WO2022140361A1 - Genetically engineered lymphocytes for adoptive cell therapy - Google Patents

Abstract

Provided herein are lymphocytes suitable for use in adoptive cell transfer or adoptive cell therapy, as well as methods of making such lymphocytes and methods of using such lymphocytes in the treatment or cancer. Disclosed herein are lymphocytes that are genetically modified to express a glucose transporter and that are in contact with one or more cytokines. Also disclosed herein are lymphocytes that are genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

Description

GENETICALLY ENGINEERED LYMPHOCYTES FOR ADOPTIVE CELL THERAPY

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63/199,376 filed December 22, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions and methods for treating cancer or a tumor in a subject and more specifically to compositions and methods for treating cancer or a tumor in a subject by modulating the immune system of the subject.

BACKGROUND

[0003] Adoptive cell transfer or adoptive cell therapy (ACT) represents a promising therapeutic approach for the treatment of cancer patients. However, ACT faces two major obstacles: i) the short-term survival of the transferred cells in the cancer patients, and ii) the hostile immunosuppressive tumor microenvironment. Additional challenges of ACT include efficient trafficking and infiltration of the tumor by transferred cells and overcoming tumor- mediated immunosuppression. Despite numerous efforts, state-of-the-art ACT therapies do not provide functional persistence within the immunosuppressive solid tumor microenvironment for long-term efficacy.

[0004] Accordingly, there is a pressing need for identifying novel ACT therapies that provide immune cells with functional persistence and/or that can overcome the immunosuppressive tumor microenvironment.

SUMMARY

[0005] In one aspect, provided is a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one embodiment, the amino acid transporter is a tryptophan transporter and the aminoacyl-tRNA synthetase is a tryptophan-tRNA synthetase. [0006] In one aspect, provided is a method of generating a genetically modified lymphocyte, the method comprising: (a) transforming the lymphocyte with a vector for the expression of a glucose transporter; and (b) cultivating the lymphocyte in the presence of one or more selected of the group consisting of IL-2, IL-7, IL- 15, and IL-21. In one embodiment, the lymphocyte is cultivated in the presence of IL-7 and IL-15. In one embodiment, the lymphocyte is cultivated in the presence of IL-21. In one embodiment, the lymphocyte is cultivated in the presence of IL-7, IL- 15, and IL-21. In some embodiments, the glucose transporter is selected from the group consisting of SLC2A1, SLC2A2, SCL2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14. In one embodiment, the glucose transporter is SCL2A3 (GLUT3).

[0007] In some embodiments, the glucose transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4. In some embodiments, the glucose transporter comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. In one embodiment, the glucose transporter comprises the amino acid sequence of SEQ ID NO:1. [0008] In some embodiments, the amino acid transporter is selected from the group consisting of SLCCL1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, and SLC1A7, and SLC7A5. In one embodiment, the amino acid transporter is SLC1A5. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one or SEQ ID NOs:5-10. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 5-7. In some embodiments, the amino acid transporter comprises the amino acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the amino acid transporter comprises the amino acid sequence of SEQ ID NO:5. In one embodiment, the amino acid transporter comprises the amino acid sequence of SEQ ID NO:6. In one embodiment, the amino acid transporter comprises the amino acid sequence of SEQ ID NO:7.

[0009] In some embodiments, the aminoacyl-tRNA synthetase is selected from the group consisting of a cytoplasmic tryptophanyl-tRNA-synthetase and a mitochondrial tryptophanyl- tRNA-synthetase. In one embodiment, the aminoacyl-tRNA synthetase is a cytoplasmic tryptophanyl-tRNA-synthetase. In one embodiment, the aminoacyl-tRNA synthetase is a mitochondrial tryptophanyl-tRNA-synthetase. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-22. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-19. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the aminoacyl-tRNA synthetase comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0010] In one aspect, provided is a lymphocyte, wherein the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more of IL-2, IL-7, IL- 15, and IL-2L In one embodiment, the lymphocyte is in contact with IL-7 and IL-15. In one embodiment, the lymphocyte is in contact with IL-21. In one embodiment, the lymphocyte is in contact with IL-7 and IL-15. In one embodiment, the lymphocyte is in contact with IL-7, IL-15, and IL-21.

[0011] In some embodiments, the lymphocyte further expresses a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0012] In some embodiments, the lymphocyte further expresses a therapeutic protein. In some embodiments, the therapeutic protein is selected from the group consisting of IL-2, IL-2 mutein, IL-15, CD40L, IL-33, and IL-12.

[0013] In some embodiments, the lymphocyte further expresses a protein that inhibits the interaction of an immunosuppressive polypeptide with its ligand. In one embodiment, the lymphocyte expresses a protein that inhibits the interaction between PD-1 and PD-L1.

[0014] Provided herein is a pharmaceutical composition comprising: (a) a lymphocyte genetically modified to express a glucose transporter, wherein the lymphocyte has been cultivated in the presence of one or more selected of the group consisting of IL-2, IL-7, IL- 15, and IL-21, and (b) a pharmaceutically acceptable carrier.

[0015] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte or a population of lymphocytes disclosed herein. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before formulation of the pharmaceutical composition for administration to the subject.

[0016] Provided herein is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte or a population of lymphocytes disclosed herein. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before formulation of the pharmaceutical composition for administration to the subject.

[0017] Provided herein is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte or a population of lymphocytes disclosed herein. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before formulation of the pharmaceutical composition for administration to the subject.

[0018] Provided herein is a method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject of a pharmaceutical composition comprising a lymphocyte or a population of lymphocytes disclosed herein. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before formulation of the pharmaceutical composition for administration to the subject.

[0019] Provided herein is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte or a population of lymphocytes disclosed herein. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before formulation of the pharmaceutical composition for administration to the subject.

[0020] In some embodiments, the subject has one or more cancers selected from the group consisting of sarcoma, carcinoma, melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, ovarian and gastric cancer.

[0021] In some embodiments, the method further comprises administering to the patient an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent. In one embodiment, the additional therapeutic agent is an immune checkpoint inhibitor.

[0022] In one embodiment, the lymphocyte is a T cell, a B cell or a natural killer cell. In one embodiment, the lymphocyte is a T cell.

BRIEF DESCRIPTION OF THE FIGURES

[0023] Figs. 1A, IB, 1C, ID, and IE illustrate that adoptive T cell transfer of GLUT3- overexpressing OT-1 T cells in tumor bearing mice generates long-term anti-tumor protection. Fig. 1A. GLUT3 -overexpressing OT-1 T cells eliminated tumors in 25% of mice bearing tumors in their right flank in two separate experiments (n=8 for each experiment). Fig. IB. The surviving four mice were challenged with an additional tumor injection in the left flank. Only one out of the four mice relapsed 22 days after the re-challenge. Fig. 1C. Naive control mice were also injected in their left flank and developed tumors as expected. OT-1 T cell persisting after a total of 50 days from the re-challenge in the left flank maintained the expression of GLUT3 (Fig. ID and Fig. IE) and produced ZFNy upon stimulation with OVA peptide (Fig. IF). [0024] Figs. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2J, 2K, and 2L illustrate that memory OTI T cells genetically manipulated to increase their glucose uptake show better survival in glucose deprived conditions and a less exhausted phenotype upon repeated stimulation with cognate peptide in vitro. Fig. 2A. GLUT3 overexpression assessed by flow cytometric detection with and Ab specific for FLAG tag. Fig. 2B. Analysis of Thy 1.1 expression in memory control MOCK T cells. Fig. 2C. Viability of T cells in culture assessed by flowcytometry with a viability dye. Fig. 2D. Analysis of memory OTI-1 cells size (pm). Fig. 2E. Population doubling level of transduced cells and control over time. MOCK: top curve. GLUT3: bottom curve. Fig. 2F. Evaluation of CD62L⁺ CD44⁺ percentage of cells in culture. Fig. 2G. Representation of 72 h time point of cells cultured or not in presence of Glc stained for AnnexinV/7AAD. (from top to bottom: Live cells= Annexin V-/ 7-AAD-, early apoptotic cells = Annexin V+77-AAD-, late apoptotic cells = Annexin V+77-AA+-, necrotic cells = Annexin V-/ 7-AAD-) (left panel). Representative dot plot of AnnexinV/7AAD staining at 72 h (right panel). Fig. 2H. Cells glucose uptake measured with luminescence-based assay. Fig. 21. Glucose uptake evaluated with the fluorescent glucose analog 2-NBDG with or without the addition of the glucose competitor 2-deoxy D-glucose (2- DG). (Average ± SD of different cultures in separate experiments, **p=0.009 MOCK vs GLUT3, *p= 0.0493 MOCK vs M0CK+ 2-DG, **p=0.0016 GLUT3 vs GLUT3+2-DG with student t test). Fig. 2J. Seahorse analysis of the basal ECAR of transduced and control T cells with a metabolic perturbation assay. (Average ± SD of different cultures in separate experiments, student t test). Fig. 2K. Apoptosis level evaluated with AnnexinV/7AAD staining over time (24, 48 and 72h) of GLUT3 (right bars) and MOCK (left bars) cells cultured in media with or without glucose (Glc). (Average ± SD of different cultures where indicated ***p=0.005, ****p<0.0001, 2way ANOVA with multiple comparison). Fig. 2L. Expression level of PD1, LAG3, TIM3 and TCF-1 upon repeated stimulation with SIINFEKL peptide (SEQ ID NO:23) for 5 days. Mean fluorescence intensity (MFI) levels are shown. MFI levels of GLUT3 overexpressing cells from each murine culture are normalized to corresponding MOCK cells. (Average ± SD different cultures in separate experiments, student t test).

[0025] Figs. 3A, 3B, 3C, 3D, and 3E illustrate that adaptive cell therapy (ACT) with memory T cells, which were engineered to overexpress GLUT3 and treated with cytokines, increase survival of tumor-bearing mice. Fig. 3A. Glucose measurement in cell culture supernatant of Bl 60 VA and B16OVA IDO after 4 days of consumption. Fig. 3B. Tumor growth curve of Bl 60 VA and Bl 60 VA IDO tumor cell line (B160VA has a slightly higher tumor volume on day 9). Fig. 3C. 1 x 10⁵ B16-OVA IDO⁺ cells were subcutaneously injected in the flank of C5B1/6 females (n= 3 to 5 mice/group). The mice were irradiated (5Gy) once the tumor was palpable to facilitate T cell grafting (day 7). On days 8 and 11 after tumor inoculation, the mice were injected intravenously with 2 x 10⁶ cells per dose of GLUT3 -expressing T cells, which had been cultivated in the presence of IL-15 and IL-7, (GLUT3, highest survival rate) or one of three controls: (1) T cells transduced with a vector without transgene (mock), (2) untransduced T cells (UTD), or (3) saline (lowest survival rate). Fig. 3D. Kaplan Meier plot of percent survival. Fig. 3E. Tumor growth in mm³ was assessed by calipering. Each trace represents one animal.

[0026] Figs. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H illustrate that OTI T cells transduced to overexpress GLUT3 confer increased overall tumor control and survival of tumor bearing mice treated with ACT. Fig. 4A. Tumor growth curve of mice treated with OTI MOCK (top curve) and GLUT3 (bottom curve) transduced T cells (n= 8 mice/group, **p=0.0065 at day 30, ****p<0.0001 at days 33, 35 and 37, ****p<0.0001, *** p= 0.0004 at day 40, ** p= 0.0021 at day 43). Fig. 4B. Kaplan Meier analysis of mice survival up to 110 days after tumor injection (GLUT3, top curve). Fig. 4C. Analysis of TMRM and MG staining of TILs, expressed as ratio between TMRM MFI geometric mean (GeoMean) vs MG MFI (GeoMean). Analysis of IFNy (Fig. 4D) and TNFa (Fig. 4E) expression from isolated TILs upon overnight stimulation with SIINFEKL peptide (SEQ ID NO:23). Shown is the pool of two different experiments. Statistic: student t test. Fig. 4F. Growth curve of right flank injected tumors of 4 survivor mice of two separate experiments. Fig. 4G. Tumor growth curve of the left flank tumor of survivors and control mice. The control mice are the four traces on the left. Fig. 4H. OTI T cells persistence in survivor mice at 50 days after the first re-challenge is assessed by CD45.1 detection by flowcytometry in lymph nodes (lymph) and spleens.

[0027] Figs. 5A, 5B, 5C, 5D, and 5E illustrate that OTI T cells gene-engineered to overexpress GLUT3 have increased levels of glucose uptake. Fig. 5A. Evaluation of transduction efficiency for GLUT3 expression by anti-Flag antibody staining and flow cytometric analysis. Fig. 5B. Viability of transduced OTI T cells as measured by staining with a viability dye and flow cytometric analysis (day 10). Fig. 5C. Evaluation of CD62L⁺ CD44⁺ percentage of cells in culture. MOCK: left bars; GLUT3 : right bars. Fig. 5D. Glucose uptake evaluated with the fluorescent glucose analog 2-NBDG with or without the addition of the glucose competitor 2- DG. (Average ± SD of different cultures, **p=0.001 MOCK vs GLUT3, **p=0.0029 MOCK vs MOCK + 2DG, *p=0.0138 GLUT3 vs GLUT3 + 2DG, student t test) (left panel). Fig. 5E. Cells glucose uptake measured with luminescence-based assay.

[0028] Figs. 6A, 6B, 6C, 6D, 6E, 6F, and 6G illustrate that OTI T cells gene-engineered to overexpress GLUT3 build a higher energy storage. Fig. 6A. Glucose cell content measured with colorimetric assay on cells lysate. Fig. 6B. Glycogen cell content evaluated with colorimetric assay (Statistic: student t test). Fig. 6C. Glycogen staining with periodic acid shiff (PAS). Fig. 6D. Intracellular staining for pGSK3p. Fig. 6E. Correlation between glycogen content and percentage of transduction. Fig. 6F. Staining with Bodipy 493/503 dye. Fig. 6G. Analysis of effector OTI T cells size (pm). Where fold change is shown, values of GLUT3 expressing cells of each culture are normalized with the values of the corresponding MOCK cells (Statistic: student t test).

