Method for modifying T cells based on small molecules

Abstract

Disclosed herein is a method of modifying a T cell during T cell activation comprising contacting the T cell with an inhibitor of prolyl hydroxylase. Also disclosed is a method of producing a chimeric antigen receptor (CAR) or T cell receptor (TCR) T cell comprising (i) modifying the T cell during activation of the T cell by contacting the T cell with an inhibitor of prolyl hydroxylase, (i) introducing a CAR or TCR transgene into the modified T cell, and (iii) harvesting the CAR or TCR T cell. In particular, the inhibitor of prolyl hydroxylase is a small molecule prolyl hydroxylase inhibitor, such as 1, 4-DPCA. The harvested CAR or TCR T cells can be used for adoptive T cell immunotherapy of cancer.

Description

Cross Reference to Related Applications

[0001] This application claims priority to 10202301612V, filed June 7, 2023, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates generally to the field of cell biology. In particular, the present disclosure relates to T cell reprogramming. BACKGROUND

[0003] Cellular immunotherapy for treating cancer is rapidly evolving. For example, chimeric-antigen-receptor T cell (CAR-T) therapy has been approved by the FDA for hematological cancers, which is currently available to patients in need. However, due to technical bottlenecks that limit the efficacy of CAR-T therapy in solid tumors and reduce the production capacity of CAR-T cells, no FDA-approved CAR-T therapy is available for solid tumors to date. Such technical limitations for conventional methods include long production duration to obtain functional CAR-T cells, which leads to T cell differentiation, reduced T cell stemness, and reduced memory T cell population during ex vivo expansion. In addition, weak immune activation of T cells and T cell exhaustion in the adverse microenvironment of solid tumors further limit the efficacy of CAR-T therapy in solid tumors. Therefore, for the purpose of immunotherapy, there is a need for a method to improve T cell expansion as well as enhance T cell stemness and memory T cell enrichment. Moreover, other desired features and characteristics will become apparent from the following DETAILED DESCRIPTION and the appended claims, taken in conjunction with the accompanying drawings and this BACKGROUND. SUMMARY

[0004] In one aspect, the present disclosure provides a method of modifying a T cell during T cell activation, comprising modifying the T cell by contacting the T cell with an inhibitor of prolyl hydroxylases.

[0005] In another aspect, the present disclosure provides a method of making a modified T cell, comprising modifying the T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases, wherein the modification comprises increasing the expansion rate of the T cell compared to an unmodified T cell.

[0006] In another aspect, the present disclosure provides a method of generating a chimeric antigen receptor (CAR) T cell, comprising: modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases; introducing a CAR transgene into the modified T cell of (a) to generate a CAR-T cell; and harvesting the CAR-T cell after (b).

[0007] In another aspect, the present disclosure provides a method of producing a T cell receptor (TCR) T cell, comprising: (i) modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases; (ii) introducing a TCR transgene into the modified T cell of (i) to produce a TCR-T cell; and (iii) harvesting the TCR-T cell after (ii).

[0008] In another aspect, the present disclosure provides a modified T cell obtained by a method as disclosed herein.

[0009] In another aspect, the present disclosure provides a CAR-T cell obtained by a method as disclosed herein.

[0010] In another aspect, the present disclosure provides a TCR-T cell obtained by a method as disclosed herein.

[0011] In another aspect, the present disclosure provides a method of treating or reducing a cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein.

[0012] In another aspect, the present disclosure provides a method of inducing a long-term anti-tumor effect in a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein.

[0013] In another aspect, the present disclosure provides a method of reducing tumor size in a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein.

[0014] In another aspect, the present disclosure provides a method of increasing memory T cell and CAR-T cell stemness in vivo in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein.

[0015] In another aspect, the present disclosure provides a method of increasing tumor infiltration of T cells in a subject having a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein.

[0016] In another aspect, the present disclosure provides a method of increasing the rate of in vivo expansion of CAR-T cells or TCR-T cells in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell as disclosed herein or a TCR-T cell as disclosed herein. Attached Figure Description

[0017] Figure 1 Cells generated after in vitro expansion were compared using conventional CAR-T methods and STEM-T methods as described herein. T cells used to generate CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from anonymous donor subjects. Figure 1 Figure A shows the T cell growth curves of CAR-T and STEM CAR-T cells over 42 days. Compared with CAR-T cells obtained from conventional methods, STEM CAR-T cells expanded faster and resulted in a greater total number of CAR-T cells. Figure 1 B and Figure 1 C shows the flow cytometry results of interferon-γ (IFN-γ) and interleukin-2 (IL-2) expression in CAR-T and STEM CAR-T cells over 20 days. STEM CAR-T cells exhibited higher or comparable levels of cytokines that mediate immune responses in vivo. Figure 1 D provides flow cytometry analysis of T cytokine 1 (TCF1) in the total number of CAR-T cells. TCF1 identifies a stem cell-like precursor CD8+ T cell population characterized by high self-renewal capacity, proliferation potential, and pluripotency. STEM CAR-T cells exhibit higher stemness compared to conventional CAR-T cells. Figure 1 E and Figure 1 F further provided information on stem cell-like memory T cells (T cells) in the total number of CAR-T cells. scm Subtypes and central memory T cells (T cells) cm The percentage of subtypes. Therefore, compared with conventional CAR-T cells, STEM CAR-T cells obtained using the STEM modification method described herein exhibit advantageous properties, including: faster in vitro expansion, greater cytokine production, improved stemness, and an increased memory T cell population.

[0018] Figure 2 The efficacy of STEM CAR-T cells and conventional CAR-T cells after six rounds of co-culturing with target tumor cells was compared. T cells used to generate CAR-T or STEM CAR-T cells were obtained from peripheral blood mononuclear cells (PBMCs) from anonymous donor subjects. Figure 2A shows the expression of the exhaustion markers PD1 and Tim3 for both regular CAR-T and STEM CAR-T cells targeting ROR1 after co-culture with MDA-MB-231 cells at round 6. T cell exhaustion refers to a condition in which T cells, after long-term sustained activation, lose their cell effector function and self-renewal capacity (e.g., killing cancer cells or virus infected cells). T cells exhausted in cancer show high levels of inhibitory receptors such as PD-1, CTLA-4, TIM-3, LAG-3, BTLA, and TIGIT, and reduced effector cytokine production such as IL-2, TNF-a, IFN-g, and GzmB. As Figure 2 A demonstrates that the reduction of PD1 and Tim3 markers in STEM CAR-T cells indicates a reduction in exhaustion of STEM CAR-T cells, allowing for long-term sustained action of STEM CAR-T cells in a subject. Figure 2 B measures CAR-T mediated cytotoxicity for both regular CAR-T and STEM CAR-T cells targeting ROR1 when co-cultured with MDA-MB-231 cells stably expressing luciferase. As Figure 2 B shows that STEM CAR-T have higher cytotoxicity than regular CAR-T cells as measured by luciferase-based cytotoxicity assay of cells. CAR-T cells were injected into NSG mice pre-injected with MDA-MB-231-LN cells, the mice bearing target tumor xenografts. Figure 2 C shows the number of CAR-T or STEM CAR-T cells in the blood of mice after 1, 2, 3, and 4 weeks post-injection. STEM CAR-T cells have a higher number compared to regular CAR-T throughout the 28 days (4 weeks) post-injection, indicating a durable in vivo persistence. Figure 2 D shows the tumor growth curve of mice injected with regular CAR-T and STEM CAR-T, respectively. Control mice were treated with vehicle, which is phosphate buffered saline (PBS). It is clear that the STEM CAR-T group shows the most reduction in tumor volume within 19 days post-infusion compared to the regular CAR-T approach in all groups. Thus, STEM CAR-T cells show reduced T cell exhaustion and cytotoxicity in vivo. In addition, STEM CAR-T cells show a significantly higher number of cell counts in the blood (C) and lead to a stronger anti-tumor efficacy in the mouse model after infusion in mice. Figure 2 C), and lead to a stronger anti-tumor efficacy in the mouse model.

[0019] Figure 3Example CAR-T cells (HER2) produced using conventional CAR-T and the STEM CAR-T method as described herein were compared for T cell exhaustion. T-cells used to produce CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. T cells exhausted in cancer show high expression levels of inhibitory receptors, such as PD1 and Tim3, which are used as markers of T cell exhaustion. STEM CAR-T cells specific for HER2 show reduced levels of exhaustion markers, indicating delayed exhaustion compared to conventional CAR-T cells.

