GBM, and as a result, impairs CAR-T cells. In a preferred embodiment of the present disclosure, stable expression of GLUT3, a high-affinity glucose transporter, in CAR-T cells restored cytokine production and killing activity. Glioblastoma (GBM) is a devastating malignant brain tumor with an annual incidence of 3.19 per 100,000 people. The advent of a combination therapy consisting of temozolomide and radiation in 2005 led to the development of tumor treating fields therapy (Novo TTF) for primary GBM, improving the prognosis of GBM patients in the clinical setting. However, the 5-year overall survival rate of GBM is still below 20%. Based on the clinical success of chimeric antigen receptor T cell (CAR-T cell) therapy in hematological malignancies, several preclinical and clinical trials of CAR-T cell therapy against GBM have been actively investigated. Among the patients treated in clinical trials, some have shown promising results such as infiltration of CAR-T cells into the tumor microenvironment (TME) and reduction of tumor volume, but most patients do not respond to CAR-T cell therapy. GBM has a metabolically harsh TME, CD8 +Impair the function of effector T cells including T cells and adoptively transfer CAR-T cells into the TME. In the brain, nervous system cells such as neurons and glial cells require glucose as an energy source, and the brain consumes 25% of the body's glucose-derived energy. Furthermore, invasive GBM cells consume more than three times as much glucose as normal neurons due to aerobic glycolysis, a characteristic metabolism of cancer cells (known as the Warburg effect). This leads to depletion of glucose in the TME of GBM. On the other hand, naive T cells shift energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to ensure increased energy demand. Therefore, intense metabolic competition via glucose occurs between activated T cells and cancer cells in the TME of GBM. In a preferred embodiment, GLUT3, encoded by SLC2A3 and physiologically expressed in neurons and glial cells, has a five-fold higher affinity for glucose than GLUT1, the major glucose transporter expressed in T cells. As a result, GBM cells expressing GLUT3 can survive and continue to proliferate while taking up sufficient glucose within the TME, while activated T cells are impaired in their function due to failure in metabolic competition. In this preferred embodiment, considering the significant glucose uptake by GBM cells via high-affinity GLUT3, expression of GLUT3 equal to or greater than that by CAR-T cells may enable complete competition with GBM cells. In one preferred embodiment of the present disclosure, a construct in which SLC2A3 is linked to a CAR is provided to induce the expression of GLUT3 in CAR-T cells.

[0047] (Cell population) In one aspect of the present disclosure, a cell population is provided that includes immune cells such as T cells with enhanced glucose uptake ability. In at least a part of the T cells included in this cell population, it is unexpectedly shown that when modified to express and / or enhance a glucose transporter, it is significantly effective against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc. (see Fig. 16, etc.). The cell population of the present disclosure is advantageously one with enhanced glucose uptake ability. Also, the cell population of the present disclosure preferably includes cells that have effector functions after being introduced into the place where they should function, particularly in the body. Such cells include T eff and the like, but are not limited thereto, and T eff progenitor cells, for example, lymphoid progenitor cells, Tnv, Tscm, Temra, etc. may also be used.

[0048] In one embodiment of the present disclosure, a cell population includes a cell population including T cells as described above, and in this cell population, it is advantageous that T cells having the characteristics of the present disclosure (for example, enhanced or expressed GLUT, effector function, or both) are about 10% or more in the cell population. In one embodiment, the proportion of T cells having the characteristics of the present disclosure in the cell population of the present disclosure can be about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0049] In one embodiment, the cell population of the present disclosure includes T cells having the property of having effector functions when introduced into the body.

[0050] In one embodiment, the cell population of the present disclosure includes the T cells in combination with a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and preferably includes a chimeric antigen receptor (CAR).

[0051] In one embodiment, in the cell population of the present disclosure, the T cells are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter.

[0052] In one embodiment, in the cell population of the present disclosure, the T cells have effector functions.

[0053] In one embodiment, the cell population of the present disclosure is the T eff , and / or T eff progenitor cells. Preferably, the cell population (or T eff progenitor cells) includes at least one type of cell such as lymphoid progenitor cells, Tnv, Tscm, Tcm, Temr, etc.

[0054] In one embodiment, in the cell population of the present disclosure, the CAR is expressed in the T cells.

[0055] In one embodiment, in the cell population of the present disclosure, the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, and preferably, GLUT3.

[0056] In one embodiment, in the cell population of the present disclosure, the T cells are human T cells.

[0057] In one embodiment, the cell population of the present disclosure is provided as a medicament or a pharmaceutical composition. Its use can be for the prevention or treatment of cancer, for the cure of cancer, for the prevention of cancer metastasis or recurrence prevention. In certain embodiments, the cell population of the present disclosure can be for preventing or treating cancer so that it does not recur.

[0058] <Glucose transporter> Cancer cells reprogram their metabolic systems to be favorable for their own cell proliferation, actively utilize the glycolytic system with low ATP production efficiency even in the presence of oxygen, and enhance glucose uptake and lactate production (Warburg effect). Therefore, because cancer cells consume a large amount of glucose and deplete it, T eff type tumor-specific T cells that require glucose as an energy source, when infiltrating the tumor, even if they receive a signal through the TCR, the Ca 2+ concentration in the cells decreases, and they become starved and exhausted.

[0059] Therefore, in one embodiment of the present disclosure, in order to promote aerobic glycolysis and enhance the competitiveness and / or fitness of immune cells in the tumor microenvironment (TME) where glucose is depleted, T cells and cell populations genetically engineered to express glucose transporters are provided.

[0060] In one embodiment, examples of the glucose transporter include GLUT1, GLUT2, GLUT3, and GLUT4. Among them, the glucose transporter with high affinity for glucose is GLUT3. Therefore, in a preferred embodiment, the glucose transporter used in the cells and cell populations of the present disclosure is GLUT3.

[0061] GLUT3 was first identified in the mouse brain and was initially defined as a neuronal glucose transporter. Subsequently, GLUT3 has been shown to be expressed in other cells that require glucose, such as mouse sperm that need energy for movement and blastocysts that are important for post-implantation development. Furthermore, GLUT3 is expressed in immune cells such as lymphocytes, monocytes, macrophages, and platelets, and is usually stored in intracellular vesicles and moves to the cell surface upon activation to maintain the metabolic switch. More recently, T cells, especially CD8 + T cells are suggested to depend not only on GLUT1 but also on GLUT3 for glucose uptake because GLUT3 is highly expressed during differentiation and activation.

[0062] T eff In T, when activating glucose transporters such as GLUT3, enhancement of effector functions such as cytotoxic activity and cytokine production ability is recognized due to glucose uptake.

[0063] In one embodiment of the present disclosure, the T cells included in the T cells or cell population of the present disclosure may include effector cells.

[0064] In one embodiment of the present disclosure, the T cells included in the T cells and cell population of the present disclosure may advantageously be human T cells.

[0065] <Chimeric antigen receptor (CAR)> In another aspect of the present disclosure, there is provided a T cell or a cell population containing such cells with enhanced glucose uptake ability, wherein the T cell contains a chimeric antigen receptor (CAR), the T cell is modified to express a glucose transporter, and / or the expression of the glucose transporter is enhanced. The CAR contained in the T cell or cell population of the present disclosure may be contained as a protein as long as it can exert its function as a CAR, or may contain a nucleic acid molecule expressing the CAR. In a representative embodiment, the CAR of the present disclosure can be expressed in T cells.

[0066] The CAR disclosed herein includes at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0067] A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. A characteristic of CARs is that, independent of MHC, they utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells towards a selected target. Antigen recognition independent of MHC can endow T cells in T cells or cell populations expressing CARs with the ability to recognize antigens independently of antigen processing, and can avoid tumor immune escape.

