Methods for reversing t-cell exhaustion and restoring stemness for cell therapies

Abstract

The present disclosure pertains to methods for reversal of exhaustion phenotypes and restoration of stem-like phenotypes of T-cells, and compositions comprising T-cells produced by such methods. Methods for reversal of T‑cell exhaustion and restoration of stemness comprise inhibiting at least a portion of polycomb repressive complexes 1 & 2 (PRC1 / 2) in the T-cell and contacting the T-cell with interleukin-15 (IL-15), IL-7, or a combination thereof. T-cells with restored stem-like phenotypes can be used as at least a portion of one or more cancer therapies, such as tumor-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR) T-cell therapy, and the like.

Description

METHODS FOR REVERSING T-CELL EXHAUSTION AND RESTORING STEMNESS FOR CELL THERAPIESCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 747763, filed January 21, 2025, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF TECHNOLOGY

[0002] The present disclosure relates to immunology and immunotherapy. More particularly, the disclosure relates to methods, compositions, and systems for reversing exhaustion in T cells, restoring immune function, and treating diseases.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with government support under Grant No. R21EB027327, awarded by the National Institute of Biomedical Imaging and Bioengineering. The government has certain rights in the invention.BACKGROUND

[0004] T cell exhaustion is a state of dysfunction that arises during chronic antigen exposure, including but not limited to cancer, chronic infection, and autoimmune or inflammatory conditions. Exhausted T cells are characterized by diminished effector function, impaired proliferative capacity, altered transcriptional and epigenetic stats, metabolic dysfunction, and sustained expression of inhibitory receptors.

[0005] Existing therapeutic approaches have largely focused on blocking individual inhibitory receptors, such as programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocytes-associated protein 4 (CTLA-4). While such approaches can transiently enhance immune responses in some contexts, they frequently fail to fully restore T cell function, are ineffective in patients, and may result in immune-related adverse events.

[0006] Accordingly, there exists a need for improved methods capable of reversing, rather than merely bypassing, T cell exhaustion and restoring durable immune functionality across a range of diseases.SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] In an aspect, the disclosure provides a method for reversal of an exhaustion phenotype and restoration of a stem-like phenotype of a T-cell in vitro, the method comprising: resting the T-cell in absence of T-cell receptor (TCR) stimulation; inhibiting at least a portion of poly comb repressive complexes 1(PRC 1) and / or polycomb repressive complexes 2 (PRC2) in the T-cell; and contacting the T-cell with at least one cytokine. In some embodiments, the at least one cytokine comprises interleukin- 15 (IL-15), interleukin-7 (IL-7), or a combination thereof.

[0009] In embodiments, the exhaustion phenotype is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the T-cell. In some embodiments, the stem-like phenotype is characterized at least in part by upregulation of TCF7 in the T-cell relative to the exhaustion phenotype.

[0010] In embodiments, the inhibiting at least a portion of PRC1 and / or PRC2 comprises inhibiting a protein component of polycomb repressive complex 2 (PRC2).

[0011] In embodiments, the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

[0012] In embodiments, the inhibiting at least a portion of PRC1 and / or PRC2 in the T-cell comprises inhibiting EZH2. In some embodiments, inhibiting EZH2 comprises contacting the T-cell with a small molecule inhibitor of EZH2.

[0013] In embodiments, the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropy 1] -6- [6-( 1 -piperaziny 1) -3 -pyridinyl] - 1 H-indole-4-carboxamide), CPI- 1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

[0014] In embodiments, the inhibiting at least a portion of PRC1 and / or PRC2 in the T-cell comprises contacting the T-cell with an RNA interference (RNAi) inhibitor of EZH2.

[0015] In embodiments, the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

[0016] In embodiments, the inhibiting at least a portion of PRC 1 and / or PRC2 in the T-cell comprises knocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4. RBBP7, or any combination thereof, by way of a nuclear genomic editing technique.

[0017] In embodiments, the nuclear genomic editing technique comprises a clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) genomic editing technique.

[0018] In embodiments, the inhibiting at least a portion of PRC 1 and / or PRC2 comprises inhibiting a protein component of polycomb repressive complex 1 (PRC1).