Figs. 7A, 7B, 7C, 7D, 7E, 7F, and 7G illustrate that GLUT3 overexpressing OTI T cells show more polarized mitochondria and improved in vitro functionality. Fig. 7A. Staining with TMRM and Mitotracker green (MG) staining, representation of TMRM high /MG high populations. Values of GLUT3 expressing cells of each culture are normalized with the values of the corresponding MOCK cells. (Statistic: student t test). Fig. 7B. Percentage of TMRM high expressing and MG high expressing populations of cells incubated or not over night with 2-DG (Statistic: 2way-ANOVA. MOCK vs MOCK+2-DG *p=0.0131, MOCK vs GLUT3 **p=0.0089, GLUT3 vs GLUT3+2-DG **p=0.0049, MOCK+2-DG vs GLUT3+2-DG *p=0.0235) Fig. 7C. Ratio between the MFI of TMRM and of MG in cells incubated or not for 4-6 hours in complete media or media without Glc. (Statistic: student t test). Fig. 7D. Quantification of intracellular reactive oxygen species (ROS). Fig. 7E. Evaluation of Mell levels with intracellular staining. Fig. 7F. Analysis of IFNg and TNFa production upon overnight stimulation with SIINFEKL peptide (SEQ ID NO:23) in media deprived of Glc, media with low Glc (0.444mM) concentration or complete media (Glc= l l. lmM). Values of GLUT3 expressing cells of each culture are normalized with the values of the corresponding MOCK cells (Statistic: student t test). MOCK: left bars; GLUT3: right bars. Fig. 7G. Mean cell division of gene engineered T cells stimulated for 5 days with irradiated B16OVA tumor cells in media deprived of Glc media with low Glc concentration or complete media. Values of GLUT3 expressing cells of each culture are normalized with the values of the corresponding MOCK cells (Statistic: student t test). MOCK: left bars; GLUT3: right bars.

[0029] Figs. 8A, 8B, 8C, and 8D illustrate that lymphocytes genetically modified to express a glucose transporter and mimicking an effector cells phenotype exhibit increased mitochondrial fitness. Fig. 8A Percentage of viable cells in culture. Fig. 8B. Population doubling level of cells. Fig. 8C. ZFNy release upon stimulation of GLUT3 -overexpressing effector T cells with an anti- CD3 antibody as compared to control (mock) in complete media or low glucose (glc) media. MOCK: left bars; GLUT3: right bars. Fig. 8D. B16-OVA IDO⁺ cells were subcutaneously injected in the flank of C5B1/6 females. The mice were irradiated once the tumor was palpable and treated with intravenous ACT of a total mock and GLUT3 -transduced T cells that had been cultured in presence of IL-7 and IL- 15 to generate a memory phenotype (MEM), or with high doses of IL-2 to generate an effector phenotype (EFF).

[0030] Figs. 9A, 9B, 9C, and 9D illustrate the efficacy of adoptive cell therapy using T cells overexpressing tryptophan transporter SLC1A5 and tryptophanyl-tRNA synthetase (TTS) in reducing tumor growth in vivo. Fig. 9A. 1 x 10⁵ B16-OVA IDO⁺ cells were subcutaneously injected in the flank of C5B1/6 females (n= 3 to 5 mice/group). The mice were irradiated (5Gy) once the tumor was palpable to facilitate T cell grafting (day 7). On days 8 and 11 after tumor inoculation, the mice were injected intravenously with 1 x 10⁶ cells per dose of (1) OT-1 untransduced T cells (UTD), (2) T cells transduced with a control vector without transgene (mock), (3) T cells engineered to express tryptophanyl-tRNA synthetase (TTS), (4) T cells engineered to express the full length form of tryptophan transporter SLC1 A5 (SLC1 A5(L), (5) T cells engineered to express a truncated form of the tryptophan transporter SLC1 A5 (SLC1 A5(S)), (6) T cells engineered to express full length tryptophan transporter and tryptophanyl -tRN A synthetase (SLC1A5(L) + TTS), (7) T cells engineered to express the truncated form of the tryptophan transporter SLC1A5 (SLC1A5(S) + TTS), and (8) saline as a control. Fig. 9B. Tumor volume over time. Each trace represents one animal. Fig. 9C. Ratio of tumor volume at day 40 and day 7. Fig. 9D. Kaplan Meier plot of percent survival.

DETAILED DESCRIPTION

[0031] Provided herein are lymphocytes that are genetically modified to express one or more proteins involved in cellular metabolism. Also disclosed are methods of making such lymphocytes as well as methods of using the disclosed lymphocytes in cellular immunotherapy to mediate a cellular immune response.

[0032] Metabolic transgenes

[0033] T cell function is linked to metabolism. Accordingly, the lymphocytes disclosed herein are genetically modified to express one or more proteins involved in the cellular metabolism of the lymphocytes, referred to herein as “metabolic proteins.” As used herein, a “metabolic transgene” refers to nucleic acid sequence that encodes such a metabolic protein. In some embodiments, the lymphocytes are genetically modified to comprise at least one, at least two, at least three, at least four, or more metabolic transgenes. In some embodiments, the lymphocytes disclosed herein are genetically modified to express one or more proteins in addition to the one or more metabolic proteins. “Genetically modified” and “genetically engineered” are used interchangeably herein. Not all cells in a population that is genetically engineered to express a protein will express the protein in a significant amount. In some embodiments, at least 25%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells genetically engineered to express a protein will express the protein at a given time.

[0034] In some embodiments, lymphocytes disclosed herein are genetically modified to express a metabolic protein, wherein the metabolic protein is a transporter protein or a synthetase. In some embodiments, the transporter protein facilitates the cellular uptake of one or more amino acids. In some embodiments, the transporter protein facilitates the cellular uptake of one or more sugars. In some embodiments, the synthetase is an ami noacyl -tRN A synthetase. [0035] In some embodiments, the metabolic protein is a protein that is naturally expressed by the lymphocyte to be genetically modified. In other embodiments, the metabolic protein is a protein that is not naturally expressed by the lymphocyte to be genetically modified. In some embodiments, the lymphocyte is genetically engineered to overexpress a metabolic protein. "Overexpressed," as used herein, means there is production of a gene product in a sample that is substantially higher than that observed in a population of control samples (e.g., an unmodified cell).

[0036] Glucose transporters

[0037] Upon T cell receptor (TCR) engagement, naive T cells switch their metabolism from housekeeping mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis to support the growth and proliferation of effector cells, leading to an increased uptake of glucose. Due to an increased dependency of activated T cells on glucose, the intensive glucose consumption by solid tumors is immunosuppressive and strongly impairs tumor infiltrating lymphocyte (TIL) functionality.

[0038] Accordingly, disclosed herein are lymphocytes that are genetically engineered to express a glucose transporter to facilitate aerobic glycolysis and to increase lymphocyte fitness in the glucose-depleted tumor microenvironment (TME). In some embodiments, the high-affinity glucose transporter is GLUT3.

[0039] GLUT3 is the third cloned glucose transporter of fourteen glucose transporters that have been described in vertebrates. GLUT3 was first identified in rodent brain samples and was originally defined as a neural glucose transporter. GLUT3 has since been shown to be expressed by other glucose demanding cells, such as murine sperm, where it ensures the necessary energy for motility, and embryonal blastocyst, where it is critical for post-implantation development. Moreover, GLUT3 is expressed in immune cells, such as lymphocytes, monocytes, macrophages and platelets, where it is normally stored in intracellular vesicles and translocates to the cell surface upon activation to sustain the metabolic switch. More recently, it has been suggested that T cells, particularly CD8⁺ T cells, rely not only on GLUT1 for Glc uptake, but also on GLUT3, as GLUT3 is highly expressed upon differentiation and activation.

[0040] Disclosed herein are lymphocytes genetically engineered to express a glucose transporter that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: l-4. In one embodiment, the glucose transporter comprises a sequence selected from the group consisting of SEQ ID NOs: l-4 (see Table 1). In some embodiments, the lymphocytes are genetically engineered to express a glucose transporter that comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In one embodiment, the glucose transporter comprises the amino acid sequence of SEQ ID NO: 1.

Table 1: Glucose transporter proteins.

[0041] In one aspect, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with one or more cytokines. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with one or more of IL-2, IL- 7, IL- 15, and IL-21. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with IL-7. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with IL-15. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with IL-2. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with IL-21. In one embodiment, the lymphocyte that is genetically engineered to express a glucose transporter is in contact with IL-7 and IL-15.

[0042] Amino acid transporters

[0043] The TME can further impair the anti -tumoral efficacy of TILs by depriving TILs of nutrients, including certain amino acids. Nutrient deprivation is the result of both the high metabolic rate of cancer cells as well as the overexpression of catabolic enzymes by cancer cells. Such enzymes include indoleamine 2,3-dioxygenase 1 (IDO-1), which is the rate limiting enzyme involved in the conversion of tryptophan to its catabolic product kynurenine. IDO-1 is overexpressed in many types of solid malignances (on both tumor cells and immune infiltrating cells), such as ovarian, endometrial, colorectal and lung cancers, and is a marker of poor prognosis. [0044] Tryptophan is an essential amino acid, and mammals are auxotrophic for this nutrient and require it from external sources. It has been shown that tryptophan is critical for T cell proliferation, and IDO-1 upregulation has been identified as a major barrier to T cell activity in tumors. IDO-1 strongly suppresses T cells immune response both by depleting Trp from the microenvironment and by increasing the levels of immune-suppressive kynurenine and its metabolites. Kynurenine is the natural ligand of aryl hydrocarbon receptor (AhR), a ligand- activated transcription factor, which has an immune-suppressive effect by promoting the polarization of resident naive T cells towards a Treg phenotype. Moreover, it has been shown that the kynurenine metabolite 3-hydroxyanthranilic acid (3- HAA) inhibits CD8⁺ T cell cytokine induced proliferation.

[0045] Accordingly, disclosed herein are lymphocytes that are genetically modified to express an amino acid transporter. Amino acid transporters are membrane transport proteins that mediate transfer of amino acids into and out of cells or cellular organelles.

[0046] In some embodiments, the amino acid transporter is a tryptophan transporter. In some embodiments, the amino acid transporter is selected from the group consisting of SLC1 Al (excitatory amino acid transporter 3), SLC1A2 (excitatory amino acid transporter 2), SLC1A3 (excitatory amino acid transporter 1), SLC1A4 (neutral amino acid transporter A), SLC1A5 (neutral amino acid transporter B(0), also known as ATB(0) or LAT1), SLC1 A6 (excitatory amino acid transporter 4), SLC1 A7 (excitatory amino acid transporter 5), and SLC7A5 (large neutral amino acids transporter small subunit 1, also known as LAT1).

[0047] In one embodiment, the amino acid transporter is SLC1 A5 (neutral amino acid transporter B(0), short name ATB(0)), which mediates the transport of tryptophan, phenylalanine, leucine and histidine with high affinity. SLC1 A5 also recognizes (but less efficiently) glutamine. Additional substrates of SLC1A5 include the non-metabolizable analog P- (±)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), the thyroid hormones T3 and T4, the dopamine precursor L-DOPA, and acid-related exogenous compounds, such as the drugs melphalan, baclofen and gabapentin. Alanine, proline and charged amino acids are not recognized as substrates.

[0048] In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:5-10 (see Table 2). In some embodiment, the amino acid transporter comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5-10. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:5-7. In some embodiment, the amino acid transporter comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5-7. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the amino acid transporter comprises an amino acid sequence of SEQ ID NO: 5.

Table 2: Amino acid transporter proteins.

[0049] In one aspect, the lymphocytes disclosed herein are genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases, which are also called tRNA ligases, covalently link an amino acid to its cognate tRNA in the first step of protein translation. In one embodiment, the amino acid transporter and the aminoacyl-tRNA synthetase recognize the same amino acid as a substrate. Disclosed herein is a lymphocyte genetically modified to express an amino acid transporter and an aminoacyl-tRN A synthetase, wherein the amino acid transporter and the aminoacyl-tRNA synthetase both recognize the amino acid tryptophan as a substrate. .Accordingly, disclosed is a lymphocyte that is genetically modified to express a tryptophan transporter and a tryptophan-tRNA synthetase. [0050] In some embodiments, the tryptophan-tRNA synthetase is the cytoplasmic enzyme tryptophanyl-tRNA-synthetase (TTS) encoded by WARSI. In some embodiments, the tryptophan-tRNA synthetase is the mitochondrial enzyme tryptophanyl-tRNA-synthetase encoded by WARS2. In some embodiments, the tryptophanyl-tRNA-synthetase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 11-22 (see Table 3). In one embodiment, the tryptophanyl-tRNA-synthetase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-22. In some embodiments, the tryptophanyl-tRNA-synthetase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, the tryptophanyl-tRNA-synthetase comprises an amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

Table 3: Tryptophanyl-tRNA-synthetase (TTS) proteins.

[0051] Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter selected from the group consisting of SLC1 Al (excitatory amino acid transporter 3), SLC1A2 (excitatory amino acid transporter 2), SLC1A3 (excitatory amino acid transporter 1), SLC1 A4 (neutral amino acid transporter A), SLC1 A5 (neutral amino acid transporter B(0), also known as ATB(O)), SLC1A6 (excitatory amino acid transporter 4), SLC1A7 (excitatory amino acid transporter 5), and SLC7A5 (large neutral amino acids transporter small subunit 1, also known as LAT1) and (2) an aminoacyl-tRNA synthetase. Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter selected from the group consisting of SLC1 Al (excitatory amino acid transporter 3), SLC1 A2 (excitatory amino acid transporter 2), SLC1A3 (excitatory amino acid transporter 1), SLC1A4 (neutral amino acid transporter A), SLC1A5 (neutral amino acid transporter B(0), also known as ATB(O)), SLC1A6 (excitatory amino acid transporter 4), SLC1 A7 (excitatory amino acid transporter 5), and SLC7A5 (large neutral amino acids transporter small subunit 1, also known as LAT1) and (2) an tryptophanyl-tRNA-synthetase. Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter selected from the group consisting of SLC1 Al (excitatory amino acid transporter 3), SLC1 A2 (excitatory amino acid transporter 2), SLC1 A3 (excitatory amino acid transporter 1), SLC1 A4 (neutral amino acid transporter A), SLC1 A5 (neutral amino acid transporter B(0), also known as ATB(O)), SLC1 A6 (excitatory amino acid transporter 4), SLC1A7 (excitatory amino acid transporter 5), and SLC7A5 (large neutral amino acids transporter small subunit 1, also known as LAT1) and (2) an tryptophanyl-tRNA-synthetase selected from the group consisting of a cytoplasmic enzyme tryptophanyl-tRNA-synthetase (TTS) encoded by WARSI and a mitochondrial enzyme tryptophanyl-tRNA-synthetase encoded by WARS2. Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) a tryptophanyl-tRNA-synthetase. Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) an tryptophanyl-tRNA- synthetase selected from the group consisting of a cytoplasmic enzyme tryptophanyl-tRNA- synthetase (TTS) encoded by WARSI and a mitochondrial enzyme tryptophanyl-tRNA- synthetase encoded by WARS2. [0052] Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) a tryptophanyl-tRNA-synthetase, wherein:

(a) the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:5-10; and

(b) the tryptophanyl-tRNA-synthetase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 11-22.