[0020] Figure 4 The cell proliferation rate of human epidermal growth factor receptor 2 (HER2)-specific STEM CAR-T cells after treatment with prolyl hydroxylase inhibitors as exemplified herein was investigated. T-cells used to produce CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. Production of STEM CAR-T cells included steps of T cell activation, engineered receptor induction, and small molecule treatment with prolyl hydroxylase inhibitors. Figure 4 A shows cell counts after treatment of T cells with exemplary prolyl hydroxylase inhibitors 1,4-DPCA, 1,4-DPCA ethyl ester, molisote, enasitide, roxadistat, IOX2, and dalcinostat. STEM CAR-T cells show more number of cells compared to DMSO-treated negative controls (i.e., conventional CAR-T cells), as illustrated by CAR-T (HER2) cell counts 12 days after treatment with prolyl hydroxylase inhibitors. Figure 4 B shows cell counts after treatment of T cells with exemplary prolyl hydroxylase inhibitors and simultaneous CD3 / 28 antibody stimulation compared to DMSO negative controls (i.e., conventional CAR-T cells). Increased cell counts after treatment with exemplary prolyl hydroxylase inhibitors confirm increased proliferation / expansion rate of STEM CAR-T cells, where T cell activation was induced by incubation with CD3 / 28 antibodies. Thus, the STEM method described herein is able to produce more number of STEM CAR-T cells for subsequent patient treatment.

[0021] Figure 5The percentage of cell populations of each T cell subset in the total number of CAR-T cells was measured in HER2-targeting STEM CAR-T 8 days after exemplary prolyl hydroxylase inhibitor treatment and CD3 / 28 antibody stimulation. T-cells used to generate CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. STEM CAR-T cells treated with exemplary prolyl hydroxylase inhibitors showed a greater number of T cell factor 1 (TCF1) positive cells and naive T cells (T n ). The number of stem central memory T cells (T scm ) and central memory T cells (T cm ) was also increased in the total CAR-T cell population. The percentage of the population of effector T cells (T eff ) and effector memory T cells (T em ) remained at low percentages relative to the total CAR-T cell population compared to DMSO-treated negative controls (regular CAR-T cells). Naive T (T n ) cells, stem-like memory T (T scm ) cells, and central memory-like (T cm ) cells exhibit high levels of stemness with the capacity for superior self-renewal, long lifespan, and unipotent differentiation compared to highly differentiated and short-lived T effector memory-like (T em ) cells and highly differentiated T effector-like (T eff ) cells. Thus, STEM CAR-T cells as described herein show improved stemness compared to regular CAR-T cells as evidenced by higher population percentages of less differentiated cell types (T n , T cm , T scm ) and lower population percentages of differentiated cell types (T em , T eff ). The high proliferation and self-renewal capacity of less differentiated cell types allows for sustained in vivo proliferation after administration, leading to improved clinical outcomes.

[0022] Figure 6 Exemplary prolyl hydroxylase inhibitors were shown to increase the proliferation and stemness of exemplary ROR1 -targeting STEM CAR-T cells while maintaining low percentages of differentiated T cells (T em and T eff ). T-cells used to generate CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. The prolyl hydroxylase inhibitor used to generate STEM CAR-T in this figure was 1,4-DPCA-ethyl-ester. As Figure 6Exemplary STEM CAR-T cells targeting Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) proliferate faster compared to conventional CAR-T cells as shown in A. Figure 6 B, Figure 6 C and Figure 6 D shows an increase in naive T cell population (T n ), stem cell-like memory T (T scm ) and T central memory-like (T cm ) cells in STEM CAR-T (ROR1) cells compared to conventional CAR-T cells, indicating an increase in less differentiated cell subtypes. Figure 6 E and Figure 6 F shows a similar low percentage of cell populations of differentiated cell types (T em and T eff , respectively) in STEM CAR-T (ROR1) cells compared to conventional CAR-T cells. For STEM CAR-T (ROR1) cells, a higher number of T cell factor 1 (TCF1) positive cells is observed in G. Thus, CAR-T cells produced by the STEM method show increased proliferation and stemness compared to conventional methods. Figure 6

[0023] Figure 7 Prolyl hydroxylase inhibitors are shown to increase proliferation and stemness of exemplary STEM CAR-T cells targeting HER2 (while maintaining a low percentage of differentiated T cells (T em and T eff ). T-cells used to produce CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. The prolyl hydroxylase inhibitor used to produce STEM CAR-T in this figure is 1,4-DPCA-ethyl-ester. As shown in A, exemplary STEM CAR-T cells targeting HER2 proliferate faster compared to conventional CAR-T cells. Figure 7 A. Exemplary STEM CAR-T cells targeting HER2 proliferate faster compared to conventional CAR-T cells as shown in A. Figure 7 B and Figure 7 C shows an increase in naive T cell population (T n ) and stem cell-like memory T (T scm ) cells in STEM CAR-T (HER2) cells compared to conventional CAR-T cells, indicating an increase in less differentiated cell subtypes. Figure 7 D, Figure 7 E and Figure 7 F shows a similar low percentage of cell populations of differentiated cell types (T cm , T em and T​eff ) of the cell population remained at low levels. For STEMCAR-T (HER2) cells, the percentage of cells positive for T-cell factor 1 (TCF1) was observed to be higher in G compared to A. Thus, CAR-T cells produced by the STE method show increased proliferation and stemness compared to conventional methods. Figure 7 G. Thus, CAR-T cells produced by the STE method show increased proliferation and stemness compared to conventional methods.

[0024] Figure 8 Increased pro-inflammatory cytokine secretion and lysis against antigen-expressing cancer cells and delayed exhaustion state in STEMCAR-T cells targeting ROR1 compared to conventional CAR-T are shown. T-cells used to produce CAR-T or STEMCAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. The prolyl hydroxylase inhibitor used to produce STEMCAR-T in this figure is 1,4-DPCA-ethyl-ester. As demonstrated in Figure 8 Long-term sustained treatment over 6 co-culture cycles, STEMCAR-T (ROR1) cells still show higher cytokine, e.g., interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha (TNFa) secretion compared to conventional CAR-T cells. At the 6th co-culture cycle, STEMCAR-T show about 2-fold enhanced cytotoxicity against triple negative breast cancer (TNBC) cells, demonstrating potent anti-tumor capacity without reduction in efficacy due to T cell exhaustion.

[0025] Figure 9 Increased pro-inflammatory cytokine secretion and lysis against antigen-expressing cancer cells and delayed exhaustion state in STEMCAR-T cells targeting HER2 compared to conventional CAR-T are shown. T-cells used to produce CAR-T or STEMCAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. The prolyl hydroxylase inhibitor used to produce STEMCAR-T in this figure is 1,4-DPCA-ethyl-ester. As demonstrated in Figure 9 Long-term sustained treatment over 6 co-culture cycles, STEMCAR-T (HER2) cells still show higher cytokine, e.g., interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha (TNFa) secretion compared to conventional CAR-T cells. At the 5th co-culture cycle, STEMCAR-T show about doubled cytotoxicity against triple negative breast cancer (TNBC) cells, demonstrating potent anti-tumor capacity without reduction in efficacy due to T cell exhaustion.

[0026] Figure 10Ex vivo T cell viability data is provided for exemplary prolyl hydroxylase inhibitors 1,4-DPCA, 1,4-DPCA ethyl ester, Molidustat, Panaxynol, Enasidenib, Roxadustat, IOX2, and Daprodustat. T cells were placed in cell culture media containing CD3 / 28 antibodies and different prolyl hydroxylase inhibitors were added for 5 days. T-cells for generating CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. Starting at day 5, antibodies and inhibitors were removed from the cell culture media. As shown in Figure 10 all prolyl hydroxylase inhibitors showed an increase in the number of viable T cells relative to the number of total viable T cells for the negative control (DMSO) group, indicating that treatment by prolyl hydroxylase inhibitors resulted in improved T cell viability. The rate of T cell proliferation was calculated for each prolyl hydroxylase inhibitor based on the number of viable T cells relative to the number of viable T cells for the DMSO treated group. Thus it can be found that T cells treated with prolyl hydroxylase inhibitors showed increased T cell viability.

[0027] Figure 11 Ex vivo CAR-T cell counts are shown for STEM CAR-T cells targeting ROR1 / HER2 / EGFR / CD19, respectively, compared to conventional CAR-T. T-cells for generating CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. For generating CAR T cells, bulk PBMCs were activated using CD3 and CD28 on day 0. Prolyl hydroxylase inhibitors (1,4-DPCA ethyl ester in this example) or an equivalent volume of DMSO were added to the cell culture on day 0. On day 3, cells were transduced with lentivirus expressing the respective CAR. Activation was continued for 5 days and control, as measured by fluorescence-activated cell sorting (FACS) percentage of transduced CAR, manual counting of CAR-T cells. Manual counting of total number of CAR-T cells was performed at the indicated days in the graph, after CD3 / 28 antibody activation and in the presence or absence of prolyl hydroxylase inhibitors (STEM CAR-T and conventional CAR-T, respectively). As shown in Figure 11 As can be seen, enhanced CAR-T cell expansion was observed in all STEM CAR-T cells compared to conventional CAR-T cells.