[0068] The intracellular T cell signaling domain of a CAR can include, for example, 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 the CAR that includes the intracellular domain of the T cell receptor, such as the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain refers to a part of the CAR that includes the intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than the antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. <T cell receptor (TCR)>

[0069] In another aspect of the present disclosure, there is provided a T cell or a cell population containing such cells with enhanced glucose uptake ability, wherein the T cell contains a T cell receptor (TCR), the T cell is modified to express a glucose transporter, and / or the expression of the glucose transporter is enhanced. The TCR contained in the T cell or cell population of the present disclosure may be included as a protein as long as it can function as a TCR, or may include a nucleic acid molecule expressing the TCR. In a representative embodiment, the TCR of the present disclosure can be one expressed in T cells.

[0070] In this specification, TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains, and there are αβ-type TCRs expressed by normal T cells and γδ-type TCRs with special functions. The α and β chain TCR molecules form a complex with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, CD3δ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens such as viral antigens that have proliferated intracellularly with viral infection and cancer antigens derived from cancer cells are presented as antigen peptides on MHC class I molecules. In addition, antigens derived from foreign microorganisms are taken up by antigen-presenting cells through endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that co-stimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules. Regarding αβ-type TCRs, each of the α and β gene products can express specificity through unique combinations. TCRs can be modified as appropriate and may contain a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain includes the intracellular domain of the T cell receptor, for example, the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain includes the intracellular domain of co-stimulatory molecules, which are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigens.

[0071] (Extracellular domain) In one embodiment, the CAR used in the T cells or T cell populations included in the T cells disclosed herein includes an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof can be appropriately selected according to the type and number of ligands on the surface of the target cells. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cells associated with a specific disease state. Therefore, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CARs of the present disclosure include those associated with tissue-specific markers, cancer cell-specific markers, viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0072] The extracellular binding domain of the CAR can be composed of a single-chain variable fragment (scFv) obtained by fusing the variable heavy and light regions of a mouse or humanized monoclonal antibody. Alternatively, an scFv derived from a Fab (obtained not from an antibody but, for example, from a Fab library) may be used. The scFv can be fused to a transmembrane domain and then to an intracellular signaling domain.

[0073] In one embodiment, the antigen-binding domain portion of the CARs of the present disclosure can target antigens including (1) allogeneic antigens such as MHC class I and MHC class II; (2) extracellular self-antigens such as TSHR (thyroid stimulating hormone receptor), DSG3 (desmoglein 3), and Cytokeratin8; (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 CARs of the present disclosure can target are not limited to these.

[0074] In one embodiment, depending on the desired antigen to be targeted, the CARs of the present disclosure can be modified to include an antigen-binding domain specific for the desired antigen target. For example, when CD19 is the target antigen, an antibody against a cancer antigen such as CD19 can be used as the antigen-binding domain in the CAR. Non-limiting examples of cancer antigens include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folate receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGE A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, midkine·BAGE, CASP-8, P-catenin, CA-125, CDK-1, ESO-1, gp75, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orfl12, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2, ART1, ART4, B-cycling, Grill, Cav-1, cathepsin B, CD74, E-Cadherin, EphA2 / Eck, Fra-1 / Fosl1, GAGE-1, ganglioside / GD2, GnT-V, pl,6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOX10, SOX11, Survivin, phCG, WT1, mesothelin, MelanA, NY-BR-1, NY-CO-58, MN(gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2, among others. In some embodiments, the effector T cells disclosed herein are engineered to express one or more CARs to recognize one or more antigens.

[0075] (Transmembrane domain) The CARs used in the T cells or T cells contained in the cell population disclosed herein can include one or more transmembrane domains fused to the extracellular domain.

[0076] In one embodiment, a linker domain derived from the extracellular domain may be linked to the transmembrane domain. The transmembrane domain may be natural or synthetic, and the natural transmembrane domain may be derived from any membrane-bound or transmembrane protein. The transmembrane regions particularly used in the present disclosure may be derived from the alpha, beta or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0077] In one embodiment, the CARs used in the T cells or T cells contained in the cell population disclosed herein can also have a spacer domain disposed between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain can preferably have a sequence that promotes the binding of the CAR to the antigen and enhances signal transduction into the cell.

[0078] (Intracellular domain) The cytoplasmic signaling domain (or intracellular signaling domain) of the CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. The intracellular signaling domain refers to the portion of the protein that transmits the effector function signal and instructs the cell expressing the CAR to perform specialized functions. The intracellular signaling domain can include any complete, mutant, or truncated portion of a given protein's intracellular signaling domain sufficient to transmit a signal that elicits or blocks an immune cell effector function.

[0079] In one embodiment, examples of intracellular signaling domains used in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signaling after antigen receptor binding.

[0080] (Alloantigens, allergens, and haptens related to rejection reactions) CARs used in the T cells or T cells contained in the cell population disclosed herein can include those related to alloantigens, allergens, and haptens related to rejection reactions.

[0081] >[Pharmaceutical uses of T cells or T cells contained in a cell population]< In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population containing such T cells, wherein the T cells are modified to express a glucose transporter and / or the expression of the glucose transporter is enhanced. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population containing such T cells, wherein the T cells contain a chimeric antigen receptor (CAR), a T cell receptor (TCR), etc., and the T cells are modified to express a glucose transporter and / or the expression of the glucose transporter is enhanced. In one embodiment of the present disclosure, the T cells in the T cells or cell population in the present disclosure can have one or more of the above-described characteristics of the T cells.

[0082] The cells, cell populations, etc. of the present disclosure can be used in immunotherapy. Immunotherapy is considered effective for diseases in which lesions with antigenicity occur (e.g., cancer, autoimmune diseases, allergies, infectious diseases, etc.), and diseases in which an abnormal immune response to a specific antigen is involved in the onset and progression of the disease state. For example, the cells, etc. of the present disclosure can be used for the treatment, therapy, or prevention of autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD), rejection, or engraftment failure during transplantation. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (SLE), antiphospholipid syndrome, polymyositis / dermatomyositis, systemic sclerosis, mixed connective tissue disease, vasculitis syndrome, type I diabetes, Graves' disease, Hashimoto's disease, idiopathic Addison's disease, autoimmune hepatitis, Goodpasture's syndrome, glomerulonephritis, autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenic purpura, autoimmune neutropenia, myasthenia gravis, pemphigus, vitiligo, idiopathic azoospermia, and the like. Examples of allergic diseases include, but are not limited to, hay fever, allergic rhinitis, bronchial asthma, atopic dermatitis, and the like. In addition, for diseases in which an abnormal immune response to a specific antigen is involved in the onset and progression of the disease state, treatment and prevention with the cells, cell populations, etc. of the present disclosure are possible.

[0083] In certain embodiments, the present disclosure provides such immunotherapy, which includes TCR-T therapy or CAR-T therapy. As used herein, "TCR-T therapy" refers to a cell therapy that utilizes modification of the T cell receptor (TCR), for example, one used for cancer treatment. As used herein, "CAR-T therapy" refers to a gene-cell therapy in which a chimeric antigen receptor (CAR) (for example, one with genetic manipulation to overcome tumor immune escape mechanisms) is introduced into a patient's T cells, the T cells are amplified and cultured in vitro, and then infused into the patient.

[0084] The cells, cell populations, etc. of the present disclosure 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 may be a combination with radiation therapy. The one or more additional agents may be any chemotherapeutic agent or may include an immune checkpoint inhibitor. Alternatively, other cancer treatments used in combination therapies include, but are not limited to, other cancer immunotherapies (e.g., immune checkpoint inhibitors), hyperthermia therapy, surgical procedures, and the like.