[0053] Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) a tryptophanyl-tRNA-synthetase, wherein:

(a) the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:5-7; and

(b) the tryptophanyl-tRNA-synthetase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or SEQ ID NO: 12.

[0054] Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) a tryptophanyl-tRNA-synthetase, wherein:

(a) the amino acid transporter comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5 and

(b) the tryptophanyl-tRNA-synthetase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or SEQ ID NO: 12.

[0055] Disclosed herein is a lymphocyte genetically engineered to express: (1) an amino acid transporter and (2) a tryptophanyl-tRNA-synthetase, wherein:

(a) the amino acid transporter comprises an amino acid comprising SEQ ID NO:5; and

(b) the tryptophanyl-tRNA-synthetase comprises an amino acid comprising SEQ ID NO: 11 or SEQ ID NO: 12.

[0056] Chimeric antigen receptors (CARs) and recombinant T cell receptors (TCRs) [0057] In some embodiments, the lymphocytes disclosed herein are engineered to express an antigen recognizing receptor. The term “antigen recognizing receptor” as used herein refers to a receptor that is capable of activating an immune cell (e.g., a T cell) in response to antigen binding. Exemplary antigen recognizing receptors may be native or genetically engineered TCRs, or genetically engineered TCR-like mAbs (Hoydahl et al. Antibodies 2019 8:32) or chimeric antigen receptors (CARs), in which a tumor antigen-binding domain is fused to an intracellular signaling domain capable of activating an immune cell (e.g., a T cell). T cell clones expressing native TCRs against specific cancer antigens have been previously disclosed (Traversari et al, J Exp Med, 1992 176: 1453-7; Ottaviani et al, Cancer Immunol Immunother, 2005 54: 1214-20; Chaux et al, J Immunol, 1999 163:2928-36; Luiten and van der Bruggen, Tissue Antigens, 2000 55: 149-52; van der Bruggen et al, Eur J Immunol, 1994 24:3038-43; Huang et al, J Immunol, 1999 162:6849-54; Ma et al, Int J Cancer, 2004 109:698-702; Ebert et al, Cancer Res, 2009 69: 1046-54; Ayyoub et al J Immunol 2002 168: 1717-22; Chaux et al, European Journal of Immunology, 2001 31 : 1910-16; Wang et al, Cancer Immunol Immunother, 2007 56:807-18; Schultz et al, Cancer Research, 2000 60:6272-75; Cesson et al, Cancer Immunol Immunother, 2010 60:23-25; Zhang et al, Journal of Immunology, 2003 171 :219-25; Gnjatic et al, PNAS, 2003 100:8862-67; Chen et al, PNAS, 2004). In one embodiment, such TCRs can be sequenced and genetically engineered into TILs for use in adoptive cell therapy. [0058] CARs typically have an antigen-binding domain that is fused to an intracellular signaling domain which is capable of activating or stimulating an immune cell. A CAR’s extracellular binding domain may be composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions of a murine or humanized monoclonal antibody. Alternatively, scFvs may be used that are derived from Fabs (instead of from an antibody, e.g., obtained from Fab libraries). The scFv may be fused to a transmembrane domain and then to an intracellular signaling domain. The CAR can be a first-generation, second generation or third-generation CAR. “First-generation” CARs include those that provide CD3(^ signals upon antigen binding. “Second-generation” CARs include those that provide both costimulation (e.g. CD28 or CD137) and activation (CD3Q. “Third-generation” CARs include those that provide multiple costimulation (e.g. CD28 and CD137) and activation (CD3Q.

[0059] In some embodiments, the lymphocytes disclosed herein are engineered to express an antigen recognizing receptor that recognizes a cancer antigen. Not-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, midkin, BAGE, CASP-8, P-catenin, CA-125, CDK-1, ESO-1, gp75, gplOO , MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orfl l2, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2, ART1, ART4, B-cycline, Glil, Cav-1, Cathepsin B, CD74, E- Cadherin, EphA2 / Eck, Fra-1 / Fosl 1, GAGE-1, Ganglioside / GD2, GnT-V, pl, 6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOXIO, SOX11, Survivin, phCG, WT1, mesothelin, melan-A, NY-BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the lymphocytes disclosed herein are engineered to express more than one antigen recognizing receptor to recognize one or more antigens.

[0060] In some embodiments, the lymphocytes disclosed herein further engineered to secrete therapeutic transgenes, including, but not limited to IL-2, IL-2 mutein, IL-15, CD40L, IL-33, and IL-12, or variants thereof. In embodiments, the lymphocytes disclosed herein are modified using the Genetic Engineering for the Enhanced Performance of T-cells technology described in PCT application publication No. WO2021/097278, which is incorporated herein by reference in its entirety. In embodiments, the lymphocytes disclosed herein are further engineered to express a transgene described in WO2021/097278, including, but not limited to the transgenes recited in the claims of WO2021/097278.

[0061] In some embodiments, the lymphocytes disclosed herein are engineered to express a protein that inhibits, blocks, or antagonizes the interaction of immunosuppressive polypeptides and/or their ligands. Immunosuppressive polypeptides that known to suppress or decrease an immune response via their binding include CD47, PD-1, CTLA-4, and their corresponding ligands, including SIRPalpha, PD-L1, PD-L2, B7-1, B7-2, and TIGIT. Such polypeptides are present in the tumor microenvironment and inhibit immune responses to neoplastic cells.

[0062] In some embodiments, lymphocytes disclosed herein are engineered to express a PD-1 variant (a PD-1 decoy) that is designed compete with endogenous PD-1. In some embodiments, the PD-1 decoy is lacking the cytoplasmic domain of PD-1. In some embodiments, the PD-1 transmembrane and intracellular signaling domains in the PD-1 decoy are replaced with a co- stimulating signaling domain of CD28 or a constitutively active IL-7 receptor, to convert possible inhibitory signal to improved T cell function.

[0063] In some embodiments, the lymphocytes disclosed herein are engineered to express one or more co-stimulatory polypeptides to stimulate or increase an immune response via their binding. Non-limiting examples of such co-stimulatory polypeptides include CD28, OX-40, 4- 1BB, CD27, and NKG2D and their corresponding ligands, including B7-1, B7-2, OX-40L, 4- 1BBL, CD70, and NKG2D ligands.

[0064] Pharmaceutical compositions

[0065] In one embodiment, provided herein is a pharmaceutical composition comprising: (a) a lymphocyte genetically modified to express a glucose transporter, wherein the lymphocyte has been cultivated in the presence of one or more selected of the group consisting of IL-2, IL-7, IL- 15, and IL-21, and (b) a pharmaceutically acceptable carrier. In one embodiment, the lymphocyte has been cultivated in the presence of IL-2. In one embodiment, the lymphocyte has been cultivated in the presence of IL-7 and IL-15. In one embodiment, the lymphocyte has been cultivated in the presence of IL-21. In one embodiment, the lymphocyte has been first cultivated in IL-2 and subsequently cultivated in IL-7 and IL-15. In one embodiment, the glucose transporter is selected from the group consisting of SLC2A1, SLC2A2, SCL2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14. In one embodiment, the glucose transporter is SCL2A3 (GLUT3). In one embodiment, the glucose transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4, or an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1.

[0066] In another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective number of a lymphocyte disclosed herein and a pharmaceutically acceptable carrier.

[0067] The pharmaceutical compositions generally comprise substantially isolated/purified lymphocytes and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions are generally formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. [0068] The terms “pharmaceutically acceptable,” “physiologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[0069] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor, can also be incorporated into pharmaceutical compositions.

[0070] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed herein are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0071] In some embodiments, the composition includes the genetically modified lymphocytes as described above and optionally a cryo-protectant (e.g., glycerol, DMSO, PEG). [0072] Methods of making genetically modified lymphocytes

[0073] The disclosure further provides a method of preparing the genetically modified lymphocytes disclosed herein. In one aspect, provided is a method comprising: (a) providing a plurality of lymphocytes; (b) introducing into the plurality of lymphocytes a nucleic acid molecule comprising one or more transgenes to obtain a plurality of genetically modified lymphocytes, wherein at least one of the one or more transgenes is a metabolic transgene; and (c) expanding the plurality of genetically modified in a cell culture medium.

[0074] In some embodiments, the method may include: (a) providing a plurality of lymphocytes; (b) introducing into the plurality of lymphocytes two or more nucleic acid molecules, each of the two or more nucleic acid molecules comprising at least one transgene, thereby obtaining a plurality of genetically-engineered lymphocytes, wherein at least one of the two or more nucleic acid molecules comprises a metabolic transgene; and (c) expanding the plurality of genetically-engineered in a cell culture medium.

[0075] The term “culturing” or “expanding” refers to maintaining or cultivating cells under conditions in which they can proliferate and avoid senescence. For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include neoantigen peptides. Stable cell lines may be established to allow for the continued propagation of cells.

[0076] Methods of making “chimeric” lymphocytes

[0077] Provided herein are method of generating genetically engineered lymphocytes that exhibit a glycolytic metabolism in combination with a memory or an effector phenotype. As used herein, these genetically engineered lymphocytes are referred to as “chimeric lymphocytes.” [0078] In some embodiments, the genetically engineered lymphocytes that exhibit a glycolytic metabolism are genetically engineered T cells that have a memory phenotype. In some embodiments, the genetically engineered lymphocytes are genetically engineered T cells that have a stem cell memory (TSCM) , central memory (TCM) phenotype.

[0079] Methods of determining whether a lymphocyte, including a T cell, exhibits a memory phenotype are known in the art. In some embodiments, the genetically engineered T cells exhibit increased longevity, improved reconstitution capacity, longer telomere, an improved in vivo dynamic proliferative nature, an enhanced potential to differentiate into multiple subsets of T cells, and increased potent antitumor as compared to effector memory (TEM) and terminal effector (TTE) T cells.

[0080] Methods of determining whether a lymphocyte, including a T cell, exhibits an effector phenotype are known in the art, and include, but are not limited to staining for CD62L and CD44 expression in a murine setting, or for CCR7 and CD45RA in a human setting.

[0081] Methods of determining whether a lymphocyte, including a T cell, exhibits a glycolytic metabolism are known in the art, and include, but are not limited to measuring the basal extracellular acidification rate (ECAR), the glycolytic reserve, spare respiratory capacity (SRC), glycolytic switch (glucose consumption as measured by determining the extracellular acidification rate), and primary energy source (amino acids versus glucose).

[0082] Accordingly, provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that has a memory phenotype.

[0083] In some embodiments, the lymphocyte is genetically engineered to express a glucose transporter and is in contact with one or more of IL-2, IL-7, IL- 15, and IL-21.

[0084] Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-2. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-7. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-15. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-21. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-7 and IL-15. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-2, IL-7, and IL-15. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-7, IL- 15, and IL-21. Provided herein is a method of making a lymphocyte that is genetically engineered to express a glucose transporter and that is in contact with IL-2, IL-7, IL- 15, and IL-21. [0085] In some embodiments, the genetically modified lymphocyte is cultivated in the presence of IL-7 and IL-15. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of IL-21. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of IL-7 IL- 15, and IL-21. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of IL-2, IL-7 IL- 15, IL-21.

[0086] In some embodiments, the lymphocyte is a T cell.

[0087] In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 50 ng/ml IL-7, IL-15, and/or IL-21. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 25 ng/ml IL-7, IL- 15, and/or IL-21. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 10 ng/ml IL-7, IL-15, and/or IL-21.

[0088] In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 50 ng/ml IL-7 and/or IL-15. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 25 ng/ml IL-7 and/or IL- 15. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 10 ng/ml IL-7 and/or IL-15.

[0089] In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 500 Ul/ml IL-2. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 1 to about 300 Ul/ml IL-2. In some embodiments, the genetically modified lymphocyte is cultivated in the presence of about 10 to about 200 Ul/ml IL-2.

[0090] Methods of cultivating cells to generate a memory phenotype

[0091] In some embodiments, provided is a method of generating a genetically engineered lymphocyte that exhibits a glycolytic metabolism and a memory phenotype, the method comprising (a) stimulating a lymphocyte with IL-2 and/or CD3/CD28 and (b) cultivating the lymphocyte in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21. In some embodiments, the lymphocyte is stimulated in IL-2 and/or CD3/CD28 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, the lymphocyte is cultivated in the presence of one or more of IL-2, IL-7, IL-15, and IL-21 between 1 and 30 days. In some embodiments, the lymphocyte is cultivated in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21 between 1 and 18 days. In some embodiments, the lymphocyte is cultivated in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21 between 1 and 15 days. In some embodiments, the lymphocyte is cultivated in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21 between 10 and 12 days.

[0092] In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with IL-2 and CD3/CD28, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, and (c) cultivating the genetically modified lymphocyte in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21. In some embodiments, the glucose transporter is GLUT3, a high affinity glucose transporter.

[0093] In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with IL-2 and CD3/CD28, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, and (c) cultivating the genetically modified lymphocyte in the presence of IL-7 and IL-15. In some embodiments, the glucose transporter is GLUT3, a high affinity glucose transporter.