[0028] Figure 12 In vivo tumor volume in different xenograft cancer models in mice after administration of CAR-T cells obtained using conventional methods or STEM modification methods as disclosed herein is provided. T-cells for generating CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject.Figure 12 A shows tumor volume over time in a DLD-1 xenograft model for colorectal cancer following treatment with CAR-T cells targeting EGFR. As can be seen from the graph, a significant reduction in tumor size was observed in the CAR-T treated groups compared to the phosphate buffered saline (PBS) treated vehicle control. In particular, CAR-T cells produced using the STEM method as described herein showed the smallest tumor volume within 16 days of injection. Figure 12 B shows tumor volume over time in a DLD-1 xenograft model for colorectal cancer following treatment with CAR-T cells targeting HER2. As can be seen from the graph, a significant reduction in tumor size was observed in the STEM CAR-T treated group compared to the regular CAR-T group and the PBS treated vehicle control group at 16 days post injection. Figure 12 C shows tumor volume over time in a MB361 xenograft model for breast cancer following treatment with CAR-T cells targeting HER2. As can be seen from the graph, a significant reduction in tumor size was observed in the STEM CAR-T treated group compared to the regular CAR-T group and the PBS treated vehicle control group at 16 days post injection. Thus, the exemplary cancer disease models demonstrate that the STEM method as described herein increases CAR-T cell anti-tumor activity in vivo.

[0029] Figure 13 CAR-T cells in a xenograft model for colorectal cancer following administration. T-cells used to produce CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. The amount of circulating CAR-T cells in the blood was quantified. As Figure 13 A shows, in a DLD-1 xenograft colorectal cancer model, the number of STEM CAR-T cells targeting EGFR following administration compared to the PBS treated vehicle control group and the regular CAR-T cell treated group. Figure 13 B provides the percentage of the population of T cm and T scm cells in the total CAR-T cells produced in vivo using the regular CAR-T method and the STEM T method in a DLD-1 colorectal cancer model. The percentage of the population of T cm and T scm cells was measured from mouse blood taken 13 days post CAR-T cell injection. The significant increase in the percentage of the population of T scm and T cm cells indicates an increase in stemness of the STEM CAR-T cells. As Figure 13As shown in C, the number of STEM CAR-T cells targeting HER2 was the most compared to the PBS-treated vehicle control and the conventional CAR-T cell group in the MDA-MB-361 breast cancer mouse model after administration. Figure 13 D provides the percentage of the population of T cm and T scm number of the population. The percentage of the population of T cm and T scm of the population was measured from the blood of mice taken 13 days after CAR-T cell injection. scm and T cm The significant increase in the population of T

[0030] Figure 14 In vivo CAR-T cell counts, tumor volume, and intratumoral CAR-T cell percentages in the immunoresistant triple negative breast cancer (TNBC) breast tumor mouse model MB231-LN treated with STEM CAR-T cells and conventional CAR-T cells are shown. T-cells used to generate CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMCs) from an anonymous donor subject. According to Figure 14 A, tumor volume growth was much slower when treated with STEM CAR T-cells compared to the conventional CAR-T cell treated group and the PBS-treated vehicle control group 19 days after injection. The number of circulating CAR-T cells counted for the STEM CAR-T group was also significantly higher than the other two groups. Figure 14 B depicts the situation of CAR-T cell subtypes within the tumor. The percentage of CAR-T cells infiltrating the tumor mass was quantified for STEM CAR-T (ROR1) and conventional CAR-T. A much higher CAR-T cell infiltration was identified for cells obtained using the STEM method as described herein, confirming the improved tumor infiltrating ability of STEM CAR-T cells. Among the CAR-T cells infiltrating the tumor, for the STEM CAR-T group, the central memory CAR-T cells (T cmThe (CD45RO+CCR7+) population showed an increase. Administration of STEM CAR-T cells also showed a small increase in the cytokine IFNy+ population of CAR-T cells within the tumor. Thus, the STEM T method as described herein leads to a stronger anti-cancer activity of CAR-T cells and increases tumor infiltration of CAR-T cells in solid tumor tissue.

[0031] Figure 15 CAR-T cell counts, tumor volume, and survival probability in a DLD-1 colorectal cancer xenograft mouse model treated with exemplary STEM CAR-T cells compared to conventional CAR-T cells are shown. T-cells used to generate CAR-T or STEM CAR-T were obtained from peripheral blood mononuclear cells (PBMC) from an anonymous donor subject. Data in this figure was obtained from the same mice. As Figure 15 As shown in A, tumor growth was reduced after injection of STEM CAR-T (EGFR1 -targeted) and the inhibitory effect persisted for more than 50 days. Figure 15 B shows the number of CAR-T cells circulating in the blood obtained from mice treated with PBS, conventional CAR-T cells, and STEM CAR-T cells. STEM CAR-T cell treated mice presented a higher number of cells in the blood compared to conventional CAR-T cells and negative control for a long-lasting period of time. Figure 15 C provides survival curves of DLD-1 colorectal cancer xenograft mouse models treated with CAR-T cells. Among the three groups, only the STEM CAR-T cell injected mice showed a high survival probability 80 days after injection. In summary, STEM-CAR-T treated mice showed persistent CAR-T cells in circulation, inhibited tumor growth, and translated the persistent immunity into higher survival rates in treated subjects. Thus, T cells modified using the STEM method as described herein demonstrated superior anti-tumor properties compared to unmodified T cells.

[0032] Definitions As used herein, the term "small molecule" refers to any organic compound with a low molecular weight that can modulate a biological process. Many drugs are small molecules. The small molecular size allows it to easily enter a cell, and thus is often used as a drug to target cellular proteins to affect molecular pathways. Typically, small molecule drugs have a size of about 1 nm or a molecular weight of less than about 500 Da.

[0033] As used herein, the term "T cell" or "T lymphocyte" refers to an important type of white blood cell and plays a central role in the adaptive immune response. T cells differentiate from hematopoietic stem cells, which are stem cells in the bone marrow. T cells can be distinguished from other lymphocytes by the presence of T-cell receptors (TCRs) on their cell surface. There are two main types of T cells: CD4+ T cells ("helper T cells") and CD8+ T cells ("cytotoxic T cells" or "killer T cells"). CD8+ T cells are capable of directly killing virus-infected cells as well as cancer cells, and utilize cytokines to recruit other types of cells when an immune response is generated. Unlike CD8+ killer T cells, CD4+ cells exert their function by further activating memory B cells and cytotoxic T cells, which leads to a greater immune response.

[0034] As used herein, the term "naive T cell" or "T n " refers to an immature T cell that has differentiated in the thymus. Naive T cells will mature after encountering their cognate antigen in the periphery. Differentiation and activation of T cells depend on signals transduced through three different receptors: TCR (including CD4 and CD8 receptors that respond to MHC-II displayed antigens and MHC-I displayed antigens, respectively), costimulatory receptors, and cytokine receptors. These signals drive naive T cells to differentiate into either effector T cells or memory T cells.

[0035] As used herein, the term "effector T cell" or "T eff " refers to a subset of T lymphocytes with a relatively short lifespan. Effector T cells actively respond to stimuli and perform immune response functions. Effector T cells can be cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (T reg ).

[0036] As used herein, the term "memory T cell" refers to a subset of T lymphocytes that are capable of mediating a faster and more effective immune response when they encounter the antigen to which they have been previously exposed. These cells are long-lived and can rapidly expand into large numbers of effector T cells to provide protection against subsequent exposure to the same antigen. Depending on the type of antigen encountered, memory T cells can be either CD4+ cells or CD8+ cells.

[0037] Memory T cells comprise several subtypes. Generally, memory T cells include stem cell memory T (T scm ) cells and central memory T (T cm ) cells, which have different specific phenotypes and functions.

[0038] As used herein, the term "central memory T cell" or "T cm"Central memory T cell" or "Tcm" refers to a subtype of memory T cells that express L-selectin, CD45RO and CCR7 and provide central immune surveillance by patrolling lymph nodes that drain peripheral tissue sites in vivo. Central memory T cells have several properties in common with stem cells, the most important being the capacity for self-renewal, which is largely due to high levels of phosphorylation of the key transcription factor STAT5.