[0085] In another aspect, there is provided a therapeutic agent comprising the T cells or cell population of the present disclosure, which diagnoses a subject's disease and selects an appropriate CAR, TCR, etc. contained in the T cells or the cell population based on the diagnosis.

[0086] In another embodiment, the present disclosure provides a pharmaceutical composition comprising any of the immune cells (e.g., T cells such as effector T cells) described herein or a cell population containing such cells and a pharmaceutically acceptable carrier. When the immune cells express a polypeptide such as a CAR or TCR, the pharmaceutical composition may further comprise an Fc-containing therapeutic agent such as a therapeutic antibody or an Fc fusion protein. The Fc-containing therapeutic agent can bind to a target antigen such as an immune cell specific for a tumor antigen, a pathogen antigen, or a self-antigen. The pathogen antigen may be a bacterial antigen, a viral antigen, or a fungal antigen.

[0087] In one embodiment, the Fc-containing therapeutic agent may be a therapeutic antibody including, but not limited to, adalimumab, ado-trastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, brentuximab, canakinumab, cetuximab, certolizumab, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumomab, infliximab, ipilimumab, labetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tositumomab, ustekinumab, mogamulizumab, and vedolizumab.

[0088] Furthermore, the present disclosure provides a kit including (i) a first pharmaceutical composition including any of the T cells described herein or a cell population including such T cells and a pharmaceutically acceptable carrier, and (ii) the Fc-containing therapeutic agent described herein and a pharmaceutically acceptable carrier.

[0089] In another aspect of the present disclosure, a method for inhibiting cells expressing a target antigen in a subject (e.g., reducing the number of such cells, inhibiting cell proliferation, and / or suppressing the activity of the cells) is provided, including administering the cells, cell population, and / or pharmaceutical composition of the present disclosure to the subject. In one embodiment, at least a portion of the cells expressing the target antigen may be placed in a low glucose environment.

[0090] In one embodiment, the subject to be treated by the methods of the present disclosure may be a human patient suffering from cancer, such as carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Exemplary target cancers include, but are not limited to, B cell-derived cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B cell-derived cancers can include B cell acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, and B cell non-Hodgkin lymphoma.

[0091] In addition to treating target diseases or disorders such as cancer or infectious disorders, the use of the T cells or cell populations of the present disclosure for the manufacture of a pharmaceutical for a desired medical treatment is also within the scope of the present disclosure.

[0092] In one embodiment, a pharmaceutical composition comprising effector T cells comprising the chimeric antigen receptor (CAR) of the present disclosure can be used for cell therapy. In cell therapy, T cells comprising the chimeric antigen receptor (CAR) or T cell receptor (TCR) etc. of the present disclosure, or a cell population comprising such T cells, can be injected as a pharmaceutical composition or as a formulation of a therapeutically effective cell population expressing the CAR or T cell receptor (TCR) etc. of the present disclosure into a subject in need thereof. The injected T cells or cell population comprising such T cells in a subject can potentially treat a target disease or disorder such as cancer or infectious disorder of that subject. The subject may be the same subject from whom the cells were obtained (autologous cell therapy), or the cells may be from another subject of the same species (allogeneic cell therapy).

[0093] In one embodiment, T cells comprising a CAR or (TCR) etc. of the present disclosure or a cell population comprising such T cells can be formulated for administration to a subject using techniques known to those skilled in the art. In one embodiment, a formulation comprising a therapeutically effective T cell comprising a CAR or (TCR) etc. of the present disclosure or a cell population comprising such T cells may contain a pharmaceutically acceptable excipient (carrier or diluent). The excipients contained in the formulation serve different purposes, for example, depending on the nature of the antigen-binding domain of the CAR of the present disclosure. Examples of commonly used excipients include, but are not limited to, the following: saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonic agents, bulking agents, and lubricants.

[0094] In this specification, when denoting gene names and their products, contrary to the normal usage, when written entirely in capital letters, it may refer to both the gene and the protein. For example, it may be used interchangeably with the FOXP3 gene and the FOXP3 protein, and when denoted as FoxP3, it refers to both the concept and the entity (the whole) of the gene or protein.

[0095] A formulation comprising a therapeutically effective T cell comprising a CAR of the present disclosure or a cell population comprising such T cells can be administered to a subject using methods and techniques known to those skilled in the art. Exemplarily, but not limited to, intravenous injection can be mentioned. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (cerebrospinal fluid), etc.

[0096] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well-known and commonly used in the art. For example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999).These methods are described in 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, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly the entirety) of these are incorporated herein by reference.

[0097] For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, for example, gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) can be used. In addition, for example, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (I996). Bioconjugate Techniques, Academic Press, etc. are described, and the relevant parts of these are incorporated herein by reference.

[0098] In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". When it is specified in this specification that it is "within the range" of "two values", the range includes the two values themselves. References such as scientific literature, patents, patent applications, etc. cited in this specification are incorporated herein by reference in their entirety to the same extent as each is specifically described.

[0099] The above has described the present disclosure by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

Example

[0100] In this example, various functions of T cells with enhanced glucose uptake ability were examined. Specifically, the products described in the examples were used for the reagents, but equivalents from other manufacturers (such as Sigma-Aldrich, Wako Pure Chemical Industries, Ltd., Nacalai, R&D Systems, USCN Life Science INC, etc.) can also be substituted.

[0101] (Example 1) Methods and Materials Creation of GLUT3 3C10-CAR construct and viral vector creation An SLC2A3 sequence (GLUT3) with a P2A sequence placed immediately above it was inserted into an anti-EGFRvIII CAR backbone plasmid (3C10 CAR-Katushka2S) having a CD8 leader sequence - anti-EGFRvIII single-chain variable region (scFv) - CD8 hinge - CD8 transmembrane domain (TM) - CD28 intracellular domain (ICD) - 4-1BB ICD - CD3z - P2A - Katushka2S structure to construct a 3C10 CAR - GLUT3 - Katushka2S lentiviral vector (Figure 1). The lentiviral vector was transfected into 293T cells together with packaging vectors psPAX2 (Addgene#12260) and pMD2.G (Addgene#12259), and lentivirus was obtained from the supernatant.

[0102] Gene transfer Healthy donor peripheral blood mononuclear cells were separated from heparinized whole blood by the Ficoll method (Ficoll-Paque PLUS, GE Healthcare), stimulated with anti-CD3 / 28 beads (Dynabeads T-Activator CD3 / CD28, Veritas), and transfected with the CAR gene at an MOI of 4 on day 1 after stimulation. Cultures were performed in the presence of 30 U / ml of IL-2 and cryopreserved on days 8 - 12 or directly used for each evaluation.

[0103] Immunostaining 1×10 6 GLUT3 3C10 CAR-T cells at a cell count of 1×10 were fixed with 4% PFA and permeabilized with methanol. Blocking was performed with 3% BSA for 1 hour, and the cells were incubated with an anti-GLUT3 antibody (ab15311) for 2 hours. After washing with 0.05% PBST, the cells were incubated with an 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 adhered onto a slide glass with Cytospin (Thermo fisher scientific), mounted with VECTASHIELD (VECTOR LABORATORIES), and observed with Keyence XZ-800.

[0104] Phenotype analysis (FCM) The antibodies used for FCM analysis are shown in Table 1.