[0094] In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with IL-2 and CD3/CD28, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, and (c) cultivating the genetically modified lymphocyte in the presence of IL-21. In some embodiments, the glucose transporter is GLUT3, a high affinity glucose transporter.

[0095] In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with IL-2 and CD3/CD28 for one day, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, (c) cultivating the genetically modified lymphocyte for 2 days in the presence of IL-2 and, (d) starting on day 3, cultivating the genetically modified lymphocyte in the presence of one or more of IL-2, IL-7, IL- 15, and IL-21.

[0096] Methods of cultivating cells to generate an effector phenotype

[0097] In some embodiments, provided is a method of generating a genetically engineered lymphocyte that exhibits a glycolytic metabolism and a effector phenotype, the method comprising (a) stimulating a lymphocyte with IL-2 and/or CD3/CD28 and (b) cultivating the lymphocyte in the presence of IL-2. In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with IL-2 and CD3/CD28 for one day, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, and (c) cultivating the genetically modified lymphocyte in the presence of IL-2. In some embodiments, the concentration of IL-2 in step (a) is 200 lU/ml. In some embodiments, the cells are cultivated for up to 15 days in step (c). In some embodiments, the cells are cultivated for up to 15 days in step (c) while the media is refreshed every other day with the addition of fresh IL-2 at 200 lU/ml. In some embodiments, provided is a method comprising (a) stimulating a lymphocyte with 200 lU/m IL-2 and CD3/CD28 for one day, (b) transducing the lymphocyte with a vector for the expression of a glucose transporter, and (c) cultivating the genetically modified lymphocyte in the presence of IL-2 for up to 15 days in while the media is refreshed every other day with the addition of fresh IL-2 at 200 lU/ml.

[0098] Lymphocytes

[0099] Lymphocytes are one of the subtypes of a white blood cell in a vertebrate's immune system and include T cells, B cells, and natural killer (NK) cells.

[00100] In some embodiments, the lymphocytes disclosed herein are peripheral blood lymphocytes (PBLs). In some embodiments, the lymphocytes disclosed herein are tumorinfiltrating lymphocytes (TILs).

[00101] In some embodiments, the lymphocytes disclosed herein are derived from CD34 hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. In certain embodiments, the lymphocytes disclosed herein are autologous, allogeneic, syngeneic, or xenogeneic. In a preferred embodiment, the lymphocytes disclosed herein are autologous. In some embodiments, the lymphocytes disclosed herein are human.

[00102] Prior to the expansion and genetic modification of the lymphocytes described herein, a source of lymphocytes from a subject is obtained. Lymphocytes can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. As described herein, any number of lymphocyte lines available in the art can be used. Lymphocytes can be obtained from a unit of blood collected from a subject using any number of techniques known to the person skilled in the art, such as the Ficoll™ separation. Circulating blood cells of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated white blood cells, red blood cells, and platelets. The cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for the subsequent processing steps. The cells may be washed with phosphate- buffered saline (PBS). Alternatively, the wash solution may lack calcium and may lack magnesium or may lack many, if not all, divalent cations. As those of ordinary skill in the art would readily appreciate, a washing step can be achieved by methods known to those skilled in the art, such as using a semiautomatic continuous flow centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CytoMate, or elHaemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca²⁺free, PBS free Mg²⁺, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells resuspended directly in a culture medium.

[00103] As described herein, lymphocytes may be isolated from peripheral blood by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugal elutriation. Lymphocytes may also be isolated from the spleen. If needed, specific subpopulation lymphocytes, such as T lymphocytes (z.e., CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺ or CD45RO⁺ T lymphocytes) can be further isolated by positive or negative selection techniques. For example, T lymphocytes may be isolated by incubation with conjugated anti-CD3 beads for a sufficient period of time (z.e., 30 minutes to 24 hours) for positive selection of the desired T lymphocytes. For the isolation of T lymphocytes from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cellular performance. Longer incubation times can be used to isolate T lymphocytes in any situation where there are few T lymphocytes compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. The person skilled in the art will recognize that multiple rounds of selection may also be used. It may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also undergo new rounds of selection.

[00104] Enrichment of a population of lymphocytes (e.g., T lymphocytes) by negative selection can be performed with a combination of antibodies directed to unique surface markers for the negatively selected cells. One method is the sorting and/or selection of cells by negative magnetic immune adherence or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present in the negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody typically includes antibodies against CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. Alternatively, the regulatory T lymphocytes are depleted by anti-C25 conjugate beads or other similar selection method.

[00105] Lymphocytes for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, freezing and the following thawing step provide a more uniform product by eliminating granulocytes and, to some extent, monocytes in the cell population. After the washing step that removes the plasma and platelets, the cells can be suspended in a freezing solution. Although many solutions and freezing parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose human albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% of dextrose, 20% serum of human albumin and 7.5% of DMSO or other suitable cell freezing medium containing for example Hespan and PlasmaLyte A. The cells may then be frozen at -80 °C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen.

[0100] The cryopreserved cells may be thawed and washed as described herein and allowed to stand for one hour at room temperature before activation using the methods of the present disclosure. As described herein, lymphocytes can be expanded, frozen, and used later. As described herein, samples may be collected from a patient shortly after the diagnosis of a particular disease as described herein, but before any treatment. The cells may be isolated from a blood sample or an apheresis of a subject before any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablatories such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit calcium-dependent calcineurin phosphatase (e.g., ciclosporin and FK506) or inhibit p70S6 kinase that is important for signaling induced by the growth factor (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321, 1991, Bierer et al., Curr. Opin. Immun., 5: 763-773, 1993). The cells may be isolated from a patient and frozen for later use together with (e.g., before, simultaneously or after) bone marrow or stem cell transplant, therapy with T lymphocyte ablation using chemotherapeutic agents such as fludarabine, radiotherapy external beam (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. As described herein, the cells may be isolated before and can be frozen for later use in the treatment after therapy with ablation of B lymphocytes, such as agents that react with CD20, for example, Rituxan.

[0101] Either before or after the genetic modification of lymphocytes (e.g., T lymphocytes) to express a desirable transgene, lymphocytes can be activated and expanded generally using methods such as those described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and the publication of US patent application. No. 20060121005.

[0102] Vectors

[0103] Transgenes can be introduced into lymphoid cells using various methods. These methods include, but are not limited to, transduction of cells using integration-competent gamma-retroviruses or lentivirus, and DNA transposition.

[0104] A wide variety of vectors can be used for the expression of the transgenes. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into a host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells. Accordingly, in certain embodiments, a viral vector is used to introduce a nucleotide sequence encoding one or more transgenes or fragment thereof into a host cell for expression. The viral vector may comprise a nucleotide sequence encoding one or more transgenes or fragments thereof operably linked to one or more control sequences, for example, a promoter. Alternatively, the viral vector may not contain a control sequence and will instead rely on a control sequence within the host cell to drive expression of the transgenes or fragment thereof. Non-limiting examples of viral vectors that may be used to deliver a nucleic acid include adenoviral vectors, AAV vectors, and retroviral vectors.

[0105] For example, an adeno-associated virus (AAV) can be used to introduce a nucleotide sequence encoding one or more transgenes or fragment thereof into a host cell for expression. AAV systems have been described previously and are generally well known in the art (Kelleher and Vos, Biotechniques, 17(6): 1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat lmmun, 13(2-3): 141-64, 1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Details concerning the generation and use of rAAV vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference in its entirety for all purposes.

[0106] In some embodiments, a retroviral expression vector can be used to introduce a nucleotide sequence encoding one or more transgenes or fragment thereof into a host cell for expression. These systems have been described previously and are generally well known in the art (Nicolas and Rubinstein, In, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494- 513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). Examples of vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and p-actin.

[0107] In some embodiments, expression of the metabolic transgene is regulated by a constitutively activated promoter. In some embodiments, expression of the metabolic transgene is regulated by an inducible promoter. In some embodiments, expression of the metabolic transgene is induced by upon activation of the lymphocyte.

[0108] Combinations of retroviruses and an appropriate packaging line may also find use, where the capsid proteins will be functional for infecting the target cells. Usually, the cells and viruses will be incubated for at least about 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for short intervals in some applications, e.g., 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are “defective,” /.< ., unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line. The host cell specificity of the retrovirus is determined by the envelope protein, env (pl20). The envelope protein is provided by the packaging cell line. Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g., MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23. Retroviruses bearing amphotropic envelope protein, e.g., 4070A, are capable of infecting most mammalian cell types, including human, dog, and mouse. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with xenotropic envelope protein, e.g., AKR env, are capable of infecting most mammalian cell types, except murine cells. The vectors may include genes that can later be removed, e.g., using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g., by including genes that allow selective toxicity such as herpesvirus TK, BCL-xs, etc. Suitable inducible promoters are activated in a desired target cell type, either the transfected cell or progeny thereof. [0109] Non-limiting examples of the vectors useful for the genetically engineered lymphocytes disclosed herein include retroviral vector SFG.MCS, and helper plasmids RD114, Peg-Pam3 (Arber et al. J Clin Invest 2015 Jan 2; 125(1): 157-168), lentiviral vector pRRL, and helper plasmids R8.74 and pMD2G (e.g., Addgene Plasmid #12259). In some embodiments, the Sleeping Beauty transposon system can be used (Deniger et al. 2016 Mol Ther. Jun;24(6): 1078- 1089). In some embodiments, transgenes can be introduced into cells via deforming a cell as it passes through a small opening, disrupting the cell membrane and allowing material to be inserted into the cell, for example, electroporation (Xiaojun et al. 2017 Protein Cell, 8(7): 514- 526), or the Cell Squeeze® method. Such electroporation methods of an RNA encoding a transgene allow for transient expression of such transgene in cells which can limit toxicity and other undesirable effects of engineered cells (Barrett et al. 2011 Hum Gene Ther. Dec; 22 (12): 1575-1586).

[0110] In some embodiments, genome-editing techniques, such as CRISPR/Cas9 systems, designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available to induce expression of the transgenes in an immune cell. In general, “CRISPR/Cas9 system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more elements of a CRISPR system may be derived from a type I, type II, or type III CRISPR system. Alternatively, one or more elements of a CRISPR system may be derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). [OHl] In some embodiments, the lymphoyctes disclosed herein are genetically modified by transfecting the lymphocyte cell with a vector (e.g., lentiviral vector) encoding one or more transgenes or a functional fragment thereof and CA9 or a functional fragment thereof. In some embodiments, one or more transgenes or a functional fragment thereof and CA9 or a functional fragment thereof can be introduced into the immune cell using one, two, or more vectors.

[0112] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising exogenous vectors and/or nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0113] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo release vehicle is a liposome (e.g., an artificial membrane vesicle).

[0114] In the case where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, bound to a liposome via a binding molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, in a complex with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, content or in a complex with a micelle, or associated otherwise with a lipid. The compositions associated with lipids, lipids/DNA or lipids/expression vector are not limited to any particular structure in solution. For example, they can be present in a bilayer structure, as micelles, or with a “collapsed” structure. They can also be simply interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0115] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; Dicetylphosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); Cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the sole solvent since it evaporates more easily than methanol. “Liposome” is a generic term that encompasses a variety of unique and multilamellar lipid vehicles formed by the generation of bilayers or closed lipid aggregates. Liposomes can be characterized as having vesicular structures with a bilayer membrane of phospholipids and an internal aqueous medium. Multilamellar liposomes have multiple layers of lipids separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and trap dissolved water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also included. For example, lipids can assume a micellar structure or simply exist as nonuniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0116] Regardless of the method used to introduce exogenous nucleic acids into a host cell, the presence of the recombinant DNA sequence in the host cell can be confirmed by a series of tests. Such assays include, for example, “molecular biology” assays well known to those skilled in the art, such as Southern and Northern blot, RT-PCR and PCR; biochemical assays, such as the detection of the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot) or by assays described herein to identify agents that are within the scope of the disclosure.

[0117] Methods of Treatment

[0118] In another aspect, provided are methods of treating a subject having a disease or disorder by administering a therapeutically effective amount of genetically modified lymphocytes described herein. In another aspect, provided are methods of treating a subject having a disease or disorder by administering a therapeutically effective amount of a pharmaceutical composition comprising genetically modified lymphocytes described herein and a pharmaceutically acceptable carrier. In some embodiments, the lymphocyte or population of lymphocytes is washed to remove cytokines before administration to the subject.

[0119] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0120] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. In relation to ATC, an “effective amount” or “therapeutically effective amount” may also be expressed a certain or a minimal number of cells that are administered to a subject and that are capable of producing a medically desirable result in the treated subject.

[0121] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc) and a human). The subject may be a human or a non-human. In some embodiments, the subject is a human. In some embodiments, the subject is immune-depleted.

[0122] Provided herein are methods of treating cancer and/or reducing the growth of a tumor, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising genetically modified lymphocytes disclosed herein.

[0123] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term "cancer" as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject that has a cancer is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, are able to out- compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0124] In one aspect, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one aspect, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

[0125] In one aspect, provided is a method of reducing tumor growth a subject in need thereof, the method comprising administering to the subject a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one aspect, provided is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

[0126] In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

[0127] In one aspect, provided is a method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one aspect, provided is a method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

[0128] In one aspect, provided is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase. In one aspect, provided is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRNA synthetase.

[0129] In some embodiments, the lymphocyte is genetically modified to express an amino acid transport selected from the group consisting of SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7, and SLC7A5. In some embodiments, the lymphocyte is genetically modified to express a tryptophan transporter. In one embodiment, the amino acid transporter is SLC1A5. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:5-10. In one embodiment, the amino acid transporter comprises one of SEQ ID NOs:5-10. In some embodiments, the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:5-7. In one embodiment, the amino acid transporter comprises one of SEQ ID NOs:5-7. [0130] In some embodiments, the aminoacyl-tRNA synthetase is a tryptophanyl-tRNA- synthetase. In some embodiments, the aminoacyl-tRNA synthetase is selected from the group consisting of a cytoplasmic tryptophanyl-tRNA-synthetase and a mitochondrial tryptophanyl- tRNA-synthetase. In one embodiment, the aminoacyl-tRNA synthetase is a cytoplasmic tryptophanyl-tRNA-synthetase. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11- 22. In one embodiment, the aminoacyl-tRNA synthetase comprises any one of SEQ ID NOs: 11- 22. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-19. In one embodiment, the aminoacyl-tRNA synthetase comprises any one of SEQ ID NOs: 11-19. In some embodiments, the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-12. In one embodiment, the aminoacyl-tRNA synthetase comprises any one of SEQ ID NOs: 11-12.