[0039] As used herein, the term "effector memory T cell" or "T em " refers to another subtype of memory T cells that express CD45RO but lack CCR7 and L-selectin expression. Unlike central memory T cells, effector memory T cells reside in the peripheral circulation and tissues due to the lack of CCR7 lymph node homing receptor. Effector memory T cells function primarily as CD8 variants and are thus primarily responsible for cytotoxic effects against pathogens. In contrast to T cm cells, T em cells express higher levels of receptors responsible for migration to inflamed tissues and have a stronger immediate effector function.

[0040] As used herein, the term "stem cell-like memory T cell," "stem cell memory T cell" or "T scm " refers to another subtype of memory T cells that express increased levels of CD95, IL-2Rβ, CXCR3 and LFA-1 compared to naive T cells. Like naive T cells, T scm cells are CD45RO, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+. Stem cell-like memory T cells exhibit the properties of long life, sustained self-renewal, rapid differentiation into effector T cells and resistance to apoptosis.

[0041] As used herein, the term "iPSC" or "induced pluripotent stem cell" refers to a pluripotent stem cell derived from skin or blood cells, for example, that has been reprogrammed back to an embryonic-like pluripotent state, which enables the development of any type of human cell from an unlimited source. As disclosed herein, the term "iPSC induced T cell," "iPSC derived T cell" or "T-iPSC" refers to a T cell that is re-differentiated from an induced pluripotent stem cell. iPSC derived T cells are phenotypically defined, expandable and functionally identical to physiological T cells.

[0042] As used herein, the term "immunotherapy" refers to a method of treating or preventing disease by stimulating the immune system to activate or suppress immune responses. The term "cellular immunotherapy" or "adoptive cell therapy" refers to a class of immunotherapy in which a patient's own immune cells are administered to the patient to help the body fight disease, such as cancer. The immune cells can be expanded ex vivo to increase the total number of cells, or engineered to target specific tumor cell types. The term "adoptive T cell immunotherapy" refers to adoptive cell therapy utilizing T cells.

[0043] As used herein, the term "CAR" or "chimeric-antigen-receptor" refers to an antigenic recombinant receptor that redirects the specificity and function of T lymphocytes and / or other immune cells in a single molecule, programming them to target tumor-associated antigens. Chimeric antigen receptors (CARs) generally consist of an extracellular domain that binds to a specific antigen on a tumor cell, a transmembrane domain, and an intracellular domain that provides a signal for T cell activation to attack the tumor cell.

[0044] As used herein, the term "CAR-T cell" or "chimeric-antigen-receptor-engineered T cell" refers to an engineered T cell that expresses a cancer-specific artificial chimeric-antigen-receptor (CAR) that can be used in adoptive T cell immunotherapy. The T cells are obtained from a patient's blood and generated ex vivo. A large number of CAR-T cells are provided to the patient by infusion to treat disease, such as cancer.

[0045] As used herein, the term "TCR-T cell" or "T cell receptor (TCR) T cell" refers to a T cell receptor (TCR)-engineered T cell that is directed to target a specific tumor marker. Both CAR-T and TCR-T cell therapies enhance the ability of T cell receptors to recognize and attack specific antigenic cellular antigens through genetic modification. CAR-Ts directly alter a portion of the T cell receptor to a specific antibody, allowing the T cell to directly attack cancer cells under the guidance of the antibody. In contrast to CAR-Ts, which recognize surface antigens, TCR-Ts are able to interact with peptide-major histocompatibility complexes (pMHCs) generated from proteolysis of intracellular antigen proteins. TCR-Ts recognize a broad range of cancers, while CAR-Ts provide more specific and effective tumor cell targeting.

[0046] The generation of CAR-T cells requires several carefully executed steps, and quality control tests are performed throughout the protocol. Briefly, the conventional CAR-T cell generation process / method referred to herein includes: (1) isolation of white blood cells from a subject; (2) enrichment of T cells from the white blood cells; (3) activation of the T cells and co-incubation of the T cells with a viral vector encoding a CAR; and (4) expansion of the CAR-T cells and final formulation.

[0047] As used herein, the term "T cell activation" refers to the process by which an antigen presenting cell (APC) activates a T cell. Methods of activating T cells for the purpose of immunotherapy are known in the art. For example, autologous antigen presenting cells (APCs) can be isolated and purified from a patient for T cell activation. Anti-CD3 antibodies can be used alone or in combination with feeder cells and growth factors, such as IL-2, which have been commonly used by those skilled in the art. Alternatively, to simplify and standardize the activation process with higher efficiency, anti-CD3 / anti-CD28 monoclonal antibody coated beads can be used.

[0048] As used herein, the term "cancer" or "malignancy" refers to a large group of diseases that can begin in almost any organ or tissue of the body, when abnormal cells grow beyond their usual boundaries, even invading neighboring parts of the body and / or spreading to other organs.

[0049] As used herein, the term "tumor" is a collection of cells / tissues that grow in a mass due to uncontrolled multiplication of abnormal or damaged cells. Tumors can be cancerous or non-cancerous (benign). Cancerous tumors spread to or invade nearby tissues, and can move to distant locations in the body to form new tumors (metastasis). As used herein, the term "solid tumor" refers to a group of tumors that generally do not contain cysts or fluid areas. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.

[0050] As used herein, the term "differentiation" refers to the process by which a stem cell changes from a less specialized cell type to a more specialized type, including the switch from proliferation to specialization. Differentiation alters the size, shape, membrane potential, metabolic activity, and response to signals of a cell. These changes are primarily due to highly controlled modifications in gene expression, and are a subject of epigenetics.

[0051] As used herein, the term "stem cell" refers to a cell that has the potential for self-renewal and to develop into many different specialized functional types of cells (differentiation) in the body. As used herein, the term "stemness" refers to the ability of a cell to self-renew and differentiate. Stem cells can be classified into the following groups according to their differentiation capacity: (1) totipotent stem cells: cells that can differentiate into all cell types; (2) pluripotent stem cells: cells that can differentiate into almost all cell types; (3) multipotent stem cells: cells that can differentiate into a related family of cell types; (4) oligopotent stem cells: cells that can differentiate into several different cells; (5) unipotent stem cells: cells that can give rise to only one cell type.

[0052] As used herein, the term "memory" refers to the population of memory T cells within the total T cell population. Therefore, the expressions "enrichment of memory" or "improved memory" describe memory T cells, such as stem cell memory T cells (T12). scm ) cells, central memory T(T) cm Cellular and effector memory T (T) em The percentage of cells in the population increases. For example... Figure 14 , 15 As exemplified in 24, STEM CAR-T cells showed enrichment of all T cell memory, characterized by an increased population percentage of Tscm and Tcm cells compared to conventional CAR-T cells.

[0053] As used herein, the term "depletion" refers to a cellular state of dysfunction and reduced responsiveness commonly observed in response to persistent antigen exposure, such as in chronic infections. In the context of this disclosure, "T-cell depletion" refers to this T-cell-related state characterized by a gradual loss of T-cell effector function and self-renewal capacity within the tumor microenvironment, thereby limiting the efficacy of immunotherapy. Therefore, T-cell depletion is often associated with poor tumor control in patients.

[0054] As used herein, the terms “prolyl hydroxylase,” “procollagen-proline dioxygenase,” or “prolyl 4-hydroxylase” refer to members of a class of enzymes called α-ketoglutarate-dependent hydroxylases (EC 1.14.11.2). Prolyl hydroxylases irreversibly catalyze the conversion of proline residues from a variety of protein substrates to (2S,4R)-4-hydroxyproline (Hyp). Such hydroxylation is the most common post-translational modification in humans.

[0055] As used herein, the term "prolyl hydroxylase inhibitor" refers to an agent that directly or indirectly inhibits enzymatic activity. For example, a prolyl hydroxylase inhibitor can be, but is not limited to, 1,4-DPCA (… ), 1,4-DPCA ethyl ester ( ), molistat ( ), Panax notoginseng extract ( ), ennastine ( ), Roxadustat ( ), Dapoxetine ( ), Desidustat ), vardusta ( ) and IOX2 ( ).

[0056] As used herein, the term "triple negative breast cancer" or "TNBC" refers to a breast cancer that, upon testing, is found to lack (or is negative for) human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), and progesterone receptor (PR). Triple negative cancers are also known as "basal-like" cancers.

[0057] As used herein, the term "reprogramming" refers to a process that sets the fate of a somatic cell and the cell reverts to a pluripotent state to reestablish the cell fate. As used herein, "T cell reprogramming" refers to a process that alters the function, phenotype, or differentiation state of a T cell to enhance their therapeutic potential.