[0105]

Table 1

[0106] Cells were washed with 4% FBS-PBS. Dead cell staining and surface staining were performed, and after washing, data were acquired using FACSymphony A3 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0107] In vitro CAR-T cell survival and function analysis (Figure 17) Evaluate the long-term survival, memory formation, and maintenance of function of each CAR-T cell. Stimulate CAR-T cells and stimulators (medium as a negative control, EGFRvIII-stimulating beads, U87d cell line) at a ratio of 1:1. After 24 hours, remove the stimulators by magnetic beads or sorting, and divide the CAR-T cells into 0 mM, 0.5 mM, and 10 mM for continuous culture. Evaluate cell survival and cell death, memory phenotypes such as CD27, CCR4, CD45RA, and inhibitory factors such as PD-1, TIM3, and LAG3 (Figure 19), and others (transcription factors, activation / senescence markers) by cell counting at pre-stimulation, day 1, day 3, and day 7 after stimulation. In addition, extract RNA from each sample and perform detailed analysis such as RNA sequencing. After a series of analyses, stimulate a part of the day 7 cells again in the same group, and perform cell survival and function analysis after repeated stimulation in the same manner.

[0108] Cytokine analysis (FCM) CART cells 1×10 5 cells and 5×10 EGFRvIII-expressing U87 cell line (U87Δ) 5 The cells were co-cultured in 1 ml of medium for 12 hours, then cultured for 6 hours in the presence of 5 μg / ml Monensin (BD 554724 BD Bioscience) to stimulate the CAR T cells. The medium used was glucose-free RPMI1640 (Wako) supplemented with 10% dialyzed FBS (Cytiva), and the glucose concentration was adjusted to 10 mM and 0.5 mM with a glucose solution (Gibco). Fixed with Fixation / Permeabilization Diluent (Invitrogen), stained with the antibodies shown in Table 1 for intracellular cytokines, data was acquired using LSRFortessa X20, and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0109] Cell cytotoxicity assay In a 96-well plate, 2×10 per well 5Target cells of the cells (Luciferase-expressing U87Δ cells) were seeded and co-cultured with CAR-T cells at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1. After 16 hours, live cells were luminesced with VivoGlo Luciferin (Promega), and luciferase activity was measured using Cytation (Promega), and cytotoxic activity was calculated.

[0110] Metabolic analysis The metabolic ability of the cells was measured according to the product protocol using an XFe24 cell flux analyzer (Bioscience) and an XF glycolysis stress kit (Agilent Technologies). The day before the analysis day, coating of a 24-well flat-bottom plate for analysis with poly-D-lysine (0.1 mg / ml) and hydration of the sensor cartridge were performed in a CO 2 free incubator. On the analysis day, after washing the cells for analysis with analysis medium (Seahorse XF RPMI medium, L-glutamine 2 mM), 2×10 5 cells were seeded per well on the analysis plate coated with poly-D-lysine and left standing at CO 2 free at 37°C for 60 minutes. After filling the sensor cartridge ports with glucose at a final concentration of 10 mM or 0.5 mM, oligomycin at a final concentration of 20 μM, FCCP at a final concentration of 10 μM, oligomycin at a final concentration of 1 μM, and 2-DG at a final concentration of 50 mM, analysis was performed using a flux analyzer, and metabolic function was evaluated from OCR·ECAR.

[0111] Evaluation of glucose uptake ability 2×10 4 GLUT3 CAR-T cells of the cells were seeded in a 96-well plate and cultured for 5 hours with 200 μl of glucose-free RPMI1640 (Wako). 1 μl of 2-NDBG (abcam) was added to each well 5 minutes before the analysis, and after washing, data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0112] Brain tumor xenograft model NSG mice were injected intracranially (right basal ganglia) with 2.5×10 luciferase-expressing U87Δ tumor cell line (U87Δ-luc). 4 After engraftment, Mock, 3C10 CAR-T cells, and GLUT3 CAR-T cells (2×10 6 cells) were administered via the tail vein. Tumor burden was monitored by in vivo imaging over time, and survival was observed.

[0113] (Method) Figure 1 shows the structure of the metabolically modified CAR (EGFRvIII_CAR_GLUT3). A single-chain antibody (asFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) was constructed and linked to CD8 hinge, CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BB ICD, and CD3z to construct the CAR. High-affinity glucose transporter (GLUT3) was encoded via the P2A sequence under the CAR.

[0114] (Results) The results are shown below Figure 2. As shown in Figure 2, enhanced GLUT3 expression on the T cell surface and glucose uptake were demonstrated. Normal CAR (3C10 CAR) and metabolically modified CAR (GLUT3 CAR) were introduced into T cells from healthy donors to obtain CAR T cells. Homogeneous GLUT3 expression on the GLUT3 CAR-T cell membrane was observed under a fluorescence microscope. Also, glucose uptake was enhanced in GLUT3 CAR-T cells compared to T cells and normal CAR-T cells (3C10 CAR-T cells).

[0115] Next, Figure 3 shows T cell amplification efficiency equivalent to that of normal CAR T cells. As shown in Figure 3, T cell proliferation efficiency was analyzed after gene introduction of the CAR following anti-CD3 / 28 bead stimulation. GLUT3 CAR-T cells had an establishment efficiency comparable to that of normal CAR-T cells (3C10 CAR-T cells).

[0116] Next, the enhancement of glycolytic ability of T cells by GLUT3 expression is shown in Fig. 4. As shown, the metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Flax analyzer. GLUT3 CAR enhanced glycolytic ability in both low-glucose and normal-glucose environments.

[0117] Next, GLUT3 CAR-T cells were shown to have a competitive advantage under low glucose (Fig. 5). As shown in Fig. 5, 3C10 CAR-T cells and GLUT3 CAR-T cells were co-cultured under normal glucose and low glucose conditions. Similar survival was observed for both under normal glucose, but significant survival of GLUT3 CAR-T cells was observed under low glucose, suggesting that it can favorably take up glucose.

[0118] Next, Fig. 6 shows that the cytokine production ability of GLUT3 CAR-T cells is enhanced. As shown in Fig. 6, after antigen stimulation of 3C10 CAR-T cells and GLUT3 CAR-T cells, the proportion of cytokine-producing cells was analyzed by FCM. A significant increase in the expression of cytokine groups (IFN-γ, IL-2, TNF-α) important for T cell maintenance / activation and exertion of anti-tumor activity was observed.

[0119] Next, Fig. 7 shows the function of GLUT3 CAR-T cells maintained even under low glucose. As shown, the cytokine production ability under normal glucose and low glucose was compared. In normal-type CAR-T cells (3C10 CAR-T cells), an extreme decrease in function was observed under low glucose. On the other hand, GLUT3 CAR-T cells showed high cytokine production ability under normal glucose and also exerted a function comparable to that of 3C10 CAR-T cells under normal glucose even under low glucose.

[0120] Next, the enhanced cytotoxic activity of GLUT3 CAR-T cells is shown in Figure 8. As shown, the cytotoxic activity was analyzed targeting the EGFRvIII-expressing cell line. The cytotoxic activity was analyzed under low glucose and high glucose conditions, and under both conditions, GLUT3 CAR-T cells showed higher cytotoxic activity compared to 3C10 CAR-T cells.

[0121] Next, the characteristics imparted to the memory phenotype of GLUT3 CAR are shown in Figure 9. As shown, 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 observed.

[0122] Next, the suppression of inhibitory molecule expression in GLUT3 CAR-T cells is shown in Figure 10. The expression of inhibitory molecules was compared under non-stimulated conditions and under stimulation with EGFRvIII antigen-positive cells. In GLUT3 CAR-T cells, suppression of the expression of PD-1, LAG3, and Tim3 was observed.

[0123] Next, the mRNA analysis (evaluation of metabolism, exhaustion, activation, and differentiation) of GLUT3 CAR-T cells is shown in Figure 11. The details of 3C10 CART and GLUT3 CAR-T cells were analyzed by mRNA expression. In GLUT3 CAR-T cells, a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors were observed, similar to FCM. Overall, the data show an enhancement of T cell effector functions such as activation and cytokine production.