[0131] In one aspect, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0132] In one aspect, provided is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0133] In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0134] In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0135] In one aspect, provided is a method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of reducing tumor- associated fibrosis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0136] In one aspect, provided is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject a lymphocyte, the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines. In one aspect, provided is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lymphocyte is genetically modified to express a glucose transporter and wherein the lymphocyte is in contact with one or more cytokines.

[0137] In some embodiments, the glucose transporter is selected from the group consisting of SLC2A1, SLC2A2, SCL2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14. In one embodiment, the glucose transporter is SCL2A3 (GLUT3). In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-15, and IL-21.

[0138] Cancers that can be treated by the compositions and methods disclosed herein include tumors that are not vascularized or are not substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (such as hematologic tumors, e.g., leukemias and lymphomas) or may comprise solid tumors. The types of cancers to be treated with the genetically engineered lymphocytes disclosed herein include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemias or malignant lymphoid tumors, benign and malignant tumors and malignancies, e.g., sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and pediatric tumors/cancers.

[0139] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or haematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous myelogenous leukemia, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin’s lymphoma (indolent and high-grade forms), myeloma Multiple, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0140] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer , lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat gland, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma of pheochromocytomas, carcinoma papillary, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma) (such as brainstem glioma and mixed gliomas), glioblastoma (also astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

[0141] The pharmaceutical compositions, as described, can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials. The precise amount of the compositions disclosed herein to be administered can be determined by a physician having account for individual differences in age, weight, tumor size, extent of infection or metastasis, and patient's condition (subject). It can generally be stated that a pharmaceutical composition comprising the lymphocytes described herein can be administered at a dose of 10⁴ to 10⁹ cells/kg body weight, e.g., 10⁵ to 10⁶ cells/kg body weight, including all values integers within these intervals. The compositions can also be administered several times at these dosages. The genetically modified lymphocytes disclosed herein can be administered using infusion techniques that are commonly known in immunotherapy see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0142] The administration of the present compositions can be carried out in any convenient way, including infusion or injection (i.e., intravenous, intrathecal, intramuscular, intraluminal, intratracheal, intraperitoneal, or subcutaneous), transdermal administration, or other methods known in the art. Administration can be once every two weeks, once a week, or more often, but the frequency may be decreased during a maintenance phase of the disease or disorder. In some embodiments, the composition is administered by intravenous infusion.

[0143] In certain cases, the cells activated and expanded using the methods described herein, or other methods known in the art wherein the lymphocytes are expanded to therapeutic levels, are administered to a patient together with (e.g., before, simultaneously or consecutively) any number of relevant treatment modalities. Also described herein, the lymphocytes can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablating agents such as CAMPATH, anti-cancer antibodies. CD3 or other antibody therapies, cytoxine, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0144] The genetically engineered lymphocytes disclosed herein can also be administered to a patient together with (e.g., before, simultaneously or after) bone marrow transplantation, therapy with T lymphocyte ablation using chemotherapy agents such as fludarabine, radiation therapy external beam (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. Also described herein, the compositions can be administered after ablative therapy of B lymphocytes, such as agents that react with CD20, for example, Rituxan. For example, subjects may undergo standard treatment with high-dose chemotherapy followed by transplantation of peripheral blood stem cells. In certain cases, after transplantation, the subjects receive an infusion of the expanded lymphocytes, or the expanded lymphocytes are administered before or after surgery.

[0145] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-cancer or anti -tumor agent. In some embodiments, the composition is administered to the subject before, after, or concurrently with the second therapeutic agent.

[0146] “ Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0147] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint modulator. Accordingly, in some embodiments, genetically engineered lymphocytes disclosed herein are administered with a checkpoint inhibitor. Checkpoint proteins interact with specific ligands that send a signal into the T cell and switch off or inhibit T cell function. By expressing high levels of checkpoint proteins on their surface, cancer cells can control the function of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response. Examples of immune checkpoint modulators include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The immune checkpoint protein Programmed Death- 1 (PD-1) is a key immune checkpoint receptor ex-pressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand- 1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibition of the PD-1/PD-L1 interaction can promote potent antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, Pembrolizumab (MK-3475), Nivolumab (MDX-1106), Cemiplimab-rwlc (REGN2810), Pidilizumab (CT-011), Spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to, Atezolizumab (MPDL3280A), Durvalumab (MEDI4736), Avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316. The checkpoint modulators may be administered simultaneously, separately, or concurrently with the genetically engineered lymphocytes disclosed herein.

[0148] In some embodiments, the method further comprises administering a therapeutically effective amount of a “chemotherapeutic agent,” which is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, methyldopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBLTMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, see, e.g., Agnew Chem. Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotics chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK®.; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’, 2’ ’-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, xeloda, gemcitabine, KRAS mutation covalent inhibitors and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additional examples include irinotecan, oxaliplatinum, and other standard colon cancer regimens.

[0149] Kits

[0150] In another aspect, provided are kits for the manufacturing, preparation, and development of the composition of genetically modified subsets of lymphocytes of the embodiments described above, comprising at least one or more containers, each with a different reagent for the manufacturing of the genetically engineered lymphocytes disclosed herein. Kits may include a set of instructions in the use of the reagents, essential information on how performing the procedures for the manufacturing.

[0151] In certain embodiments, kits are provided for the preparation and development of a pharmaceutical composition comprising a therapeutically effective amount of the composition of lymphocytes described above and a pharmaceutically acceptable carrier. The kit may comprise at least one, or more containers, each with a different reagent. Kits may include instruction for the manufacturing, for the therapeutic regimen to be used, and periods of administration. In more complex embodiments, the kits may comprise further therapeutic elements, e.g. checkpoint modulators, in accordance with this disclosure.

[0152] The composition or the pharmaceutical composition described herein can be provided in a kit. In one embodiment, the kit includes (a) a container that contains the composition and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit. For example, kits may include instruction for the manufacturing, for the therapeutic regimen to be used, and periods of administration. In an embodiment, the kit includes also includes an additional therapeutic agent (e.g., a checkpoint modulator). The kit may comprise one or more containers, each with a different reagent. For example, the kit includes a first container that contains the composition and a second container for the additional therapeutic agent.

[0153] The containers can include a unit dosage of the pharmaceutical composition. In addition to the composition, the kit can include other ingredients, such as a solvent or buffer, an adjuvant, a stabilizer, or a preservative.

[0154] The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

EXAMPLES

[0155] Methods

[0156] Provided below are the methods for Examples 1-7.

[0157] Cloning strategies

[0158] The gene strings of the transgenes were ordered from Addgene and cloned in frame in retroviral (pMSGV) or lentiviral (pRRL) vectors available in the lab. The vectors amplification was performed in Stellar competent cells (E. coli HST08) and purified with plasmid mini/maxi- prep kit (400250/220020, Genomed) upon confirmative sequencing. The codon optimized gene strings of murine TTS, SLC1 A5(L), SLC1 A(S) (242 amino acids were removed in the N terminal part of the transporter) and GLUT3 were cloned in frame in pMSGV retroviral vector under the 5’LTR promoter using the cloning site (BamhI/Sall for TTS, Mlul/Hindlll for GLUT3 3 and both versions of SLC1 A5). Tag encoding sequences were added to the 5’ end of the sequences encoding TTS and the transporter, namely Myc tag (EQKLISEEDL) for TTS, 3 FLAG tag (DYKDDDDK) for GLUT3, and V5 tag (GKPIPNPLLGLDST) for SLC1 A5.

[0159] Retroviral supernatant production

[0160] Phoenix-Eco (ATCC CRL-3214), a second-generation retrovirus producer cell line for the generation of helper-free ecotropic and amphotropic retroviruses, were seeded in a T150 flask (Corning, 430825) for a total number of 10 x 10⁶ cells/flask in RPMI 1640 Glutamax medium (Invitrogen) with the addition of heat inactivated (HI- for 30 min at 56 °C) 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (P/S) (referred as RPMI complete medium). The following day, when the cells reached 70-80% confluency, they were transfected with a polymer-based reagent (Turbofect) and the plasmid mix (14 pg of Phoenix-ECO plasmid, 21 pg of the plasmid of interest) according to the manufacturer protocol. In details, 107 pl of Turbofect were gently resuspended in 2 ml of RPMI medium supplemented with 10 mM Hepes used to afterwards dilute 21.4 pg of transfer plasmid and 14.3 pg of Phoenix-Eco packaging vector. After 30 min of incubation at RT the DNA mixture was added on top the cells and the volume was adjusted up to a total of 30 ml. The medium was refreshed after 24 h and the retroviral supernatant was harvested upon 48 and 72 h of transfection and concentrated through an ultracentrifugation of 2 h at 24000 g in 38.5 ml tubes, then resuspended in a total volume of 400 pl of RPMI Glutamax supplemented with 10% HI FBS, 1% P/S, 1% sodium pyruvate, 1% non- essential amino acids, 0.1% 2-mercaptoethanol (referred as mouse T cells complete medium), snap frozen on dry ice and kept at -80 °C until the T cell transduction day.

[0161] Primary murine T cells isolation, stimulation, transduction and culture

[0162] C57B1/6 CD45.1 mice and OT-1 C57B1/6 CD45.1 mice were homozygous inbreed.

OT-1 mice contain transgenic inserts for mouse Tcra-V2 and Tcrb-V5 genes. The transgenic T cell receptor was designed to recognize ovalbumin peptide residues 257-264 (OVA257-264) in the context of H2Kb (CD8 co-receptor interaction with MHC class I). This results in MHC class I-restricted, ovalbumin-specific, CD8⁺ T cells (OT-1 cells). That is, the CD8 T cells of this mouse primarily recognize OVA257-264 when presented by the MHC I molecule.

[0163] Upon euthanasia with high doses of CO2 and harvest of the spleen, the organ was smashed on a strainer of 40 pm size (352340, Corning) using a 2 ml syringe plunger (01227, Becton Dickinson) and Roswell Park Memorial Institute medium (RPMI) Glutamax medium (Invitrogen). Upon red blood cells lysis of the collected splenocytes, T cells were negatively selected using the opportune kit (EasySep Mouse T cells Isolation Kit, Stem Cell) according to the manufacturer’s protocol. Isolated T cells were then resuspended in mouse T cells complete medium and stimulated with anti-CD3/anti-CD28 dynabeads (2: l=beads: T cells ratio) (Invitrogen), 50 lU/ml of human recombinant IL-2 (Glaxo) or murine IL-2 and seeded at 1 x 10⁶ cells/ml in a cell culture treated 24 well plate. The day after the cells were transduced in a 24 well not treated, previously incubated ON at 4 °C with 20 pg/ml of a recombinant human fibronectin fragment, blocked with a solution of PBS with 2% of bovine serum albumin (BSA) for 30 min and centrifuged for 90 min at 2000 g at 32 °C with the retroviral supernatant. The ratio of transduction was 1 x 10⁶ cells with 400 pl of viral supernatant. Starting from the third day of culture, the cells were maintained at a concentration of 0.5 x 10⁶/ml with the addition of human IL-15 and human IL-7 at 10 ng/ml. The cells were manually counted over time with Neubauer chamber with trypan blue dilution to distinguish live and dead cells. Alternatively, cells were counted with NucleoCounter®200 (Chemometec) counting machine to automatically assess cell number, viability and size (in pM). Population doubling level was calculated as Log (number of cells counted/number of cells previously seeded). [0164] To transduce primary murine T cells to co-express TTS and SLC1A5(L)/(S), two rounds of transduction were performed respectively 24 h and 48 h upon activation with a mixture of the two concentrated virus particles (1 x 10⁶ cells with 200 pl of TTS and 200 pl of SLC1A5(L)/(S) transgene viral particles).

[0165] Prior to all the in vitro and in vivo experiments, transporter expressing T cells were normalized for equivalent cell surface expression.

[0166] Murine cell lines

[0167] B16 cell line was purchased from ATCC (ATCC CRL-6475) and genetically manipulated to express OVA peptide presented on MHC class I molecules. B16 wt and OVA cells were kept in culture Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% HI FBS and 1% P/S and kept at a maximum of 80% confluency. B16-OVA cell lines were genetically manipulated to express murine indoleamine-pyrrole 2,3-dioxygenase 1 (IDO-1) by incubating 2.5 x 10⁵ cells with either 1.5, 0.75, 0.3 ml of viral supernatant to generate Bl 6-0 VA expressing IDO-1 at high, medium and low levels. Three days later, transduced cells were selected with 0.1 pg/ml of Puromycin and expanded.

[0168] Intracellular and extracellular staining for flow cytometry

[0169] The cells were collected in a 96 well V shaped bottom plate (3849, Coming), washed with Facs Buffer (PBS with the addition of 2% HI FBS and 2mM of EDTA - 15575020 ThermoFisher Scientific) and incubated with the antibody mixture on ice at 4°C for 30 min. The antibodies used were anti-murine CD3 (clone 10A7), anti -murine CD62L phycoerythrin (PE) (clone Mel 14, eBioscience), anti- murine CD44 allophycocyanin (APC) (clone A20), antimurine PD1 Brilliant Violent 711 (135231, Biolegend), anti- murine TIM3 Pacific Blue (119723, Biolegend), anti- murine LAG3 PercpCy5.5(125226, Biolegend), , anti-Thyl. l APC (), anti- CD45.1 APC (clone A20). For distinguishing the live versus dead cells, the Live/dead Fixable Aqua dead staining kit (L34957, ThermoFisher Scientific) and Live/Dead fixable near red kitl (L10119, ThermoFisher Scientific). For degranulation assay, anti-CD107a conjugated with fluorochrome PE (12-1079-41, ThermoFisher) was used.