[0058] As used herein, the term "modifying" refers to a process as disclosed herein that induces a change in the properties of a T cell. The change includes, for example, increasing the expansion rate of the modified T cell compared to the unmodified T cell, improving the ex vivo stemness of the modified T cell compared to the unmodified T cell, increasing the T memory cell population in the modified T cell compared to the unmodified T cell, increasing the antigen-specific cytotoxicity of the modified T cell compared to the unmodified T cell, and decreasing the exhaustion of the modified T cell compared to the unmodified T cell. DETAILED DESCRIPTION

[0059] The present disclosure provides a small molecule-based method of T cell modification to induce T cell reprogramming, resulting in increased T cell stemness and enrichment of memory T cell populations. The methods described herein generally apply to T cells and can be combined with existing T-cell based cell therapies, such as CAR-T or TCR-TCR-T. The methods described herein effectively shorten the ex vivo production timeline. The resulting modified T cells show a persistent anti-tumor effect and improved T cell in vivo proliferation and survival. The modification methods as described herein have the potential to overcome existing obstacles for cell immunotherapies in solid tumors, including the lack of in vivo persistence and durable anti-tumor responses due to rapid T cell exhaustion in the tumor microenvironment.

[0060] Accordingly, in one aspect, the present disclosure provides a method of modifying a T cell. In one example, the method of modifying a T cell is an in vitro or ex vivo method. In another example, the methods described herein can occur during T cell activation. In some examples, the method comprises contacting the T cell with an inhibitor of prolyl hydroxylases.

[0061] In one example, the time period for modifying the T cells can be about 1 day, about 3 days, about 4 days, about 5 days, about 6 days, and about 7 days. In some further examples, the duration of modifying the T cells can be about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 2 to 3 days, about 2 to 4 days, about 3 to 4 days, about 2 to 6 days, about 1 to 7 days, about 2 to 7 days, about 3 to 6 days, about 3 to 7 days, about 4 to 6 days, about 4 to 7 days, about 5 to 6 days, and about 5 to 7 days. In one example, the duration of modifying the T cells can be about 1 to 5 days. In another example, the duration of modifying the T cells can be less than one week.

[0062] As used herein, the term "inhibitor" generally refers to an agent that directly or indirectly slows, inhibits, or interferes with the enzymatic activity of a prolyl hydroxylase enzyme. The inhibitor can include, but is not limited to, a compound, a small molecule drug, an enzyme, an antibody, a nucleic acid, a protein, a polymer, or a combination thereof. As used herein, the term "prolyl hydroxylase," "procollagen-proline 4-dioxygenase," or "prolyl 4-hydroxylase" refers to a member of a class of enzymes known as a-ketoglutarate-dependent hydroxylases (EC number: 1.14.11.2). Prolyl hydroxylases irreversibly catalyze the hydroxylation of proline residues from a variety of protein substrates to (2S,4R)-4-hydroxyproline (Hyp). Such hydroxylation reactions are the most common post-translational modification in humans. Thus, one of skill in the art will appreciate that an agent that inhibits or reduces the hydroxylation activity of a prolyl hydroxylase, regardless of the mechanism of action, will be suitable for the purposes described herein.

[0063] In some examples, the inhibitor of prolyl hydroxylase is a small molecule prolyl hydroxylase inhibitor. As Figure 1 illustrated in the examples provided herein, a small molecule prolyl hydroxylase inhibitor can be included in a cell culture medium, e.g., a T cell culture medium, to allow the inhibitor of prolyl hydroxylase to come into contact with the T cells. Other ways of bringing the inhibitor into contact with the T cells, in addition to the examples provided herein, are well known in the art.

[0064] In some further examples, the inhibitor of prolyl hydroxylase is a small molecule inhibitor. By convention, small molecule drugs have a size of about 1 nm or a molecular weight of less than about 500 Da. Small molecule inhibitor drugs of prolyl hydroxylase have been actively developed due to their clinical relevance for treating diseases, e.g., chronic kidney disease. For example, the prolyl hydroxylase inhibitor can be, but is not limited to, 1,4-DPCA (Doxylamine) (Doxylamine), 1,4-DPCA ethyl ester (Doxylamine), Molidustat (GSK1278867A), Panaxynol (GSK1278867A), Fedradustat (GSK1278867A), Enasidenib (AG-221), Roxadustat (FG-4592), Daprodustat (GSK1278867A), and GSK1278867A. ), 1,4-DPCA ethyl ester ( ), Molidustat ( ), Panaxynol ( ), Enasidenib ( ), Roxadustat ( ), and Daprodustat (GSK1278867A). ), Desidustat ), vardusta ( ) and IOX2 ( In one instance, a small molecule inhibitor of prolyl hydroxylase is 1,4-DPCA-ester (…). As described herein, for example, the concentration of the small molecule prolyl hydroxylase inhibitor can be about 0.5 µM, about 0.75 µM, about 1 µM, about 1.25 µM, about 1.5 µM, about 1.75 µM, about 2 µM, about 2.25 µM, about 2.5 µM, about 2.75 µM, or about 3 µM. In some instances, the concentration range of the small molecule prolyl hydroxylase inhibitor can be about 1.25 µM to about 1.75 µM, about 1 µM to about 2 µM, about 0.75 µM to about 2.25 µM, about 0.5 µM to about 2.5 µM, or about 0.25 µM to about 2.75 µM. In one instance, the concentration of the small molecule prolyl hydroxylase inhibitor is about 1 µM. In addition to the exemplary concentrations and concentration ranges provided herein, the appropriate amount of a small molecule prolyl hydroxylase inhibitor can be determined within the capabilities of a person skilled in the art, taking into account the characteristics of the selected inhibitor, the teachings of this disclosure, and cellular conditions.

[0065] In another aspect, this disclosure provides a method for preparing modified T cells. The method, as described herein, includes modifying T cells during T cell activation by contacting them with an inhibitor of prolyl hydroxylase. In one example, the modification includes increasing the rate of T cell proliferation compared to unmodified T cells. For example, as... Figure 11 As shown, the in vitro proliferation rate of isolated T cells was measured after treatment with an inhibitor of prolyl hydroxylase. T cells in all treatments expressing different CARs showed increased proliferation compared to a control group serving as conventional CAR-T cells. Figure 10 Treatment with eight exemplary prolyl hydroxylase inhibitors also showed a significant increase in T cell viability, which in turn promoted an increased T cell proliferation rate.

[0066] In another example, modified T cells comprised an increased population of memory T cells within the total T cell population compared to unmodified T cells. For example... Figure 5 As shown, for example, after treatment with seven exemplary prolyl hydroxylase inhibitors, 12 days post-treatment, naive T cells (T cells) n ), stem cell-like memory T cells (T scm ) and central memory T cells (T cm) population is higher than the DMSO treated control. In another example, the modification of the T cells includes improving T cell stemness in the ex vivo T cells compared to unmodified T cells. As used herein, stemness refers to the ability of a cell to self-renew and differentiate. As shown in Figure 5 For example, TCF1 expression is higher than the DMSO treated group at D5 and D8 post treatment after treatment with the seven exemplary prolyl hydroxylase inhibitors.

[0067] In another example, the modification of the T cells includes increasing antigen specific cytotoxicity of the modified T cells compared to unmodified T cells. As shown in Figure 2 B, for example, which provides a comparison between the modified STEM CAR-T cells and the conventional unmodified CAR-T cells in mediating cytotoxicity in cancer cells. The modified cells show almost double cytotoxicity compared to the unmodified cells, and this effect is proportional to the increase in the ratio of effector cells (E) to target cells (T). Thus, the modification methods as disclosed herein provide effective improvement in cytotoxicity against target cells, e.g., cancer cells.

[0068] In another example, the modified T cells include reducing T cell exhaustion in vivo compared to unmodified T cells. As shown in Figure 2 A, the ROR1 -targeting CAR-T cells modified with the methods as described herein (STEM CAR-T cells) show a reduction in exhaustion markers, e.g., PD1 and Tim3, after 6 rounds of co-culture with tumor cells. Figure 8 and 9 Additional examples of modified STEM CAR-T cells (targeting ROR1 and HER2, respectively) are provided. Both the ROR1 -targeting and HER2-targeting modified T cells show increased proinflammatory cytokine secretion and lysis of antigen-expressing cancer cells and delayed exhaustion state after long-term sustained treatment over 5 to 6 co-culture cycles. Thus, the modification methods as described herein reduce T cell exhaustion in vivo compared to unmodified T cells.

[0069] The methods described herein are generally applied to T cells and can be combined with existing T-cell based cell therapies. In one example, the modified T cells obtained using the methods disclosed herein can be used in adoptive T cell immunotherapy. In another example, the modified T cells obtained using the methods disclosed herein can be used in chimeric antigen receptor (CAR) T cell therapy. In yet another example, the modified T cells obtained using the methods disclosed herein can be used in T cell receptor (TCR) T cell therapy.