[0124] Next, Figure 12 shows that not only 3C10CAR but also when loaded onto CD19 CAR, the same effect can be obtained, that is, its universality is proven. GLUT3 was loaded onto CD19CAR. It was confirmed that the same effect as 3C10 CAR was obtained. In addition, CD19 was expressed in pancreatic cancer cell lines, indicating that it is also effective against pancreatic cancer.

[0125] As shown in Fig. 13, it was shown that rapid tumor eradication was achieved in the intracranial xenograft model. Anti-tumor activity was observed in the U87Δ intracranial xenograft model. In GLUT3 CAR-T cells, more rapid tumor eradication was observed than in 3C10 CAR-T cells. Also, rejection was confirmed in tumor challenge.

[0126] (Example 2: Enhancement of Glucose Transporter Expression) To achieve enhanced expression of the glucose transporter, a CAR construct that constantly expresses GLUT3 was designed (the same as stbl-GLUT3 = GLUT3 CAR in Fig. 12).

[0127] Using the cells transfected with the CAR in Fig. 12, their functions and anti-tumor effects were analyzed.

[0128] Fig. 15 shows the results of evaluating the anti-tumor effect of GLUT3 CAR-T cells in a similar mouse model. While CR was not obtained with Mock T cells or 3C10 CAR-T cells, 4 cases of CR were obtained with GLUT3 CAR-T.

[0129] Fig. 16 shows the results of the tumor challenge. That is, it shows the situation of mouse death in the GLUT3 CAR-T cell group. Tumor (U87d) was re-transplanted into the cured mice in the previous experiment to evaluate the rejection ability ("surrogate for memory").

[0130] Fig. 17 shows that long-term survival is enhanced after various stimulations. Each CAR-T cell was observed over time under each condition. As a result, under low glucose, normal CAR-T (3C10) does not show effective cytokine production, while GLUT3 CAR-T exhibits effective cytokine production ability. However, GLUT3 CAR-T differentiates (terminal differentiation, loss of stemness) into CCR7-negative Tem or Temra over time (day 3 and day 7) due to activation by excessive glucose uptake and undergoes apoptosis.

[0131] In addition, when CAR-T cells (established with normal glucose) are rapidly (day 0) placed under low glucose conditions of 0 mM or 0.5 mM, the activation state is high, and GLUT3 CAR-T, which is highly glucose-dependent, is easily prone to apoptosis.

[0132] Figure 18 shows enhanced long-term survival after stimulation. The anti-tumor effect, survival, and adverse events are examined in a system similar to the aforementioned mice. In Experiment 1, GLUT3 CAR-T shows equivalent anti-tumor activity. In Experiment 2, apoptosis and over-differentiation occur due to over-activation of GLUT3 CAR-T by sugar exposure, resulting in the loss of anti-tumor activity. In Rechallenge, GLUT3 CAR-T cells reject the tumor. In Experiment 4, CAR-T cell engraftment and tumor T cell infiltration, which are superior in GLUT3 CAR-T, are observed in proportion to the tumor effect. Additionally, Experiment 3 is an experiment in a liver tumor model suggesting immunosuppression by a low glucose and high lactate environment, and it is expected to obtain the same results as Experiment 2.

[0133] Figure 19 shows data (in vitro data) indicating enhanced long-term survival after stimulation. On the left, at each time point, the staining data of 7-AAD and Annexin V, which reflect cell death and apoptosis, are shown. Cell death in each CAR-T cell was examined using Annexin V and 7-AAD. In the unstimulated state, although a difference in glucose concentration was observed, no significant difference was found among CAR-T cells.

[0134] (Example 3: Example of CAR-T) The following shows further demonstration examples with CAR-T.

[0135] (Materials and Methods) (Cell Line) The 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 Dulbecco's Modified Eagle Medium (D-MEM, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The EGFRvIII-packaged lentiviral vector was introduced into U-87 MG and U-251 MG cells, and the transduced cell lines were named U-87 MGΔ and U-251 MGΔ, respectively. The SUP-T1, NALM6, and AsPC-1 cells were purchased from ATCC and cultured in RPMI 1640 (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% FBS and 1% penicillin / streptomycin. The CD19 cleavage vector was introduced into NALM6 cells to obtain the CD19-expressing AsPC1 cell line.

[0136] (Flow cytometry assay) The flow cytometry assay was performed as previously described (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. Cancer Cell;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.l and 43. Tanegashima T, Togashi Y, Azuma K, et al (2019) Immune Suppression by PD-L2 against Spontaneous and Treatment-Related Antitumor Immunity. Clin Cancer Res;25(15):4808-19.). Briefly, cells were washed twice with FACS buffer and Fc blocking was performed for 10 minutes at 4°C in the dark using an Fc receptor blocking solution (BioLegend, San Diego, CA). Cells were washed twice with FACS buffer, an antibody targeting cell surface molecules was added, and the cells were incubated for 20 minutes at 4°C in the dark. Subsequently, the cells were washed twice with FACS buffer.For intracellular antigen staining, cells were incubated in a fixation / permeabilization solution (BD Biosciences, San Jose, CA) for 1 hour at room temperature and then washed twice with wash buffer (BD Biosciences) according to the manufacturer's instructions. An antibody targeting intracellular antigen was added and incubated for 20 minutes at 4°C in the dark, followed by two washes. For the apoptosis assay, cells were washed once with FACS buffer and once with Annexin binding buffer. Subsequently, cells were stained with Annexin V and 7-AAD for 15 minutes at room temperature. After washing, flow cytometry (FCM) analysis was performed using an LSRFortessa X-20 cytometer (BD Biosciences) and analyzed with FlowJo ver.10 software (BD Biosciences). The staining solutions were prepared according to the manufacturer's instructions.

[0137] (Cytokine staining) Intracellular cytokine staining was performed as previously described (40. Kumagai S, et al (2020) at Immunol;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 medium for the last 5 hours of 6-hour T cell stimulation to retain cytokines intracellularly. After staining cell surface markers, Cytofix / Cytoperm reagent (BD Biosciences) was added and incubated at 4°C in the dark for 20 minutes. Then, the cells were washed twice with the wash buffer, and antibodies targeting cytokines were added. Incubated at 4°C in the dark for 20 minutes. After washing, FCM analysis was performed using an LSRFortessa X-20 cytometer (manufactured by BD Biosciences) and analyzed with FlowJo ver.10 software (BD Biosciences). The antibodies used for cell staining are summarized in Table 2. The preparation of the antibodies for staining was carried out according to the manufacturer's instructions.

[0138]

Table 2

[0139] (Cytotoxicity assay) Total 1×10 5Luciferase-expressing cells (U-87 MGΔ, U-251 MGΔ, AsPC-1, and NALM6) were co-cultured with CAR-T cells at the indicated ratios in 200 μL of RPMI 1640 medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with low glucose (0.5 mM) or high glucose (10 mM) in a flat-bottom 96-well white plate. After 24 hours of culture, Bio-Glo (Promega, Madison, WI) was added to each well, and luminescence was measured using a multiplate reader, Cytation5 (Agilent Technologies, Santa Clara, CA). % specific lysis was calculated using the following formula: % specific lysis = [(experimental lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis)] × 100.

[0140] (Immunofluorescence staining) After washing, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with cold methanol for 10 minutes at -20°C. The cells were blocked with 3% BSA / PBS for 60 minutes at room temperature. After blocking, the cells were incubated with the primary antibody for 2 hours at 4°C and the secondary antibody for 1 hour at 4°C. Next, DAPI was added and incubated for 5 minutes at room temperature. After washing, the stained cells were fixed onto slides with Cytospin and analyzed with a BZ-X710 (KEYENCE, Osaka, Japan).