[0170] For intracellular staining, the cells were fixed and permeabilized for 30 min at RT with the Fix/Perm buffer set kit according to the manufacturer protocol (88-8824-00, eBioscience) and stained with the antibody mixture resuspended in permeabilization buffer on ice at 4°C for 30 min. The antibodies used were anti-FLAG APC (MCA4764A647, Biorad), anti-murine fFNy PercpCy5.5, anti-murine TNFa FITC, Bodipy 493/503 (D3992, Thermofisher), anti-Mcll (12- 9047-41, Invitrogen), anti-murine TCF1 (2203S, Bioconcept) and secondary anti-rabbit (4412S, Bioconcept), anti-murine TOX (130-120-716, Miltenyi Biotec GmbH), and secondary anti-rabbit Alexa Fluor 488 (4412, Cell Signaling). For pGSK3b flow staining the Fix Buffer I (557870, BD), Phospho Flow Perm Buffer III (558050, BD) and Phospho-GSK-3P (Ser9) (5558, Cell Signaling).

[0171] For Annexin V/7AAD staining the Apoptosis detection kit was used (640930, Biolegend) according to the manufacturer technical suggestions. For cell proliferation the Cell Trace Violent (c34557, Invitrogen) reagent was used according to the manufacturer protocol.

For mitochondrial staining, Tetramethylrhodamine-Methyl Ester-Perchl orate (TMRM) (T668, Invotrogen) and Mitotracker Green FM (M7514, Invitrogen) were incubated in cell cultures for 30 minutes at 37C. For oxidative stress detection, Cell ROX Green reagent (C10444, Invitrogen) was used as per manufacturer indications.

The stained samples were kept on ice and acquired with the LSRII or Canto machines at the UNIL Flow Cytometry Facility.

[0172] T cells stimulation

[0173] 1 x 10⁵ T primary murine OT-1 T cells were stimulated by co-culture with target cell lines (B16-OVA, B16-OVA IDO⁺) at 1 : 1 ratio. Where indicated, OT-1 T cells were stimulated with the addition in culture of SIINFEKL peptide (SEQ ID NO:23) of Ovalbumin (OVA) at 0.1 pg/ml. Upon 24 h and 48 h of co-culture or peptide stimulation, 100 pl of cell culture supernatant were collected and frozen at -80 °C for cytokines release analysis.

[0174] Proliferation assessment with CSFE trace assay

[0175] B16-OVA and L1210 cell lines were lethally irradiated with 33 Gray (Gy) with a ¹³⁷Caesium source irradiator (LISA-1) according to safety protocols. 1 x 10⁵ T cells were stained with Carboxyfluorescein succinimidyl ester (CFSE), according to the manufacturer suggested protocol, and seeded with 1 x 10⁵ irradiated tumor cell line. Upon 24 h and/or 48 h of co-culture, T cells proliferation analysis was evaluated by CFSE dilution analysis with flow cytometry. [0176] Enzyme-Linked Immunosorbent Assay (ELISA) for cytokine detection

[0177] In the human setting, 5 x 10⁴ primary UTD and transduced T cells were co-cultured with 5* 10⁴ target cells per well in 96-well round bottom plates, in duplicate, in a final volume of 200 pL RPMI media. After 24 h, the co-culture supernatants were harvested and tested for presence of IFN-y. In the murine setting, 1 x 10⁵ OT-1 T cells were co-cultured with 1 x 10⁵ B16-OVA cell line for 24 h or with OVA peptide (0.1 pg/ml) ON and 100 pl of supernatant were collected and kept at -80 °C until the day of analysis IFNg and TNFa released levels. Upon appropriate dilution (1/300-1000), the collected supernatant was analyzed with ELISA according to the manufacturer protocol.

[0178] Cytotoxicity with IncuCyte System

[0179] Cytotoxicity assays were performed using the IncuCyte System (Essen Bioscience). Briefly, 1.5 x 10⁴ target cells were seeded 18 h before the co-culture set up, in flat bottom 96 well plates. The following day, rested T cells (no cytokine addition for 48 h) were counted and seeded at 3xl0⁴ cells/well, at a ratio 1 :2 = target: T cells in complete media. Cytotox Red reagent (Essen Bioscience) was added at a final concentration of 125 nM in a total volume of 200pl. The total number of red cells or total red area/well was obtained by using the software provided by the Incucyte manufacturer.

[0180] Radiolabeled CH) amino acids uptake

[0181] To measure the uptake of ³H glutamine and ³H tryptophan, 0.25 x 10⁶ wild -type, and Slcla5(L) and Slcla5(S) gene-engineered T cells, previously stimulated ON with OVA peptide (0.1 pg/ml) were resuspended in 50 pl of HBSS Buffer in a 96 well V shaped bottom plate. A solution at 10 pCi of either ³H Gin and ³H Trp was added to the cells suspension for a final concentration of 5 pCi and incubated for 10 min at RT. In addition, 10 mmol/L BenSer inhibitor was added as internal control. The samples were then washed for 3 times at 1800 rpm for 3 min and lysed with 50 pl solution at 1% Triton for few min. 40 pl of cell lysate were added to 160 pl of MicroScint-40 (cocktail for liquid scintillation counting) and transferred in a 96 well white flat bottom plate ready for the acquisition at the scintillation reader. [0182] Glucose uptake

[0183] 2-(N-(7-Nitrobenz-2-oxa-l,3-diazol-4-yl) Amino)-2-Deoxyglucose (2NBDG) is a fluorescent analogue of glucose which was used to measure the glucose uptake by OT-1 T cells transduced with mock vector or with GLUT3. 5 x 10⁵ T cells were incubated for 30 min in glucose free media (12633-012, Invitrogen) to normalize the Glc uptake rate across different samples, then incubated with 0.2 mM of 2NBDG resuspended in 100 pl of Glc free media for 30 min at 37 °C. Upon extensive washes with PBS the cells were analyzed with flow cytometry for green fluorescence (FL-1 channel). Data are presented as mean fluorescence intensity (MFI). Alternatively, the glucose uptake luminescence-based assay was used as per the manufacturer instructions (JI 341, Promega).

[0184] Glycogen and glucose quanti fication

[0185] Intracellular glycogen levels were measured using the glycogen colorimetric assay kit (K648, Biovision) following the manufacturer’s instructions. Briefly, cells were homogenized in water on ice, boiled for 10 min, spun at 18000 g for 10 min and supernatants were analyzed for glycogen content. Results were normalized by cell number. For intracellular glucose content in T cells, the background values obtained with the glycogen colorimetric assay kit (K648, Biovision) are shown. For tumor cell lines, glucose levels in the supernatant were measured using the glucose assay kit (ab6533, Abeam) according to the manufacturer’s directions.

[0186] Periodic Acid Schiff Staining

[0187] A suspension of 1-5 x 10⁵ T cells was seeded on a poly-L Lysine slides, fixed with 4% paraformaldehyde, incubated in solution of periodic acid (100524, Merck) 1% for 5 minutes at room temperature, rinsed in distilled water and dipped in Schiff reagent (109033, Merck) for 20 minutes. The slides were than stained with Harris’s Hematoxylin (HHS16, Sigma Aldrich). The control slides were treated with a solution of amylase 0.1% (10080, Fluka) for 30 minutes at 37 °C prior to the staining. The stainings were performed at the Pathology Institute of the University of Lausanne. The slides were scanned with a Nanozoomer Slide Scanner at 40 x magnification with NDP view 2 software. The pictures were analyzed with a customized macro (Area/ min and max gray value/ Mean Gray value/ Limit to threshold/ display label/ Add to overlay) applied on FIJI software [0188] In vivo experiments with syngeneic tumor models

[0189] C57B1/6 CD45.2 females were purchased from Harlan Laboratories and housed in a dedicated animal facility. 1 x 10⁵ B16-OVA IDO⁺ cells were subcutaneously injected in the flank of 8 weeks old animals. Once the tumor was palpable after 7 days either the ACT with intravenous injection of the indicated T cell numbers was performed, or the mice were irradiated with 5Gy prior to ACT. The second round of ACT was performed at the days indicated in the text. The tumor growth was then monitored over time by calipering.

[0190] During experimentation, all animals were monitored at least every other day for signs of distress. Mice were euthanized at end-point by carbon dioxide overdose and, where indicated, tumors, spleens and peripheral blood were collected.

[0191] Ex vivo analysis of syngeneic tumor models

[0192] At terminal point solid tumor mass was excised from the mice, weighted, cut into small pieces with a scalpel, passed through 40 mm pore cell strainers and centrifuged for 5 min at 1500 rpm to pellet the cells. The cells were then resuspended in 3 ml of PBS and a Ficoll was performed to eliminate dead cells and tumoral debris with a centrifugation at 2000 rpm for 20 min with acceleration 2 and brake 7. Upon extensive washes, the obtained cells were stained with anti-CD45.1 APC (clone A20) Ab to assess the adoptively transferred T cells infiltration. Additionally, the spleens of the mice were also resected, smashed and passed through 40 mm pore cell strainers. Upon red blood cells lysis, the cells were stained with anti-CD45.1 APC Ab and analyzed by flow cytometry. Moreover, peripheral blood of the animals was obtained, and stained with same Ab upon red blood cells lysis.

[0193] Cell lines and culture conditions

[0194] Ovalbumin (OVA)-expressing B16-F10 murine melanoma (B16.OVA) cell lines were cultured in Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 pg/mL streptomycin sulfate and routinely tested for mycoplasma contamination. Primary murine T cells were cultured in RPMI 1640-Glutamax media supplemented with 10% FBS, 100 U/mL penicillin, 100 pg/mL streptomycin sulfate, 1 mM Pyruvate, 50 pM P-mercaptoethanol (T cell medium) and cytokines as described in the experiments. [0195] Transmission Electron Microscopy

[0196] Electron microscopy pictures were taken at the Electron Microscopy Facility at University of Lausanne. Briefly, the cells were fixed in glutaraldehyde solution (EMS, Hatfield, PA) 2.5% and in osmium tetroxide 1% (EMS) with 1.5% of potassium ferrocyanide (Sigma, St. Louis, MO) in phosphate buffer (PB 0.1 M [pH 7.4]) for 1 h at RT. The cells were then washed with water and embedded in agarose (Sigma, St Louis, MO, US) 2% in water, dehydrated in acetone solution (Sigma, St Louis, MO, US) at graded concentrations (30%-40 min; 70% - 40 min; 100% - 2xlh), infiltrated in Epon resin (EMS, Hatfield, PA, US) at graded concentrations (Epon 33% in acetone-2h; Epon 66% in acetone-4h; Epon 100%-2x8h) and finally polymerized for 48h at 60°C in an oven. Ultrathin sections of 50 nm thick were cut using a Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria), picked up on a copper slot grid 2x1 mm (EMS, Hatfield, PA, US) coated with a polystyrene film (Sigma, St Louis, MO, US). The sections were than stained with uranyl acetate (Sigma, St Louis, MO, US) 4% in H2O for 10 min, rinsed several times with water followed by Reynolds lead citrate in water (Sigma, St Louis, MO, US) for 10 min and rinsed several times with water. Micrographs were taken with a transmission electron microscope FEI CM 100 (FEI, Eindhoven, The Netherlands) at an acceleration voltage of 80kV with a TVIPS TemCamF416 digital camera (TVIPS GmbH, Gauting, Germany).

[0197] Mouse strains

[0198] Mice were housed at an animal facility. All in vivo experiments were conducted in accordance and approval from the Service of Consumer and Veterinary Affairs (SCAV) of the Canton of Vaud. Female C57BL/6 mice aged 6-8 weeks were purchased from Harlan (Harlan, Netherlands) and CD45.U OT-1 TCR mice (C57BL/6-Tg(TcraTcrb)l lOOMjb/J) were bred inhouse.

[0199] Tumor inoculation and adoptive T cell transfer

[0200] C57BL/6 mice (typically aged 8 weeks) were injected subcutaneously with 1 x 10⁵

B16.OVA tumor cells. Once the tumors were palpable (day 7), the mice received 5 Gy of sub- lethal total body irradiation (TBI) and were grouped (n > 5 mice/group). The mice were then treated by intravenous transfer of 0.5-2 x 10⁶ CD8⁺CD45.1⁺OT-1 T cells, gene-engineered or not to over-express GLUT3. Some mice received a second T cell transfer (as indicated, but typically 2-3 days later). For rechallenge experiments, 1 x 10⁵ B16-OVA IDO+ cells were subcutaneously injected in the left flank of the mice. Mice were carefully monitored, and tumor length (L; greatest longitudinal measurement) and width (W; greatest transverse measurement) were measured by caliper every 2-3 days. Tumor volumes (V) were calculated using the formula: V = (L x W2)/2. Mice were sacrificed once tumors reached 1000 mm³, or according to regulations if they became distressed or moribund.

[0201] Statistical Analysis

[0202] Student t test or Mann-Whitney u test were used to evaluate differences among cell types population doubling level, cytokine release, antigen expression, killing capacity. Two-way ANOVA with post-hoc Turkey test was used to evaluate significant differences in tumor growth in vivo. All the statistical analysis were performed with GraphPad Prism 4.0 (GraphPad Software). Starting from p< 0.05 the difference was considered significant.

[0203] Example 1: Generation of OT-1 T cells genetically engineered to overexpress a functional glucose transporter

[0204] To demonstrate that T cells can be engineered to preferentially use glucose as a primary energy source, the codon-optimized sequence of the high affinity glucose transporter GLUT3 was cloned in an MSGV based retroviral vector with a FLAG tag at the N-terminus for transduction assessment. Primary OT-1 T cells were successfully transduced to overexpress GLUT3 with an average expression level of 45% ± 8.4 SD in six different experiments as evaluated with an anti -FLAG antibody. OT-1 T cells express a TCR that recognizes ovalbumin peptide residues 257-264 (OVA257-264) in the context of a CD8 co-receptor interaction with MHC class I.