[0070] Accordingly, in a further aspect, the present disclosure provides a method of producing a chimeric antigen receptor (CAR) T cell. In one example, the method comprises modifying a T cell as disclosed herein. In another example, the method comprises modifying a T cell by contacting the T cell with an inhibitor of prolyl hydroxylases during T cell activation. Methods of activating T cells are known in the art. For example, autologous antigen presenting cells (APCs) can be isolated and purified from a patient for T cell activation. Anti-CD3 antibodies can be used alone or in combination with feeder cells and growth factors, such as IL-2, which have been commonly used by those skilled in the art. In one example of a method as described herein, T cell activation can be induced by anti-CD3 and / or anti-CD28 antibodies. Alternatively, to simplify and standardize the activation process with higher efficiency, anti-CD3 / anti-CD28 monoclonal antibody-coated beads can be used. In another example, other cytokines, such as IL17 or IL15, can also be used.

[0071] In another example, a method of producing a chimeric antigen receptor (CAR) T cell comprises modifying a T cell by contacting the T cell with an inhibitor of prolyl hydroxylases during T cell activation and introducing a CAR transgene into the modified T cell to produce a CAR-T cell. Methods of designing a CAR transgene and methods of introducing a CAR transgene into a T cell are known in the art. The CAR transgene comprises a target sequence that allows recognition and clearance of tumor cells by the CAR-T cell. A variety of target sequences can be used in cellular immunotherapy, such as C-type lectin-like molecule-1 (CLL-1), CD19, CD20, B-cell maturation antigen (BCMA), HER2, ROR1, and EGFP1. The present disclosure provides, for example, modified STEM CAR-T cells targeting HER2 for the treatment of breast cancer. The present disclosure provides, for example, modified STEM CAR-T cells targeting EGFR for the treatment of colorectal cancer. Depending on the disease to be treated and the tumor genotype specifically targeted, those skilled in the art will be able to design a suitable CAR transgene. The production process of a CAR-T cell simply comprises: (1) isolating white blood cells from a subject; (2) enriching T cells from the white blood cells; (3) activating the T cells and co-incubating the T cells with a viral vector encoding a CAR. In one example, a lentivirus is used to introduce the CAR transgene into the modified T cell. In another example, the step of modifying the T cell and the step of introducing the CAR transgene are performed simultaneously or sequentially. Those skilled in the art will be able to follow the teachings of the present disclosure and available protocols for introducing a CAR transgene into a modified T cell as described herein. Figure 9 Figure 13 In another example, a method of producing a chimeric antigen receptor (CAR) T cell comprises modifying a T cell by contacting the T cell with an inhibitor of prolyl hydroxylases during T cell activation and introducing a CAR transgene into the modified T cell to produce a CAR-T cell. Methods of designing a CAR transgene and methods of introducing a CAR transgene into a T cell are known in the art. The CAR transgene comprises a target sequence that allows recognition and clearance of tumor cells by the CAR-T cell. A variety of target sequences can be used in cellular immunotherapy, such as C-type lectin-like molecule-1 (CLL-1), CD19, CD20, B-cell maturation antigen (BCMA), HER2, ROR1, and EGFP1. The present disclosure provides, for example, modified STEM CAR-T cells targeting HER2 for the treatment of breast cancer. The present disclosure provides, for example, modified STEM CAR-T cells targeting EGFR for the treatment of colorectal cancer. Depending on the disease to be treated and the tumor genotype specifically targeted, those skilled in the art will be able to design a suitable CAR transgene. The production process of a CAR-T cell simply comprises: (1) isolating white blood cells from a subject; (2) enriching T cells from the white blood cells; (3) activating the T cells and co-incubating the T cells with a viral vector encoding a CAR. In one example, a lentivirus is used to introduce the CAR transgene into the modified T cell. In another example, the step of modifying the T cell and the step of introducing the CAR transgene are performed simultaneously or sequentially. Those skilled in the art will be able to follow the teachings of the present disclosure and available protocols for introducing a CAR transgene into a modified T cell as described herein.

[0072] ​In a further example, a method of producing a chimeric antigen receptor (CAR) T cell comprises modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases, introducing a CAR transgene into the modified T cell to produce a CAR-T cell, and harvesting the resulting CAR-T cell. In one example, modifying the T cell during T cell activation increases the number of CAR-T cells harvested. The harvested CAR-T cells can be preserved, for example, by cryopreservation, or infused into a patient in need thereof.

[0073] For example, a chimeric-antigen-receptor (CAR) cassette is introduced by lentivirus after 1-3 days of stimulation. CAR-T cells are collected after 2 days for injection. The entire production process takes less than 1 week. The method utilizes a low dose and sustained relatively short period of a small molecule prolyl hydroxylase inhibitor compound, resulting in minimal cost and allowing easy scale-up of standard operating procedures in the production process.

[0074] In another aspect, the present disclosure provides a method of producing a T cell receptor (TCR) T cell. Like CAR-T cell therapy, engineered T cell receptor (TCR) therapy involves treating cancer cells with activated T lymphocytes from a patient. Both strategies provide T cells with new receptors to enable more efficient targeting of cancer cells. In one example, the method comprises modifying a T cell as disclosed herein. In another example, the method comprises modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases. Methods of activating T cells are known in the art. For example, autologous antigen presenting cells (APCs) can be isolated and purified from a patient for T cell activation. Anti-CD3 antibodies can be used alone or in combination with feeder cells and growth factors, such as IL-2, which have been commonly used by those skilled in the art. In one example of a method as described herein, T cell activation can be induced by anti-CD3 and / or anti-CD28 antibodies. Alternatively, to simplify and standardize the activation process with higher potency, anti-CD3 / anti-CD28 monoclonal antibody-coated beads can be used. In another example, other cytokines, such as IL17 or IL15, can also be used.

[0075] In another example, a method of producing a T cell receptor (TCR) T cell comprises modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases and introducing a TCR transgene into the modified T cell to produce a TCR-T cell. Methods of designing TCR transgenes and introducing TCR transgenes into T cells are known in the art. Similar to CAR-T, the TCR transgene comprises a target sequence that allows recognition and clearance of tumor cells by the TCR-T cell. Depending on the disease to be treated and the tumor genotype to be specifically targeted, the skilled person will be able to design a suitable TCR transgene. The skilled person will also be able to follow the teachings of the present disclosure and available protocols for introducing TCR transgenes into modified T cells as described herein.

[0076] In a further example, a method of producing a T cell receptor (TCR) T cell comprises modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases, introducing a TCR transgene into the modified T cell to produce a TCR-T cell, and harvesting the obtained TCR-T cell. In one example, modifying the T cell during T cell activation increases the number of harvested TCR-T cells. The harvested TCR-T cells can be preserved, for example, by cryopreservation, or infused into a patient in need thereof. In a further example, the harvested CAR-T or TCR-T cells can be used in adoptive T cell immunotherapy.

[0077] In some examples of the methods as described herein, the harvested CAR-T or TCR-T cells show reduced T cell exhaustion after repeated tumor antigen stimulation compared to unmodified T cells. T cell exhaustion refers to a condition in which T cells lose their ability to kill certain cells, e.g., cancer cells or virus-infected cells, after long-term sustained activation. Exhausted T cells in cancer show high expression levels of inhibitory receptors, e.g., PD-1, CTLA-4, TIM-3, LAG-3, BTLA, and TIGIT, and reduced effector cytokine production, e.g., IL-2, TNF-a, IFN-g, and GzmB. The reduction in T cell exhaustion can be characterized by changes in the expression levels of T cell exhaustion markers compared to unmodified T cells. In one example, the reduced T cell exhaustion is characterized by a reduction in the expression of the T cell exhaustion markers PD-1 and TIM-3 compared to unmodified T cells. In another example, the reduced T cell exhaustion is characterized by a reduction in the upregulation of the T cell exhaustion markers PD-1 and TIM-3 compared to unmodified T cells. As Figure 2 A and Figure 3 As demonstrated in the Examples, for example, a reduction in exhaustion markers was observed in STEM CAR-T cells after long-term sustained exposure to tumor cells.

[0078] In some examples of the methods as described herein, the harvested CAR-T or TCR-T cells show increased cytokine release levels compared to unmodified CAR-T or TCR-T cells. T cells act as the primary effector cells in cellular immunity, producing cytokines to mediate inflammation and to regulate other types of immune cells in an immune response. Activation and proliferation of CAR-T and TCR-T cells release primary cytokines, such as IL1, IFN-gamma, and TNF, which induce activation of other immune cells, such as macrophages, DCs, and monocytes. These cells then produce excess secondary cytokines, such as IL6, IL10, and IL5. Cytokines can regulate the growth, apoptosis, activation, and differentiation of target cells. Significant correlations between cytokine concentrations and cancer patient outcomes have been reported. In one example, the cytokines released by the harvested CAR-T or TCR-T cells can be, but are not limited to, IFN-gamma, IL-2, antigen Ki67, and TNF alpha.