[0019] In embodiments, the protein component of PRC 1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (R1NG1) and YY1 transcription factor (YY1) binding protein (RYBP), RING1A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

[0020] In embodiments, the inhibiting at least a portion of PRC 1 and / or PRC2 in the T-cell comprises inhibiting RING1A. In some embodiments, inhibitin RING1A comprises contacting the T-cell with a small molecule inhibitor of RING1A.

[0021] In embodiments, the small molecule inhibitor of RING1A comprises PRT4165 (2-(3-pyridinylmethylene)- 1 H-indene- 1 ,3(2H)-dione).

[0022] In embodiments, the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

[0023] In embodiments, the nuclear genomic editing technique comprises a CRISPR-Cas9 genomic editing technique.

[0024] In embodiments, the method further comprises contacting the T-cell with interleukin-2 (IL-2).

[0025] In an aspect, the disclosure provides a method for preparation of a tumorinfiltrating lymphocyte (TIL) therapy for a subject, the method comprising: isolating a T-cell from a tumor of the subject and, after isolating the T-cell from the tumor of the subject and before administering the T-cell to the subject, reversing an exhaustion phenotype of the T-cell and restoring a stem-like phenotype of the T-cell. In some embodiments, reversing an exhaustion phenotype and restoration of a stem-like phenotype of the T-cell comprises: resting the T-cell in vitro in absence of T-cell receptor (TCR) stimulation; inhibiting at least a portion of poly comb repressive complexes 1(PRC 1) and / or polycomb repressive complexes 2 (PRC2) in the T-cell; and contacting the T-cell with at least one cytokine. In some embodiments, the at least one cytokine comprises interleukin- 15 (IL-15), interleukin-7 (IL-7), or a combination thereof.

[0026] In an aspect, the disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject the TIL therapy of the present disclosure; optionally wherein the cancer comprises melanoma, renal cell carcinoma, or both.

[0027] In embodiments, the method further comprises administering to the subject an effective amount of IL-2.

[0028] In an aspect, the disclosure provides a method for preparation of a chimeric antigen receptor (CAR) T-cell therapy, the method comprising: resting a CAR T-cell in absence of TCR stimulation; inhibiting at least a portion of PRC 1 and / or PRC2 in the CAR T-cell; and contacting the CAR T-cell with at least one cytokine. In some embodiments, the at least one cytokine comprises interleukin- 15 (IL- 15), interleukin-7 (II -1), or a combination thereof. In an embodiment, CAR T-cell therapy comprises the CAR T-cell disclosed herein.

[0029] In an aspect, the disclosure provides a method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cell of the present disclosure.

[0030] In yet another aspect, the disclosure provided a composition comprising isolated T cells modified ex vivo to reverse an exhaustion phenotype in the isolated T cells. In embodiments, the modification comprises resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the isolated T cells with an inhibitor effectivein inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine. In some embodiments, the at least one cytokine comprises interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

[0031] In an embodiment, the exhaustion phenotype in the isolated T cells is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the isolated T-cells relative to reference T cells or nonexhausted T cells. In some embodiments, the modification is characterized at least in part by upregulation of TCF7 relative to the exhaustion phenotype levels of TCF7 in the isolated T cells before the modification.

[0032] In an embodiment, the inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC1 / 2) in the isolated T-cells comprises inhibiting a protein component of Polycomb Repressive Complex 2 (PRC2). In some embodiments, the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 Polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

[0033] In some embodiments, the inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC1 / 2) in the isolated T-cells comprises inhibiting EZH2. In some embodiments, inhibiting EZH2 comprises contacting the isolated T-cell with a small molecule inhibitor of EZH2. In embodiments, the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438: N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide), CPI-1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l -(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

[0034] In some embodiments, the inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC 1 / 2) in the isolated T-cells comprises knocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique. In anembodiment, inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC1 / 2) in the isolated T-cells comprises contacting the isolated T-cells with an RNA interference (RNAi) inhibitor of EZH2. In some embodiments, the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

[0035] In embodiments, the inhibiting at least a portion of Polycomb Repressive Complex 1 and / or Polycomb Repressive Complex 2 (PRC1 / 2) in the isolated T-cells comprises inhibiting a protein component of Polycomb Repressive Complex 1 (PRC1). In certain embodiments, the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), RING1A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), Polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