[0141] (Immunohistochemical (IHC) staining) IHC staining was performed on 5-μm-thick sections of formalin-fixed paraffin-embedded specimens. Antigen retrieval was performed using a citrate buffer (pH 6.0) in a steamer, followed by deparaffinization and rehydration. The slides were incubated with the primary antibody for 16 hours and the HRP-labeled secondary antibody for 1 hour, and then developed with a diaminobenzidine substrate. The anti-CD3ε antibody (clone: SP7, catalog number: ab16669) (Abcam, Waltham, MA) was used for primary staining. The stained slides were counterstained with hematoxylin.

[0142] Hematoxylin and eosin (HE) staining was performed according to the standard protocol. Deparaffinized and hydrated slides were added to the hematoxylin solution and incubated for 4 minutes. After washing, the slides were incubated in the eosin solution for 2 minutes. The stained slides were scanned at magnifications of 40x and 400x using a BZ-X710 (manufactured by Keyence). Two pathologists independently evaluated the stained slides.

[0143] (ELISA method) A total of 2.0×10 5 U-87 MGΔ cells and 2.0×10 5 CAR-T cells were co-cultured in a 24-well plate. After 24 hours of culture, the supernatant was collected and subjected to ELISA to measure cytokine concentrations. ELISA kits for IFN-γ, IL-2, and TNFα (R&D Systems, Minneapolis, MN) were used according to the manufacturer's instructions. Cytokines in mouse serum were analyzed by a highly sensitive LUMINEX assay (Merck Millipore, Burlington, MA) according to the manufacturer's instructions.

[0144] (CAR-T cell production) Peripheral blood was collected from healthy individuals, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL). T cells were separated from PBMCs by negative selection using MojoSort (BioLegend). The isolated T cells were stimulated with anti-CD3 / 28 beads (Thermo Fisher Scientific, Waltham, MA) at a T cell:bead ratio of 1:1 (day 0). After 24 hours of stimulation (day 1), the T cells were transduced with CAR lentivirus at an MOI of 3 and cultured in 30 U / ml IL-2. Half of the medium was replaced after 24 hours. After 5 days, the CD3 / 28 beads were removed. During culture, half of the medium was replaced on days 4 and 10, and the concentration of T cells was adjusted to 0.7×10 6 cells / mL. The developed CAR-T cells were collected on day 10 and used for subsequent analysis.

[0145] (Animal model) Female NSG mice (6 weeks old) were purchased from Jackson Laboratory. Before any painful procedures, an anesthetic was injected intraperitoneally to prevent pain. Using a stereotaxic frame, tumor cells were inoculated at a position 2 mm to the right of bregma, 3 mm posterior, and 3 mm deep from the surface of the brain. A total of 2.0×10 4 cells were injected into 5 mL of PBS over 1 minute, after which the 1-mm needle was removed and the needle was left in place for an additional 1 minute before being removed. Four days after tumor implantation, tumor growth was confirmed by bioluminescence imaging (BLI), and random assignment to each treatment group was performed. On day 5, 1.0×10 6 CAR-T cells were injected via the tail vein. Tumor growth after CAR-T cell injection was monitored by BLI twice a week. Animal housing and experiments were conducted in accordance with the guidelines of the National Cancer Center Animal Committee after approval by the National Cancer Center Animal Experiment Ethics Review Committee.

[0146] (Glucose concentration measurement) Tissue samples (5×5 mm) were washed to remove adherent blood. The samples were minced and centrifuged at 4°C for 10 minutes to collect the tumor interstitial fluid. The glucose concentration of the interstitial fluid was measured using a Multiskan GO (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0147] (Metabolic analysis) OCR (unit: pmol / min) and ECAR (unit: mpH / min) were evaluated using a Seahorse XF-24 Extracellular Flux Analyzer (Agilent Technologies). CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, On-d GLUT3 EGFRvIII CAR-T cells) stimulated with EGFRvIII beads (ACRO Biosystems, Beijing, China) for 24 hours were resuspended in glucose-free, non-buffered RPMI-1640 medium (Agilent Technologies) and plated in Seahorse cell plates (2.0×10 5 cells per well) coated with poly-L-lysine (BD Bioscience). Perturbation profiling of the utilization of metabolic pathways by CAR-T cells was achieved by the addition of glucose (10 mM or 0.5 mM), oligomycin (1 μM), 2-deoxy-D-glucose (50 mM) (all from Agilent Technologies). Experiments using the Seahorse system were performed under the following assay conditions: 3 minutes of mixing, 2 minutes of waiting, and 3 minutes of measurement. Subsequently, the values of metabolic parameters were calculated.

[0148] (RNA Sequencing and Subsequent Analysis) CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, On-d GLUT3 EGFRvIII CAR-T cells) sorted by BD FACSymphony S6 (BD Biosciences) were stimulated with EGFRvIII beads at a ratio of 1:1 with 30 U / ml IL-2 supplementation under low glucose (0.5 mM) or high glucose (10 mM) conditions. CAR-T cells were harvested on days 3 and 7, and RNA was extracted from them using the RNeasy kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from the isolated RNA using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA). RNA-seq libraries were subjected to next-generation sequencing (paired-end reads) of 150 bp from both ends using Novaseq X plus (Illumina, San Diego, CA). For expression profiling by RNA-seq data, paired-end reads were aligned to the hg38 human genome assembly by 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, uncovers the cell / tissue type-dependent action of sulforaphane. J Biol Chem;299(6):104810.).

[0149] (Statistical analysis) GraphPad Prism 9 (GraphPad Software, San Diego, CA) was used for statistical analysis. The relationships between groups were compared by t-test or one-way analysis of variance. Survival rates were analyzed by the Kaplan-Meier method and statistically compared by the log-rank test. A P-value < 0.05 was considered statistically significant.

[0150] (Results) (GBM has a low glucose concentration in the TME, which impairs the function of CAR-T cells.) Evidence is accumulating that low glucose levels in the TME are a potential barrier to CAR-T cell therapy in solid tumors, but how the actual glucose concentration and low glucose state in the TME affect the function of CAR-T cells has not yet been clarified [Peng JJ, Wang L, Li Z, et al (2023) Metabolic challenges and interventions in CAR T cell therapy. Sci Immunol; 8(82): eabq3016]. We examined the glucose concentration in the interstitial fluid of surgical specimens and serum (Figure 20a). The glucose concentration in the interstitial fluid of GBM specimens was approximately 10-fold lower than that in serum [< 0.5 mM (mean 0.217 mM)] (Figure 20b). Although glucose concentrations are generally low in various types of cancers such as non-small cell lung cancer and colorectal cancer, GBM has the lowest glucose concentration in the TME. This indicates that the low glucose concentration in the TME is a characteristic of GBM (Figure 20c).

[0151] Next, we investigated how a low-glucose environment affects the functions of CAR-T cells, such as cytokine production. CAR-T cells targeting the GBM-related antigen EGFRvIII (conv EGFRvIII CAR-T cells) were co-cultured with a human GBM cell line (U-87 MGΔ) expressing EGFRvIII under low-glucose conditions (0.5 mM) that mimicked the GBM TME. Stimulation with U-87 MGΔ led to a significant decrease in the production of cytokines (IFN-γ, IL-2, TNFα) even after a short-term (16-hour) exposure under the low-glucose environment (Figs. 20d, e). To further examine the functional changes of CAR-T cells under low-glucose conditions, CAR-T cells were stimulated for 16 hours under low-glucose (0.5 mM) or high-glucose (10 mM) conditions, and the gene expression profiles were analyzed. The gene expression profiles were significantly changed by exposure to the low-glucose conditions (Fig. 22a). Gene sets related to the cell cycle, differentiation, and cytokine production were significantly decreased, while gene sets related to the cellular response to glucose starvation and intrinsic apoptosis were increased under the low-glucose conditions (Figs. 20f, g). Thus, the effector functions of CAR-T cells are immediately impaired when exposed to low-glucose conditions such as those in the GBM TME.