[0079] In the methods of the disclosure, the T cells can be isolated T cells, derived from stem cells, or iPSC induced T cells. In one example, the T cells are isolated from a sample from a subject. The subject can be a healthy subject or a subject receiving cellular immunotherapy. The subject can be at risk of being diagnosed with cancer, or has been diagnosed with cancer, or is being treated for cancer.

[0080] The T cells can be obtained from blood, hematopoietic stem cell-derived lymphoid progenitor cells, embryonic stem cells (ESC), or induced pluripotent stem cells (iPSC). The sample used for T cell isolation can be, but is not limited to, a blood sample or a surgically removed tumor sample. In one example, the sample comprises peripheral blood mononuclear cells (PBMCs).

[0081] In another aspect, the disclosure provides a modified T cell obtained by the methods described herein. In another aspect, the disclosure provides a CAR-T cell obtained by the methods described herein. In another aspect, the disclosure provides a TCR-T cell obtained by the methods described herein. In a further aspect, the disclosure provides a modified T cell as disclosed herein, a CAR-T cell as disclosed herein, or a TCR-T cell as disclosed herein, for use in therapy.

[0082] In another aspect, the disclosure provides a method of treating or reducing cancer in a subject. The treatment or reduction outcome in a subject can be evaluated according to established clinical criteria. For example, the evaluation can include, but is not limited to: overall function, quality of life (QOL), pain, cognition, fatigue, and objective measures, such as tumor size and overall survival (OS) and progression-free survival (PFS).

[0083] In another aspect, the present disclosure provides a method of inducing long-term anti-tumor effects in a subject having a cancer.

[0084] In another aspect, the present disclosure provides a method of reducing tumor size in a subject having a cancer.

[0085] In another aspect, the present disclosure provides a method of increasing memory T cell and CAR-T cell stemness in a subject.

[0086] In another aspect, the present disclosure provides a method of increasing tumor infiltration of T cells in a subject having a cancer.

[0087] In another aspect, the present disclosure provides a method of increasing the rate of in vivo expansion of CAR-T cells or TCR-T cells in a subject.

[0088] Methods as described herein include administering to a subject in need thereof a therapeutically effective amount of a modified T cell as described herein, administering a therapeutically effective amount of a CAR-T cell as described herein, or administering a therapeutically effective amount of a TCR-T cell as described herein. As used herein, the term "pharmaceutically effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying cause, prevent symptoms and / or their underlying cause from occurring, and / or ameliorate or remedy damage caused or related to the disease state (e.g., reduce infection). A person of skill in the art is able to determine a pharmaceutically effective amount of a CAR-T or TCR-T cell as described herein, a composition or pharmaceutical composition as disclosed herein, depending on considerations such as the disease state, body size, frequency and route of administration. Any pharmaceutical or medical composition described herein can be administered with acceptable pharmaceutical excipients and / or additives and / or carriers. Such additional components are well known in the art.

[0089] Administration of the CAR-T cells or TCR-T cells can be by intravenous infusion. One of skill in the art will appreciate that other routes of administration can be suitable for delivering the cells intact to a subject. Administration can be a single (monodose) administration or repeated administrations. In one example, the CAR-T cells or TCR-T cells are formulated as a single administration unit. In another example, the CAR-T cells or TCR-T cells are formulated as multiple administration units, for example two, three or four units per day or per week. The amount and frequency of administration can be determined by one of skill in the art as appropriate depending on, for example, the disease state and / or the condition of the subject.

[0090] In some examples, the modified cells, CAR-T cells or TCR-T cells as described herein can be used in combination with other agents. For example, anti-cancer agents or therapeutic agents for controlling symptoms or agents that improve the efficiency of delivery of the cells. In some examples, the modified cells, CAR-T cells or TCR-T cells as described herein can be used in combination with other anti-cancer therapies.

[0091] In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for treating cancer. In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for inducing long-term anti-tumor effect in a subject having cancer. In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for reducing tumor size in a subject having cancer. In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for increasing memory T cells and CAR-T cell stemness in a subject. In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for increasing tumor infiltration of T cells in a subject having cancer. In another aspect, the disclosure provides use of the modified T cells as disclosed herein, the CAR-T cells as disclosed herein or the TCR-T cells as disclosed herein in the manufacture of a medicament for increasing the rate of in vivo expansion of CAR-T cells or TCR-T cells in a subject.

[0092] In methods or uses as disclosed herein, the subject can have a cancer, or be at risk of having a cancer. In some examples, the cancer is a benign or malignant cancer. Methods and clinical criteria for determining whether a cancer is a benign or malignant cancer are known to those of skill in the art. For example, the cancer can include, but is not limited to, leukemia, myeloma, sarcoma, melanoma, lymphoma, breast cancer, colon cancer, bladder cancer, prostate cancer, lung cancer, kidney cancer, pancreatic cancer, liver cancer, uterine cancer, ovarian cancer, or testicular cancer. In some examples, the cancer is a solid tumor cancer. For example, the cancer can include, but is not limited to, melanoma, sarcoma, melanoma, lymphoma, breast cancer, colon cancer, bladder cancer, prostate cancer, lung cancer, kidney cancer, pancreatic cancer, liver cancer, uterine cancer, ovarian cancer, or testicular cancer. In some examples, the cancer can be: B-lymphoma cells (RAJI) and breast cancer cells (TNBC: MDA-MB231-LN, HER2+: MDA-MB-361, SkBr3, BT-474).

[0093] The methods provided in the present disclosure are advantageous compared to the prior art because the methods as disclosed herein identify prolyl hydroxylase inhibitors that can promote T cell stemness and inhibit T cell exhaustion. The methods described herein allow for the application of prolyl hydroxylase inhibitors as described herein during CAR-T production to generate modified CAR-T cells with higher proliferation potential and higher percentage of memory T cells compared to conventional CAR-T cells.

[0094] Accordingly, applications of the methods disclosed herein include, but are not limited to, the use in research and product development for the optimization of cell immunotherapy models. Customized modified T cells (e.g., STEM CAR-T cells or TCR-T cells) can be produced as commercial products for hospitals, pharmaceutical companies, or individuals.

[0095] The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, the terms "comprising," "including," containing", and the like, shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have taken on a meaning that follows accepted usage by those skilled in the technical areas in which the disclosure is being discussed. As used herein, such terms and expressions are used where appropriate to convey the intended meaning to those skilled in the art, and are not to be taken in a literally or restricted sense, which is stated to be understood by those skilled in the art to which the claimed disclosure pertains. Accordingly, it is to be understood that although the present disclosure has been made specifically with respect to the preferred embodiments and optional features, modifications and variations of the disclosure herein disclosed that are obvious or apparent to those skilled in the art are deemed to be within the concept and scope of the disclosure.

[0096] It should be further understood that the exemplary embodiments are only examples and are not intended to limit the scope, applicability, or configuration of the disclosure in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the disclosure, it being understood that various changes can be made in the function and arrangement of elements and method steps described in the exemplary embodiments without departing from the scope of the disclosure as set forth in the appended claims.

[0097] Experimental Section Example 1: Conventional and STEM CAR-T cell production Day 0: T cell activation and modification 1. Thaw peripheral blood mononuclear cells (PBMCs) or purified T cells.

[0098] 2. Determine the number of cells needed.

[0099] 3. For every 1 x 10 6 cells, resuspend the cells in 1 mL T cell activation media (contains: 1 mL T cell media (Stemcell Technology ImmunoCult™-XF T Cell Medium, Cat# 10981); 10 ng / mL human IL2; 10 μL of CD3 / CD28 beads (Miltenyi Biotec T Cell TransAct CD3 / CD28 beads, Cat# 130-128-758). To obtain STEM CAR-T cells, 2 μΜ 1,4-DPCA-ester or any prolyl hydroxylase inhibitor can be added to the T cell activation media on day 0 of T cell activation. Regular CAR T cells are produced without the addition of a prolyl hydroxylase inhibitor, or treated with the vehicle for the prolyl hydroxylase inhibitor, which is DMSO.

[0100] 4. Incubate the cells in a tissue culture incubator at 37 °C, 5% CO2 for 5 days.

[0101] Day 3: CAR viral transduction Add 100 μL of concentrated viral supernatant to the activated and modified T cells along with 6 μg / mL of polybrene. One skilled in the art can adjust the amount of viral supernatant. Alternative methods of CAR viral transduction can be used.