[0205] Experiments were performed to evaluate the GLUT3 overexpression with an anti- GLUT3 specific antibody, confirming that GLUT3 -transduced T cells showed higher expression of the transporter (29.9% ± 3.4 SD) as compared to cells transduced with a control vector without transgene (15.1% ± 4.5 SD vs, p=0.002 comparing GLUT3- and mock-transduced cells with student t test). The functionality of the overexpressed transporter was evaluated through the incorporation of the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-l,3-diazol-4-yl) Amino)-2 -Deoxy glucose (2-NBDG) with in presence or absence of the glucose competitor 2- Deoxyglucose (2DG). GLUT3 -transduced T cells showed higher 2-NBDG uptake as compared to mock-transduced T cells (a 1.422 ± 0.16 SD fold change), indicating that the overexpressed GLUT3 is functional.

[0206] Overexpression of GLUT3 did not affect cell viability, proliferation, apoptotic levels, cell size, level of degranulation, and cytotoxic capacity against Bl 6-0 VA tumor cells (data not shown).

[0207] GLUT3 expressing cells exhibited a glycolytic phenotype. Specifically, GLUT3- transduced T cells had higher basal extracellular acidification rate (ECAR) (33.7 mPH/min ± 2.45 SD) compared to both mock-transduced T cells (20.2 mPH/min ± 7.42 SD) and untransduced T cells (22.6 mPH/min ± 8 SD). GLUT3 -transduced T cells has also a lower glycolytic reserve (1.84 ± 0.41 SD) compared to mock-transduced T cells (2.3 ± 0.46) and untransduced T cells (2.76 ± 0.08). Moreover, GLUT3 -transduced T cells showed no spare respiratory capacity (SRC) (-34.7 ± 4.9 SD) in contrast to mock-transduced T cells (88.8 ± 23 SD) and untransduced T cells (85.6 ± 48.1 SD). When stimulated with OVA peptide, GLUT3- transduced T cells exhibited a similar glycolytic switch (glucose consumption as measured by determining the extracellular acidification rate) compared to mock T cells. Finally, when analyzing the amino acids levels remaining in the cell culture supernatant upon 9 days of culture, a tendency of higher concentration of Gin, Asn, Ser, Thr, Leu, He, Phe and Trp remaining in the cell culture supernatants of GLUT3 -transduced T cells compared to mock-transduced and untransduced T cells was observed, indicating a preference for the usage of glucose instead of amino acids as primary energy source for the GLUT3 -transduced T cells.

[0208] Next, the ability of GLUT3 -transduced OT-1 T cells to promote long-term anti-tumor protection was examined in a mouse model. For this experiment, OT-1 CD45.1 T cells were isolated, stimulated and transduced to overexpress the GLUT3 transporter. CD45.2 mice were injected in their right flank with 10⁵ B16OVA IDO cells. After 7 days, the tumors were palpable and the mice were irradiated with a low dose of x-rays (5Gy). The next day, the mice were treated with an intravenous injection of 0.5-2 x 10⁶ gene manipulated OT-1 T cells and the tumor growth was monitored over time. Upon tumor treatment, the cured mice were rechallenged with an injection in their left flank of 10⁵ B16OVA IDO cells. Tumor growth was monitored over time. [0209] GLUT-3-overexpressing OT-1 T cells completely eliminated tumors in 25% of the mice in two separate experiments (n=8 for each experiment) (Fig. 1A). The surviving four mice were re-challenged with an additional tumor injection in the left flank as indicated in Fig. 1A. Only one of the four mouse that were re-challenged relapsed 22 days after the re-challenge (Fig. IB). As a control, naive control mice were also injected with tumor cells in their left flank and developed tumors as expected (Fig. 1C). After a total of 50 days from the re-challenge in the left flank, the three survivor mice were sacrificed the absence of tumors in both sides was visually confirmed. The persistence of OT-1 T cells was confirmed in both spleens and draining lymph nodes (axillar and inguinal) (Fig. ID). The persisting OT-1 T cells maintained the expression of GLUT3 (Fig. IE) and cytokine (IFNy) production upon stimulation with OVA peptide (Fig. IF).

[0210] This data demonstrates that adoptive T cell transfer of GLUT3 -overexpressing OT-1 T cell in tumor bearing mice promotes long-term anti-tumor protection.

[0211] Example 2: Central memory GLUT3-T cells demonstrate superior survival upon glucose deprivation and a less exhausted phenotype under conditions of stress

[0212] Murine CD8⁺ T-cell expansion in the presence of the common gamma chain cytokines IL-7 and IL- 15 (OTI T cells isolation and stimulation with CD3/CD28 beads and 50 lU/ml of IL- 2, followed by transduction with viral constructs on day L followed by culturing in presence of 10 ng/ml of IL-7 and IL- 15), as compared to IL-2 only, generates a greater proportion of central memory T cells (TCM; CD44⁺ CD62L⁺) characterized by superior expansion and cytokine production levels. Given the higher intake of glucose by GLUT3 -engineered T cells, it was examined whether this would limit the generation of TCM cells under our IL-7/IL-15 expansion protocol. To evaluate this, OTI T cells were transduced to express GLUT3 (Fig.2A) or control vector Thy 1.1 (Fig. 2B) and expanded in the presence of IL-7/IL-15. No differences were observed in the viability (Fig. 2C), cell size (Fig. 2D), population doubling over time (Fig. 2E), or proportion of TCM phenotype (CD44⁺ CD62L⁺, Fig. 2F) for GLUT-3- versus MOCK-T cells expanded in the presence of IL-7/IL-15.

[0213] As for GLUT3-TEM cells, GLUT3-TCM (central memory) cells showed increased levels of glucose uptake as compared to the MOCK-TCM cells, and this was inhibited in the presence of 2-DG (Figs. 2H and 21). A higher basal extracellular acidification rate (ECAR) was observed for the GLUT3-TCM cells upon metabolic flux analysis with the Seahorse XF analyzer (Fig. 2 J), indicative of higher lactate secretion resulting from greater levels of glycolysis than for the MOCK-TCM cells. Increased energy stores acquired by GLUT3-T cells may support their viability. Indeed, by Annexin V and 7AAD staining a significantly lower proportion of necrotic GLUT3- than MOCK-T cells was observed after 48 and 72 hours in the presence of glucose, and even more pronounced in the absence of glucose (Fig. 2G and Fig. 2K). No differences in cytotoxicity of GLUT3- versus MOCK-TCM T cells were observed as measured by both an Incucyte target killing assay, % CD 107a upregulation, or IFNg release (data not shown).

Notably, however, under conditions of stress (daily stimulation with the SIINFEKL peptide (SEQ ID NO:23) over 5 days), significantly lower expression levels of PD-1 and LAG-3 was observed by GLUT3-T cells, while there were no significant differences in TIM-3 expression levels (Fig. 2L). The GLUT3-T cells also maintained higher expression levels of the transcription factor TCF-1 (Fig. 2L), but there were no differences in TOX expression (data not shown).

[0214] In sum, this data indicates that GLUT3-T cells are less susceptible to necrosis and to exhaustion than MOCK-T cells.

[0215] Example 3: Central memory GLUT3-T cells confer enhanced tumor control and survival

[0216] While T effector cells depend heavily on aerobic glycolysis to satisfy their energy needs, quiescent memory T cells primarily rely on OXPHOS to generate ATP for energy. To produce “chimeric” cells, which exhibit a glycolytic metabolism in combination with a memory phenotype, naive T cells isolated from a murine spleen were stimulated with IL2 and CD3/CD28 beads for one day. In the next day, the cells were transduced with a vector encoding GLUT3, a high affinity glucose transporter, cultivated for 2 days IL-2 and, starting on day 3, exposed to IL- 15 and IL-7.

[0217] To increase stringency, the B16-OVA tumor cell line was genetically modified to express the immunosuppressive enzyme indoleamine 2-3 dioxygenase- 1 (IDO-1) as it has been previously been demonstrated to inhibit glucose uptake by T cells. Indeed, a trend for higher glucose consumption by B16-OVA IDO⁺ tumor cells than B16-OVA tumor cells was observed (Fig. 3A), but both tumor-types grew at a similar rate upon subcutaneous injection in mice (Fig.

3B)

[0218] In a first experiment, C57B1/6 females were subcutaneously injected with B16-OVA IDO⁺ tumors and irradiated. Myelo-ablative total body irradiation facilitates the engraftment of the transferred T cells. On days 8 and 11 after tumor injection, the mice received were injected with 1 x 10⁶ (for a total of 2 x 10⁶ ) untransduced OT-1 T cells, GLUT3 transduced OT-1 T cells, or T cells transfected with a control vector not encoding for any transgene (mock) (Fig. 3C). Tumor growth was monitored over time and survival was evaluated.

[0219] At day 45 after tumor injection, 80% of the mice that received GLUT3 -transduced T cells had survived, compared to 20% of the mice that had received untransduced T cells (but treated with IL-7 and IL- 15) or mock (Fig. 3D). Moreover, treatment of tumor-bearing mice with GLUT3 -transduced T cells efficiently controlled the tumor growth of three out of five mice (the tumor volume was below 200 mm³ after 36 days) (Fig. 3E).

[0220] In a second experiment, B16-OVA IDO⁺ tumor-bearing mice were treated by 5 Gy total body irradiation (day 7), and then treated the next day by intravenous injection with a significantly smaller amount of 5 x 10⁵ T cells and monitored over time. A significant improvement in tumor control by OT-1 TCR GLUT3-T cells was observed as compared to OT-1 TCR MOCK-T cells (Fig. 4A). There was also a significant increase in survival for GLUT3-T cell treated mice (Fig. 4B), with 25% of the mice alive and apparently cured at day 110.

[0221] Under the same experimental conditions, the tumor infiltrating lymphocytes (TILs) were characterized on day 30 post-tumor injection. A trend of higher TMRM/MG ratio (Fig.

4C), and of higher IFNg (Fig. 4D), and TNFa (Fig. 4E) expression was observed upon in vitro restimulation by GLUT3-TILs than MOCK-TILs.

[0222] From two separate in vivo studies, surviving mice (2/8 from each, Fig. 4F) were rechallenged. For this, the 4 survivor mice, together with 3 control naive mice, received an injection in the left flank of 1 x 10⁵ B16-OVA IDO+ cells. Mouse health and tumor growth were monitored over time. As a control, previously untreated mice received tumor cell injections.

Remarkably, only 1 of 4 re-challenged mice relapsed (starting on day 22, Fig. 4G) indicative of a strong memory recall by GLUT3-T cells. At day 50 post-rechallenge the mice were sacrificed, and autopsy revealed no tumor presence on either flank. Persistence of the transferred GLUT3-T cells were confirmed in both the spleen and draining lymph nodes (axillar and inguinal) of the mice surviving rechallenge (Fig. 4H). Further, it was shown that the T cells continued to express the transgene (data not shown). Together these data demonstrate superior tumor control by GLUT3-T cells, a 25% cure rate and 75% protection from re-challenge, indicative of a strong memory recall response.

[0223] Example 4: GLUT3 overexpression promotes glucose uptake and energy storage by effector T cells

[0224] Retroviral vectors encoding the Thy 1.1 marker (control T cells) were built, as well as codon-optimized GLUT3 including a Flag tag. Splenic derived OT1 CD8⁺ T cells were anti- CD3/CD28 bead-activated in presence of 200 lU/ml of IL2, transduced the following day, and then expanded in the presence of 200 lU/ml of IL-2 for 9-12 days to generate predominantly effector T cells GLUT3-TEM (effector memory) (CD44⁺ CD62L"). Transduction efficiencies for GLUT3 (GLUT3-T cells) were quite variable amongst donors, ranging from as low as 10% to over 60% as measured by Flag tag staining and flow cytometric analysis (Fig. 5A) as compared to for Thy 1.1, at -40-60% transduction efficiency (MOCK-T cells, data not shown) . There were no differences in viability amongst the transduced T cells (Fig. 5B), nor were there differences in phenotype following expansion (Fig. 5C).

[0225] Subsequently, the cells were cultured in the presence of fluorescently labeled glucose analog, 2-(N-(7-Nitrobenz-2-oxa-l,3-diazol- 4-yl)Amino)-2-Deoxyglucose (2-NBDG), as an indicator for glucose uptake by the MOCK- versus GLUT3-T cells. As shown in Fig. 5D, 2- NBDG uptake was significantly higher for GLUT3- T cells and this effect could be blocked in the presence of 2 -Deoxy -D-glucose (2-DG). Higher glucose uptake was shown through a second luminescence-based method (Fig. 5E). Due to the high variability in transduction efficiency for GLUT3 expression (Fig. 5A), the data for GLUT3-T cells were normalized with respect to the MOCK-T cells generated from the same donor.

[0226] Further, a direct significant fold-change increase in glucose cell content for GLUT3- versus MOCK-T cells was observed (Fig. 6A). Additionally, an increase in glycogen content (i.e., energy storage) for GLUT3-T cells was observed as evaluated by both direct analysis of cell lysates (Fig. 6B) and periodic acid shiff (PAS) immunohistochemical staining (Fig 6C). As a PAS staining control, the engineered T cells were treated with amylase to disrupt glycogen. The increased glycogen content in GLUT3 was further confirmed with transmission electron microscopy (data not shown).

[0227] The levels of phosphorylated glycogen synthase kinase-3 p (pGSK-3P) were examined, a serine/threonine protein kinase responsible for the phosphorylation and consequent inactivation of glycogen synthase (GS): the signaling activity of GSK-3P is inhibited upon phosphorylation at the Ser9 level, liberating GS from its inhibition. Higher levels of pGSK-3p were observed in GLUT3 overexpressing T cells, which is a further indication of increased production of glycogen (Fig. 6D). Notably, glycogen content of the GLUT3-T cells was directly proportional to transduction efficiency (Fig. 6E). As glucose can also be converted to fatty acids, Bodipy 493/503 staining was performed. A significant fold-change increase in MFI for GLUT3- T cells was observed (Fig. 6F). Notably, GLUT3 overexpression was further associated with a significant increase in the diameter of the T cells (Fig. 6G), despite no significant difference in phenotype from MOCK-T cells (Fig. 5C).

[0228] In summary, OT1 T cells engineered to overexpress the GLUT3 transporter have increased glucose uptake, as well as higher levels of both glycogen and fatty acid content.