[0152] (Overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells.) Among the SLC2 family of glucose transporters (GLUT), GLUT3, encoded by SLC2A3, is the most affinity and is predominantly expressed in neurons and glial cells with high glucose requirements [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, we investigated whether the expression of GLUT3 in CAR-T cells could promote the activity of CAR-T cells by promoting glucose uptake in the low-glucose TME of GBM. SLC2A3 was fused to the conventional anti-EGFRvIII CAR construct via a self-cleaving P2A sequence to enable simultaneous stable expression of CAR and GLUT3 (GLUT3 EGFRvIII CAR) (Figure 21a). GLUT3 was expressed on the membrane of T cells transfected with GLUT3 EGFRvIII CAR (GLUT3 EGFRvIII CAR-T cells) (Figure 21b), and its expression level was approximately twice that of the parental anti-EGFRvIII CAR-T cells (conv EGFRvIII CAR-T cells) and comparable to that of the GBM cell line U-87 MGΔ, but no change was observed in the expression level of GLUT1 (Figure 21c and Figure 23a). The glucose uptake measured by a glucose uptake assay using the glucose analog 2-NBDG was greater in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells (Figure 21d). Therefore, the glycolysis and glycolytic capacity of GLUT3 EGFRvIII CAR-T cells were significantly improved (Figure 21e and Figure 23b). Further metabolic analysis showed that GLUT3 EGFRvIII CAR-T cells had a higher oxygen consumption rate (OCR) high , extracellular acidification rate (ECAR) highIt was confirmed that it shifted more energetically in the

[0153] state (Figure 21f). In a glucose competition assay in which GLUT3 EGFRvIII CAR-T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were co-cultured with conv EGFRvIII CAR-T cells at a ratio of 1:1, the number of GLUT3 EGFRvIII CAR-T cells relative to the conventional ones increased at a low glucose level (0.5 mM) glucose level, but was comparable under high glucose (10 mM) conditions, suggesting that GLUT3 EGFRvIII CAR-T cells are substantially competitive in terms of glucose uptake, especially in a low glucose environment (Figures 23c, d). Therefore, stable GLUT3 expression by CAR-T cells promotes glucose uptake, makes CAR-T cells competitive in terms of glucose uptake, and enhances the metabolic fitness of CAR-T cells in a limited glucose environment.

[0153] Since the metabolic fitness of CAR-T cells is improved by the expression of GLUT3, its effector function and safety profile were investigated. Therefore, we examined the effector function of GLUT3 EGFRvIII CAR-T cells in both in vitro and in vivo assays. Compared with combo EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells significantly increased cytokine production (IFN-γ, IL-2, TNFα) at both low glucose level (0.5 mM) and high glucose level (10 mM) (Figures 21g, h and Figure 24a). Cytokine production was CD8 + T cell subset and CD4 +Enhanced in both T cell subsets (Figure 24b, c). GLUT3 EGFRvIII CAR-T cells also showed increased cytotoxicity against U-87 MGΔ cells and U-251 MGΔ cells, another EGFRvIII-expressing GBM cell line, under both low glucose and high glucose conditions (Figure 21i, Figure 24d, e). The expression of exhaustion-related molecules including PD-1 and Tim-3 after antigen stimulation was higher in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells (Figure 21j, k and Figure 24f, g). In addition, enhanced effector function due to stable GLUT3 expression was similarly observed in CAR-T cells targeting a different antigen, CD19 (GLUT3 CD19 CAR-T cells). This indicates that enhanced metabolic fitness by GLUT3 expression is universal for all CARs regardless of the single-chain variable fragment (scFv) used or the antigen targeted (Figure 25a-c). Therefore, with stable GLUT3 expression, CAR-T cells can be activated in a low glucose level TME despite the presence of an exhausted phenotype.

[0154] (Discussion) Enhancing the antitumor effect by improving the metabolic fitness of CAR-T cells in the tumor microenvironment (TME) is a new concept for accelerating the clinical application of CAR-T cell therapy for refractory solid tumors. Among the clinical trials of CAR-T cells for glioblastoma multiforme (GBM), some have failed to demonstrate clinical utility. However, immunological monitoring of these clinical trials has revealed the detection of abundant CAR-T cells 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. Nat Med;30(4):1001-12.]. Considering the presence of CAR-T cells in the TME, antigen loss and migration disorders can be excluded as the causes of failure, and it can be assumed that specific conditions in the GBM TME are involved in inducing CAR-T cell dysfunction.Since effector T cells perform glycolysis for survival and activation, glucose is an essential 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 that a significantly low glucose environment is detected in the TME of GBM, the low glucose levels in the TME of GBM must be a major metabolic stress for CAR-T cells. Indeed, conv EGFRvIII CAR-T cells are in a dysfunctional state (producing less cytokines and expressing high levels of exhaustion markers). Furthermore, since the PD-1 signal disrupts the PI3K / Akt / mTOR signal and further impairs the glycolytic pathway, effector T cells may enter 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].

[0155] Considering that nervous system cells such as neurons and glial cells, and GBM, especially GBM expressing EGFRvIII, meet their high glucose requirements using GLUT3, a high-affinity glucose transporter [Vannucci SJ, Maher F, Simpson IA (1997) Glucose transporter 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 tumourigenesis 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.], it is highly likely that overwhelming nutritional competition can be achieved by highly expressing GLUT3 in CAR-T cells. In fact, it has been demonstrated that the GLUT3 EGFRvIII CAR construct developed by the present inventors enhanced glucose uptake and effector functions such as cytokine production and cytotoxicity in GLUT3 EGFRvIII CAR-T cells.

[0156] (Example 3: Example in precursor cells of other effector T cells) Perform the same experiment with other T cells. For example, unpurified naive T cells contain Tnv, Tcm, Tem, Temra, etc., and any fraction can be made to express CAR (for example, constitutively expressed GLUT3 CAR).

[0157] In this example, a CAR (CAR, constitutively expressed GLUT3 CAR) is introduced into any purified and concentrated T cell memory fraction. In another experiment, CAR-T cells in which any T cell memory fraction is concentrated are induced under culture conditions.

[0158] Experimental procedure: · From T cell sources such as peripheral blood, bone marrow, and umbilical cord blood, T eff precursor cells such as Tnv, Tscm, Tcm, or T that have already differentiated and mainly exhibit effector functions eff and Temra fractions are purified and concentrated by FACS sorting or magnetic beads based on the CD45RA + CCR7 + CD95 - CD45RA + CCR7 + CD95 + CD45RA - CCR7 + CD45RA - CCR7, and CD45RA + CCR7 - Based on this, they are purified and concentrated. Each fraction is stimulated with CD3 / 28 beads or the like, and a CAR is introduced using a lentiviral vector. · As another method, after gene transfer into bulk T cells, the types and amounts of cytokines are adjusted (use of IL-7 and IL-15 or use of platelet lysate) to induce suppression of T eff differentiation and preferential proliferation of Tnv, Tscm, and Tcm to obtain CAR-T cells with a predominance of precursor cells. · In vitro evaluation; These cells are co-cultured with the U87d tumor cell line or EGFRvIII beads, and after 1 day, 3 days, and 7 days, the proliferation by CFSE staining and cell counting, and the activation and exhaustion states by flow cytometry are evaluated. · In vivo evaluation: These cells are administered to the aforementioned U87d intracranial drug - making mice, and the antitumor effect is evaluated by the antitumor activity and survival by BLI.