[0102] Day 5 - Day 12: Conventional CAR-T and STEM CAR-T cell expansion 1. Collect all cells and perform a cell count with a small portion of the cells; 2. Centrifuge remaining cells at 300 x g for 10 minutes to wash away CAR virus and CD3 / CD28 beads. Completely aspirate supernatant.

[0103] 3. Resuspend cells in 1 mL T cell expansion media (contains: 1 mL T cell media (Stemcell Technology ImmunoCult™-XF T Cell Medium, Cat# 10981); 10 ng / mL human IL7; 10 ng / mL human IL15) for every 0.25 x 10 6 6

[0104] 4. Continue T cell expansion at 0.25 x 10 6

[0105] 5. On days 7 and 10, count and reseed cells with fresh T cell expansion media at 0.25 x 10 6

[0105]

[0106] 6. From day 7 to day 12, when cells reach sufficient quantity, both regular CAR-T cells and STEM CAR-T cells can be harvested and used.

[0107] Example 2: STEM-T cell modification / reprogramming and expansion STEM-T technology as disclosed herein is generally used to modify T cells, which can be applied to T cell-based adoptive immunotherapy, such as CAR-T or TCR-T therapy. Exemplary protocols for STEM-T cell modification / reprogramming and expansion are provided below.

[0108] Day 0: T cell activation 1. Thaw PBMCs or purified T cells.

[0109] 2. Determine the number of cells needed.

[0110] 3. Resuspend cells in 1 mL T cell expansion media (contains: 1 mL T cell media (Stemcell Technology ImmunoCult™-XF T Cell Medium, Cat# 10981); 10 ng / mL human IL7; 10 ng / mL human IL15) for every 1 x 10 6 ​Cells, resuspend cells at 0.25 x 10

[0111] 4. Incubate cells in tissue culture incubator at 37 °C, 5% C02 for 5 days.

[0112] Day 5 - Day 12: T cell expansion 1. Collect all cells and perform cell count with a small portion of cells.

[0113] 2. Centrifuge remaining cells at 300 x g for 10 minutes to wash away CD3 / CD28 beads. Completely aspirate supernatant.

[0114] 3. For every 0.25 x 10 6 Cells, resuspend cells in 1 mL T cell expansion media (contains: 1 mL T cell media (Stemcell Technology ImmunoCult™-XF T Cell Medium, Cat # 10981); 10 ng / mL human IL7; 10 ng / mL human IL15; or optionally 2 μΜ 1,4-DPCA-ester or any prolyl hydroxylase inhibitor. Addition of 1,4-DPCA-ester or any prolyl hydroxylase inhibitor during the T cell expansion step is optional.

[0115] 4. Continue T cell expansion at 0.25 x 10 6 Cells / mL T cell expansion media.

[0116] 5. On days 7 and 10, count and reseed cells at 0.25 x 10 6 Cells / mL with fresh T cell expansion media. Optionally, 2 μΜ 1,4-DPCA-ester or any prolyl hydroxylase inhibitor can be added to the T cell expansion media at this step.

[0117] 6. From day 7 to day 12, STEM T cells are ready to be harvested and used when cells reach sufficient quantity.

[0118] Example 3: Co-culture experiments and exhaustion assays: Cancer cells were seeded in 24-well plates for 1 day to form a monolayer. The culture medium of cancer cells was then removed and CAR T cells were subsequently added to the cancer cells for 72 hours. Co-cultured CAR T cells were subsequently used for flow cytometry to check PD1 and Tim3 expression of the depleted cell population, which is PD1+Tim3+.

[0119] Example 4: Co-culture cytotoxicity assays: Co-culture cytotoxicity assay uses luciferase-based killing assay, CAR T cells were incubated with cancer cells stably expressing luciferase at the indicated effector to target (E:T) ratios. After 48 hours of co-culture, 1x luciferase substrate luciferin was added to the cells and chemiluminescent signal was detected by a GlowMAX Explorer (Promega). This measurement was used to indicate cell viability against cancer cells in the absence of co-culture. The percentage of cytotoxicity of the cells was calculated using the following formula:

[0120] Example 5: CAR T-cell and STEM CAR-T cell production Anonymous human healthy donor peripheral blood mononuclear cells (PBMCs) were used to produce regular CAR-T cells and STEM CAR-T cells. To produce CAR-T cells, at day 0, ImmunoCult TM -XF T Cell Expansion Medium (Stemcell Technologies; Cat# 10981) or any other suitable medium for culturing T cells, using 10 pL T Cell Transact activation batch of PBMCs containing CD3 and CD28, for 5 days. To produce STEM T-cells, prolyl hydroxylase inhibitor was added to the culture medium at day 0. An equal volume of DMSO was provided to the culture of regular T cells at day 0. At day 3, cells were transduced with 100 pL CAR lentivirus in the presence of 6 pg / mL polybrene. At day 5, CD3 / CD28 agonist beads were removed by washing the cells twice in lx PBS. Cells were further expanded in ImmunoCult TM -XF T Cell Expansion Medium, maintaining a density of 250,000 cells / mL from day 5. Subsequently, regular CAR T cells and STEM CAR T cells were subcultured every 3 days under the same conditions.

[0121] CAR-T cells are ready for use on day 7.

[0122] To characterize the expansion rate and differentiation profile of CAR-T cells, manual cell counts were performed and recorded at the indicated days.

[0123] Example 6: Mouse xenograft models NSG mice were injected with 2 x 10 6 MDA-MB-231 cells, 5 x 10 6 MDA-MB-361 cells, or 2 x 10 6 DLD-1 cells suspended in 50 μL phosphate buffered saline (PBS) to establish human breast cancer or colon cancer models. CAR T cells targeting ROR1, HER2, or EGFR1 were injected at 2 x 10 6 , 0.5 x 10 6 , and 2.5 x 10 6 cells in 200 μL PBS into the relevant mouse models, respectively. Mice were measured for tumor volume twice a week. Blood was collected from mice through retro-orbital sinus once a week for FACS analysis. Data are shown in the description Figures 12-15

[0124] The embodiments set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the compositions, systems, and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Modifications of the above-described ways of practicing the disclosure can be made by those skilled in the art without departing from the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. All references cited herein are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0125] Many modifications and variations of this application can be made without departing from its scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and this application is limited only by the terms of the appended claims and the full range of equivalents that are properly applied to the terms of those claims.​

Claims

1. A method of modifying a T cell during T cell activation comprising modifying the T cell by contacting the T cell with an inhibitor of prolyl hydroxylases.

2. A method of making a modified T cell comprising modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases, wherein the modification comprises increasing the rate of expansion of the T cell compared to an unmodified T cell.

3. The method of claim 1 or 2, wherein the inhibitor of prolyl hydroxylases is a small molecule prolyl hydroxylase inhibitor.

4. The method of any one of claims 1-3, wherein the modified T cell is used in adoptive T cell immunotherapy.

5. A method of generating a chimeric antigen receptor (CAR) T cell comprising: modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases; introducing a CAR transgene into the modified T cell of (a) to generate a CAR-T cell; and harvesting the CAR-T cell after (b).

6. The method of claim 5, wherein steps (a) and (b) are performed simultaneously.

7. The method of any one of the preceding claims, wherein the period of time for modifying the T cell is about 1 to 5 days.

8. A method of generating a T cell receptor (TCR) T cell comprising: (i) modifying a T cell during T cell activation by contacting the T cell with an inhibitor of prolyl hydroxylases; (ii) introducing a TCR transgene into the modified T cell of (i) to generate a TCR-T cell; and (iii) harvesting the TCR-T cell after (ii).

9. The method of claim 8, wherein steps (i) and (ii) are performed simultaneously.

10. The method of any one of claims 5-9, wherein the harvested CAR-T cell or TCR-T cell is used in adoptive T cell immunotherapy.

11. The method of any one of claims 1-10, wherein the inhibitor of prolyl hydroxylases is selected from the group consisting of 1,4-DPCA, 1,4-DPCA ethyl ester, molisote, panaxynol, enasidenib, Roxadustat, IOX2, Daprodustat, and combinations thereof.

12. A modified T cell obtained by the method of any one of claims 1-4.

13. A CAR-T cell obtained by the method of any one of claims 5-7 and 10-11.

14. A TCR-T cell obtained by the method of any one of claims 8-11.

15. A method of treating or reducing cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

16. A method of inducing a long-term anti-tumor effect in a subject having cancer comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

17. A method of reducing tumor size in a subject having cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

18. A method of increasing memory T cell and CAR-T cell stemness in vivo in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

19. A method of increasing tumor infiltration of T cells in a subject having cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

20. A method of increasing the rate of in vivo expansion of CAR-T cells or TCR-T cells in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cell of claim 13 or the TCR-T cell of claim 14.

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