[0036] In some embodiments, inhibiting at least a portion of Polycomb Repressive Complex 1 in the isolated T-cells comprises inhibiting RING1A. In certain embodiments, inhibiting RING1A comprises contacting the isolated T-cells with a small molecule inhibitor of RING1A. In an embodiment, the inhibiting at least a portion of PRC 1 / 2 in the isolated T-cells comprises contacting the isolated T-cells with a small molecule inhibitor of RING1A. In embodiments, the small molecule inhibitor of RING1 A comprises PRT4165 (2-(3-pyridinylmethylene)-lH-indene-l,3(2H)-dione). In an embodiment, the inhibiting at least a portion of PRC1 / 2 in the isolated T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

[0037] In some aspects, the isolated T cells are further modified to express a recombinant receptor. In an embodiment, the recombinant receptor is an engineered T cell receptor (TCR). In an embodiment, the recombinant receptor is specific for a tumor antigen.

[0038] In certain embodiments, the isolated T cells are native, naturally occurring T cells, autologous T cells, or T cells with specificity for and activity against a tumor. In some embodiments, the native, naturally occurring T cells are obtained from resected tumors, and wherein the T cells are expanded ex vivo prior to the modification. In someembodiments, the T cells with specificity for and activity against a tumor are peripheral blood derived-T cells genetically modified to express a receptor that recognizes and responds to tumor.

[0039] In embodiments, the isolated T cells are selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, gamma delta T cells, a combination of CD4+ and CD8 T+ cells, memory T cells, cytokine-induced killer cells, and combinations thereof. In an embodiment, the isolated T cells are a combination of CD4+ and CD8+ cells.

[0040] In yet another aspect, the disclosure provided compositions and methods as disclosed herein for use in a method of treating a disease or pathological condition in a subject. In some embodiments, the method comprises administering to the subject an effective amount of one or more compositions of the present disclosure. In some embodiments, the disease or the pathological condition is a tumor or cancer, optionally a solid tumor or hematopoietic malignancy. In some embodiments, the method further comprises administering to the subject one or more of anticancer agents and / or one or more chemotherapeutic agents. In some embodiments, the disease or the pathological condition is a bacterial and / or parasitic infection. In an embodiment, the method further comprising administering to the subject one or more anti-bacterial or anti-parasitic agent.

[0041] In another aspect, the disclosure provides a kit comprising the compositions disclosed herein.

[0042] In yet another aspect, the disclosure provides a method for treating a cancer in a subject in need thereof. In some embodiments, the method comprises administering to the subject the TIL therapy disclosed herein or any one or more of the compositions disclosed herein. In an embodiment, the cancer comprises melanoma, renal cell carcinoma, or both. In some embodiments, further comprises administering to the subject an effective amount of IL-2.

[0043] In another aspect, the disclosure provides a method for preparation of a chimeric antigen receptor (CAR) T-cell therapy. In some embodiments, the method comprises resting CAR T-cells in absence of TCR stimulation; inhibiting at least a portion of PRC1 / 2 in the CAR T-cell; and contacting the CAR T-cells with at least one cytokine. In some embodiments, the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof. In an embodiment, the CAR T-cell therapy comprises the CAR T-cell.

[0044] Aspects of the disclosure provide a method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cells disclosed herein.

[0045] In yet another aspect, the disclosure provides an in vitro method of reversing T cell exhaustion comprising modifying isolated T cells. In some embodiments, the modification comprising resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the T cells with an inhibitor effective in inhibiting at least a portion of polycomb repressive complexes 1 and / or 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.DESCRIPTION OF THE DRAWINGS

[0046] The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0047] FIG. 1A shows a diagram of precursor exhausted, intermediate exhausted, and exhausted effector T cells and changes in transcription factors Tim3 and Tcf7 as a T cell can transition from a precursor exhausted to an exhausted effector T cell with silencing of Tcf7, according to aspects of the disclosure.

[0048] FIG. IB shows an experimental setup for CRISPR screen for identification of factors involved in silencing of Tcf7, according to aspects of the disclosure.