[0159] Results: · When introduced into bulk T cells or differentiated T eff Compared with the case of introduction into Temra, in CAR-T cells introduced into precursor cells such as Tnv, Tscm, Tcm, etc. of T eff or in which Tnv, Tscm, Tcm are preferentially amplified, the ability to divide and proliferate is high, and long-term division is expected to be observed. Also, the expression of PD1, TIM3, LAG3 is low, and it is expected to be resistant to exhaustion.

[0160] (Example 4: Example of TCR) The same experiment is carried out in an example of introducing TCR instead of CAR. For example, TCR can also be introduced into fractions such as T eff , Tnv, Tcm, Tem, Temra, etc.

[0161] In this example, TCR (constitutively expressed GLUT3 TCR) is introduced into any purified and concentrated T cell memory fraction. In another experiment, under culture conditions, TCR-T cells in which any T cell memory fraction is concentrated are induced.

[0162] Perform the various in vitro and in vivo evaluations shown in Examples 1 to 3.

[0163] (Example 5: Prevention, reinfection prevention, and treatment of bacterial infections) In this example, the prevention, reinfection prevention, and / or treatment of bacterial infections are verified.

[0164] (Method) In the prevention of bacterial infections (for example, bacterial conjunctivitis, etc.), for subjects without bacterial infections, the T cells of the present disclosure modified with the infectious causative antigen of bacterial infections are administered.

[0165] In the prevention of recurrence and / or treatment of bacterial infections (for example, bacterial conjunctivitis, etc.), for subjects who had and / or have bacterial infections, the T cells of the present disclosure are administered.

[0166] After administration, prognosis observation of bacterial infections in the subject is carried out by techniques commonly used in this technical field.

[0167] (Example 6: Prevention, reinfection prevention, and treatment of parasitic infections) In this example, prevention of parasitic infections, prevention of reinfection, and / or treatment are verified.

[0168] (Method) In the prevention of parasitic infections (e.g., acanthamoeba keratitis, etc.), the T cells of the present disclosure modified with the infection-causing antigen of the parasitic infection are administered to subjects without the parasitic infection.

[0169] In the prevention of recurrence and / or treatment of parasitic infections (e.g., acanthamoeba keratitis, etc.), the T cells of the present disclosure are administered to subjects who had and / or have the parasitic infection.

[0170] After administration, prognosis observation of parasitic infections in the subject is carried out by techniques commonly used in this technical field.

[0171] (Example 7: Prevention of infection, prevention of onset, prevention of reinfection, and treatment of viral infections) In this example, verification of prevention of infection, prevention of onset, prevention of reinfection, and / or treatment of viral infections is carried out.

[0172] (Method) In the prevention of viral infectious diseases (including, but not limited to, tuberculosis, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, whooping cough, Japanese encephalitis, meningococcal infection, Salmonella infection, pathogenic Escherichia coli, Toxoplasma, Zika virus, herpes simplex virus type 1, EBV / Epstein - Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza (virus), MARS, rabies, diphtheria, etc.), for subjects without viral infectious diseases, administer the T cells of the present disclosure modified with the infectious - cause antigen of the viral infectious disease.

[0173] In the prevention of recurrence and / or treatment of the above - mentioned viral infectious diseases, administer the T cells of the present disclosure to subjects who had and / or have viral infectious diseases.

[0174] After administration, perform prognostic observation of viral infectious diseases in the subjects by techniques commonly used in the art.

[0175] (Example 8: Prevention, recurrence prevention, and treatment of allergy) In this example, verify the prevention, recurrence prevention, and / or treatment of allergy.

[0176] (Method) In the prevention of allergy (e.g., allergic conjunctivitis), for subjects without the causative antigen of allergy, administer the T cells of the present disclosure modified with the allergy - infectious - cause antigen.

[0177] In the prevention of recurrence and / or treatment of allergy (e.g., allergic conjunctivitis), administer the T cells of the present disclosure to subjects who had and / or have the causative antigen of allergy.

[0178] After administration, the prognosis of allergies in the subject is observed by techniques commonly used in the art.

[0179] (Example 9: Prevention, recurrence prevention, and treatment of autoimmune diseases) In this example, the prevention, recurrence prevention, and / or treatment of autoimmune diseases are verified.

[0180] (Method) In the prevention of autoimmune diseases (e.g., autoimmune uveitis), the T cells of the present disclosure modified with an antigen causing autoimmune disease infection are administered to subjects who do not have an allergenic antigen.

[0181] In the prevention of recurrence and / or treatment of autoimmune diseases (e.g., autoimmune uveitis), the T cells of the present disclosure are administered to subjects who had and / or have an antigen causing autoimmune disease.

[0182] After administration, the prognosis of autoimmune diseases in the subject is observed by techniques commonly used in the art.

[0183] (Note) As described above, the present disclosure has been exemplified using preferred embodiments of the present disclosure. However, it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that patents, patent applications, and other documents cited in this specification should be incorporated by reference into this specification as if their contents were specifically described herein. This application claims priority to Japanese Patent Application No. 2024-001532 filed with the Japan Patent Office on January 9, 2024, the contents of which are incorporated by reference in their entirety into this specification.

Industrial Applicability

[0184] According to the present disclosure, even in an environment where glucose is depleted, such as the tumor microenvironment (TME), it is possible to provide cells that are competitive with cancer cells without starvation or exhaustion, and therefore, it is possible to develop a strategy for improving the effect of cell-based immunotherapy, which is expected to be applied in the medical field.

Claims

1. An effector T cell comprising a chimeric antigen receptor (CAR), modified to express a glucose transporter and / or having enhanced expression of the glucose transporter, wherein the T cell exhibits effector function under low glucose conditions (0.5 mM) that is at least equivalent to that under normal glucose conditions (10 mM).

2. The T cell of claim 1, wherein the CAR is expressed on the T cell.

3. The T cell of claim 1 , wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4.

4. An effector T cell comprising a chimeric antigen receptor (CAR) that has been modified to express a glucose transporter and / or has enhanced expression of a glucose transporter, wherein the T cell exhibits effector function under low glucose conditions (0.5 mM) that is at least equivalent to that under normal glucose conditions (10 mM), and the glucose transporter is GLUT3.

5. Effector T cells transfected with the GLUT3 gene.

6. The T cell of claim 1, which is a human T cell.

7. The T cell described in claim 5, which is a human T cell.

8. A cell population comprising effector T cells that comprise a chimeric antigen receptor (CAR) and have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions (0.5 mM) that is at least equivalent to that under normal glucose conditions (10 mM).

9. The cell population of claim 8, wherein the CAR is expressed in the T cells.

10. The cell population of claim 8 , wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4.

11. A cell population comprising effector T cells that have been modified to express a glucose transporter, including a chimeric antigen receptor (CAR), and / or have enhanced expression of the glucose transporter, wherein the T cells exhibit effector function under low glucose conditions (0.5 mM) that is at least equivalent to that under normal glucose conditions (10 mM), and the glucose transporter is GLUT3.

12. A cell population containing effector T cells into which the GLUT3 gene has been introduced.

13. The cell population of claim 8 , wherein the T cells are human T cells.

14. The cell population described in claim 12, wherein the T cells are human T cells.

15. A pharmaceutical composition comprising a T cell according to any one of claims 1 to 7 or a cell population according to any one of claims 8 to 14.

16. The pharmaceutical composition of claim 15 for treating or preventing cancer.

17. The pharmaceutical composition according to claim 16, which is for the treatment of cancer.

18. The pharmaceutical composition of claim 17, wherein the cancer cure is confirmed by tumor eradication.

19. The pharmaceutical composition of claim 16, which is for long-term action against cancer.

20. The pharmaceutical composition according to claim 16, which is for preventing metastasis or recurrence of cancer.

21. The pharmaceutical composition according to claim 16, which is for preventing cancer recurrence.

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