[0229] Example 5: Enforced GLUT3 expression by CD8⁺ T cells is associated with increased mitochondrial polarization and higher effector function in vitro

[0230] Next, the in vitro fitness of effector GLUT3-T cells was assessed. First, the mitochondrial membrane potential and mass was assessed by Tetramethylrhodamine-methyl ester (TMRM) and Mitrotracker Green (MG) staining, respectively. GLUT3 overexpression was associated with an increase in the proportion of TMRM^hlgh/MG^hlgh T cells (Fig. 7A). Notably, this increased proportion of TMRM^hlgl7MG^hlgh T cells upon GLUT3 overexpression was not observed in the presence of 2-DG (/.< ., upon competition for glucose uptake, Fig. 7B). Glucose depletion induced a drop in TMRM/ MG ratio significantly higher for MOCK-T cells, but there was no significant change for GLUT3-T cells (Fig. 7C).

[0231] CD8⁺ memory T cells engage glycogen metabolism to support their memory status maintenance, particularly decreasing the levels of reactive oxygen species (ROS). Consistent with their higher glycogen content, GLUT3-T cells showed reduced levels of ROS (Fig. 7D). [0232] Finally, increased glucose metabolism stabilizes the antiapoptotic Bcl-2 family protein Mcl-1 and increases the threshold for cell death in primary lymphocytes. Similarly, it was observed that GLUT3-T cells, together with their improved glucose utilization, showed higher levels of Mcl-1 (Fig. 7E).

[0233] Next, effector function was assessed by stimulating the engineered T cells overnight with the ovalbumin SIINFEKL peptide (SEQ ID NO:23) and measured IFNg and TNFa production in the presence of increasing concentrations of glucose (no Glc;0mM, low Glc;0.444mM, complete media; 1 l.lmM). A higher production of both IFNg and TNFa by GLUT3-T cells was observed in all experimental conditions tested (Fig. 7F). In addition, the GLUT3-T cells demonstrated significantly higher proliferative capacity upon 5 days of coculture with B16-OVA tumor cells cultured in low glucose media, and a trend for higher proliferation in no glucose and complete media (Fig. 7G).

[0234] In sum, it was shown that over-expression of GLUT3 not only promotes glucose uptake and higher energy storage, but also sustains higher mitochondrial polarization and effector function.

[0235] Example 6: Lymphocytes genetically modified to express a glucose transporter and exhibiting an effector cell phenotype reduce tumor growth in vivo

[0236] Next, the ability of GLUT3 -overexpressing T cells to reduce tumor growth in vivo was determined for T cells exhibiting an effector phenotype (Examples 4-6) or a memory phenotype (Examples 2-3) at the moment of the adoptive transfer. Cells were cultivated in the presence of high doses of IL-2 (200 lU/ml). The cells were cultured for up to 12 days in presence of IL-2 at 200 lU/ml, The media was refreshed every other day with the addition of fresh IL-2 and maintained at a cells concentration of 0.5-1 x 10⁶ cells/ml. Effector OT-1 T cells were successfully transduced to overexpress GLUT3. The genetic manipulation did not affect the viability (Fig. 8A) and the population doubling levels (Fig. 8B) of the cells as compared to control cells. 1 x 10⁵ B16-OVA IDO⁺ cells were subcutaneously injected in the flank of C5B1/6 females (n= 6 mice/group). The mice were irradiated (5Gy) once the tumor was palpable and treated with intravenous ACT of a total of 0.5 x 10⁶ OT-1 mock and GLUT3 -transduced T cells that had been cultured in presence of IL-7 and IL- 15 to generate a memory phenotype (Examples 2-3), or with high doses of IL-2 to generate an effector phenotype (Examples 4-6). A trend for improved control of tumor growth could be shown for both types of GLUT3 overexpressing T cells as compared to the controls. (Fig. 8D). [0237] GLUT3 -overexpressing effector T cells showed a significantly higher increase in IFNy production upon stimulation with an anti-CD3 antibody for 48 h in media comprising a low concentration of glucose as compared to complete media (Fig. 8C), demonstrating that the overexpression of GLUT3 confers an advantage under low glucose conditions.

[0238] In sum, it was shown that T cells exhibiting an effector phenotype or a memory phenotype at the moment of the adoptive transfer reduced tumor growth in vivo.

[0239] Example 7: Lymphocytes genetically engineered to co-express of a tryptophan transporter and a tryptophanyl-tRNA synthetase promote a significant reduction of tumor growth

[0240] Next, it was demonstrated that T cells expressing both a tryptophan transporter and tryptophanyl-tRNA synthetase have a functional and survival advantage in presence of IDO- 1 (which converts tryptophan into kynurenine and causes Trp depletion) within the context of an immune-suppressive TME.

[0241] The full length (2873 bp, coding region, 541 amino acids, NCBI Reference Sequence NM_005628.3, encodes SEQ ID NO:5) and spliced short variant (1737 bp, coding region 313 amino acids, NCBI Reference Sequence NM 001145144.2, encodes SEQ ID NO:6) of the amino acid transporter SLC1A5 was cloned in a retroviral MSGV derived backbone tagged with the V5 sequence at the C terminal part of the transporters.

[0242] Primary OT-1 T cells were efficiently transduced as evaluated with a V5 antibody and by the increased uptake of Gin and Trp. The co-transduction did not affect the viability, the ratio of memory/effector cells in the culture, or the population doubling level of the cells (data not shown).

[0243] The therapeutic potential of memory OT-1 T cells expressing SLC1 A5 and tryptophanyl-tRNA synthetase (TTS) was evaluated in mice bearing Bl 6-0 VA IDO⁺ tumors. C57B1/6 CD45.2 females were subcutaneously injected with B16-OVA IDO⁺ cell line, and once the tumor was palpable the animals were irradiated to favor the engraftment of the T cells. On days 8 and 11 after the tumor injection, mice were injected with 1 x 10⁶ per injection of (1) OT-1 untransduced T cells (UTD), (2) T cells transduced with a control vector without transgene (mock), (3) T cells engineered to express tryptophanyl-tRNA synthetase (TTS), (4) T cells engineered to express the full length form of tryptophan transporter SLC1 A5 (SLC1 A5(L), (5) T cells engineered to express a truncated form of the tryptophan transporter SLC1 A5 (SLC1 A5(S)), (6) T cells engineered to express full length tryptophan transporter and tryptophanyl -tRN A synthetase (SLC1A5(L) + TTS), (7) T cells engineered to express the truncated form of the tryptophan transporter SLC1A5 (SLC1A5(S) + TTS), and (8) saline as a control (Fig. 9A).

[0244] T cells expressing both the full length tryptophan transporter and tryptophanyl-tRNA synthetase were more effective in controlling tumor growth than T cells expressing the length tryptophan transporter or the tryptophanyl-tRNA synthetase alone: 40 days after tumor injection, four of five mice showed tumor volume below 200 mm³ (one unexpectedly died shortly after the beginning of the experiment) (Fig. 9B). T cells expressing the full-length version of the tryptophan transporter performed better than T cells expressing the truncated transporter protein. Calculation of the tumor growth rate by comparing the ratio of the tumor volume calipered at day 40 after tumor challenge compared to the beginning of the experiment (day 7) showed that a striking and more potent tumor control was provided by T cells expressing both a tryptophan transporter and a tryptophanyl-tRNA synthetase compared to all the other treatments (Fig. 9C). Moreover, the animal survival was monitored up to 47 days and a visible improvement in percentage of surviving mice subjected to ACT with SLC1A5(L) + TTS was observed, with 4 out of 5 animals surviving when treated with the combinatorial strategy (Fig. 9D).

[0245] These results demonstrate that that lymphocytes genetically modified to co-express an amino acid transporter and the corresponding aminoacyl-tRNA synthetase are effective in reducing tumor growth and increasing survival in tumor-bearing mammals in vivo.

Claims

We claim:

1. A lymphocyte, wherein: the lymphocyte is genetically modified to express a glucose transporter; and the lymphocyte is in contact with one or more of IL-2, IL-7, IL-15, and IL-21.

2. The lymphocyte of claim 1, wherein the lymphocyte is in contact with IL-7 and IL-15.

3. The lymphocyte of any one of claims 1 or 2, wherein the lymphocyte is in contact with IL-21.

4. A method of generating a genetically modified lymphocyte, the method comprising: a. transforming the lymphocyte with a vector for the expression of a glucose transporter; b. cultivating the lymphocyte in the presence of one or more selected of the group consisting of IL-2, IL-7, IL-15, and IL-21.

5. The method of claim 4, wherein the lymphocyte is cultivated in the presence of IL-2.

6. The method of claims 4 or 5, wherein the lymphocyte is cultivated in the presence of IL-7 and IL-15.

7. The method of any one of claims 4-6, wherein the lymphocyte is cultivated in the presence of IL-21.

8. The method of claim 4, wherein the lymphocyte is first cultivated in IL-2 and subsequently cultivated in IL-7 and IL-15.

9. A pharmaceutical composition comprising:

(a) a lymphocyte genetically modified to express a glucose transporter, wherein the lymphocyte has been cultivated in the presence of one or more selected of the group consisting of IL-2, IL-7, IL-15, and IL-21, and

(b) a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein the lymphocyte has been cultivated in the presence of IL-2.

11. The pharmaceutical composition of any one of claims 9 or 10, wherein the lymphocyte has been cultivated in the presence of IL-7 and IL-15.

12. The pharmaceutical composition of any one of claims 9-11, wherein the lymphocyte has been cultivated in the presence of IL-21.

13. The pharmaceutical composition of claim 9, wherein the lymphocyte has been first cultivated in IL-2 and subsequently cultivated in IL-7 and IL-15. The lymphocyte of any one of claims 1-3, the method of any one of claims 4-8, or the pharmaceutical composition of any one of claims 9-13, wherein the glucose transporter is selected from the group consisting of SLC2A1, SLC2A2, SCL2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14. The lymphocyte, the method, or the pharmaceutical composition of claiml4, wherein the glucose transporter is SCL2A3 (GLUT3). The lymphocyte of any one of claims 1-3, the method of any one of claims 4-8, or the pharmaceutical composition of any one of claims 9-13, wherein the glucose transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:l-4. The lymphocyte, the method, or the pharmaceutical composition of claim 16, wherein the glucose transporter comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. The lymphocyte, the method, or the pharmaceutical composition of claim 17, wherein the glucose transporter comprises the amino acid sequence of SEQ ID NO: 1. A lymphocyte, wherein the lymphocyte is genetically modified to express an amino acid transporter and an aminoacyl-tRN A synthetase. The lymphocyte of claim 19, wherein the amino acid transporter is a tryptophan transporter and the aminoacyl-tRNA synthetase is a tryptophan-tRNA synthetase, The lymphocyte of claim 19, wherein the amino acid transporter is selected from the group consisting of SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7, and SLC7A5 The lymphocyte of claim 21, wherein the amino acid transporter is SLC1A5. The lymphocyte of any one of claims 19 or 20, wherein the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:5-10. The lymphocyte of claim 23, wherein the amino acid transporter comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs:5-7. The lymphocyte of claim 24, wherein the amino acid transporter comprises the amino acid sequence of any one of SEQ ID NOs:5-7. The lymphocyte of any of claims 19-25, wherein the aminoacyl-tRNA synthetase is selected from the group consisting of a cytoplasmic tryptophanyl-tRNA-synthetase and a mitochondrial tryptophanyl-tRNA-synthetase. The lymphocyte of claim 26, wherein the aminoacyl-tRNA synthetase is a cytoplasmic tryptophanyl-tRNA-synthetase. The lymphocyte of any of claims 19-25, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-22. The lymphocyte of claim 28, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-19. The lymphocyte of claim 29, wherein the aminoacyl-tRNA synthetase comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 11-12. The lymphocyte of claim 30, wherein the aminoacyl-tRNA synthetase comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. A pharmaceutical composition comprising

(a) the lymphocyte of any one of claims 1-3 or 14-31 and

(b) a pharmaceutically acceptable carrier. The lymphocyte of any one of claims 1-3 or 14-31, the method of any one of claims 4-8 or 14-18, or the pharmaceutical composition of any one of claims 9-18 or 32, wherein the lymphocyte further expresses a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The lymphocyte of any one of claims 1-3 or 14-31, the method of any one of claims 4-8 or 14-18, or the pharmaceutical composition of any one of claims 9-18 or 32, wherein the lymphocyte further expresses a therapeutic protein. The lymphocyte, the method, or the pharmaceutical composition of claim 34, wherein the therapeutic protein is selected from the group consisting of IL-2, IL-2 mutein, IL- 15, CD40L, IL-33, and IL-12. The lymphocyte of any one of claims 1-3 or 14-31, the method of any one of claims 4-8 or 14-18, or the pharmaceutical composition of any one of claims 9-18 or 32, wherein the lymphocyte further expresses a protein that inhibits the interaction of an immunosuppressive polypeptide with its ligand. The lymphocyte, the method, or the pharmaceutical composition of claim 36, wherein the lymphocyte expresses a protein that inhibits the interaction between PD-1 and PD- Ll. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 9-18 or 32-37. A method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 9-18 or 32-37. A method of reducing cancer sternness in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 9-18 or 32-37. A method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject of the pharmaceutical composition of any one of claims 9-18 or 32-37. A method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 9-18 or 32-37. The method according to any of claims 38-42, wherein the subject has one or more cancers selected from the group consisting of sarcoma, carcinoma, melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, ovarian, and gastric cancer. The method according to any of claims 38-43, the method further comprising administering to the patient an additional therapeutic agent. The method according to claim 44, wherein the additional therapeutic agent is a chemotherapeutic agent. The method according to claim 44, wherein the additional therapeutic agent is an immune checkpoint inhibitor. The lymphocyte of any one of claims 1-3, 14-31, or 33-37, the method of any one of claims 4-8, 14-18, or 33-46, or the pharmaceutical composition of any one of claims 9-18 or 32-37, wherein the lymphocyte is a T cell, a B cell or a natural killer cell. The lymphocyte or the method of claim 47, wherein the lymphocyte is a T cell.