[0049] Mouse Model: in vitro chronic stimulation of mouse CD8+T-cells, recapitulating features of tumor exhaustion, including silencing of key memory transcription factor Tc / 7. The model was utilized to identify genes involved in epigenetically “locking down” the Tc / Zoff state in exhausted T-cells.

[0050] FIG. 1C shows example results demonstrating that targeted inhibition of PRC1 / 2, when paired with conditions promoting T-cell memory differentiation (rest from TCR stimulation, culture in IL- 15), can reverse exhaustion and restore a stem-like phenotype in exhausted T-cells, according to aspects of the disclosure. The figure shows experimental results from treatment of exhausted T cells rested in vitro in the presence of IL- 15 or IL-2 (10 or 100 U / mL) as well as absence (_Ezh2i) or presence (+Ezh2i) of the Ezh2 inhibitor Tazemetostat (Taz), showing 7c ZYFP+cells as a function of treatmentconditions, i.e., differential reactivation of key memory transcription factor Tcf7. Thus, rest in combination with PRC inhibition and IL- 15 broadly restores sternness in previously exhausted T-cells.

[0051] FIG. ID shows example flow cytometry results of exhausted T cells rested for 8 days in the presence of various small molecule Polycomb inhibitors: Tazemetostat (Ezh2i), PRT4165 (Ringli), and UNC3866 (CBX4i). Rest in the Ezh2 inhibitor Tazemetostat (Ezh2i) and IT- 15 reactivated Tc / 7-YFP in terminally exhausted CD8+ T-cells, according to aspects of the disclosure. Rest in Ezh2i in combination with Ringli led to the greatest increase in Tcf7 reactivation. Data are representative flow cytometry plots from three technical replicates per drug condition. The cells were exhausted according to an in vitro exhaustion protocol (FIG. 2A or FIG. 2D) and rested in 50 ng / mL of IL- 15.

[0052] FIG. IE shows example results illustrating that CRISPR / Cas9 KO of PRC2 or other PRC subunits, followed by rest in IL- 15, restores a 7c / 77CD62L+memorylike state in exhausted CD8+T-cells, according to aspects of the disclosure. Representative flow cytometry plots from three technical replicates per condition. NTC is a non-targeting control.

[0053] FIG. IF shows example results illustrating that 4 days of rest in Ezh2i (Taz) with IL- 15 accelerates reactivation of key transcription factors TCF1 (encoded by Tcf ) and Foxol compared to a DMSO carrier control, according to aspects of the disclosure. In various examples and embodiments, one can evaluate TCF1 (protein) expression, TcJ7 expression (e.g., messenger RNA, mRNA), or both.

[0054] FIG.1G shows example results from unsorted, in vitro exhausted T cells, illustrating that 4 days of rest in IL-15 with Ezh2i (Taz), RINGli (PRT4165), or both increases the percentage of Tcf7- YFP+ / CD62L+stem-like cells (CD62L expression), according to aspects of the disclosure. Representatives flow cytometry plots from three technical replicates per drug condition.

[0055] FIG.1H shows example results in sorted terminally exhausted and sorted intermediate exhausted T cells showing that 4 days of rest in IL- 15 with either Ezh2 inhibitor (Ezh2i) Tazemetostat, Ringl inhibitor PRT4165 (Ringli), or both increases expression of Slamf6+, according to aspects of the disclosure.

[0056] FIG. II shows example results showing that reprogrammed cells, when re-stimulated with anti-CD3 and anti-CD28 (aCD3 / 28) antibodies, IL-2, and IFNa for 4days, retained higher ' / c / Z-YI’P and lower TIM3, indicating reduced exhaustion differentiation, according to aspects of the disclosure.

[0057] FIG. 1J shows examples results showing that TCF1 reactivation potential can be restored by inhibition of chromatin regulators, according to aspects of the disclosure. Results are from a drug screen to see if inhibiting chromatin regulators would influence reactivation potential. A hit from this screen included the Ezh2 inhibitor tazemetostat (“Taz”), which blocks H3K27 methylation. When Taz is added during the rest phase, it drastically increases TCF1 reactivation in both populations.

[0058] FIG.2A shows a diagram showing chronic stimulation drives exhaustion differentiation in T cells and shows an ex vivo exhaustion assay according to aspects of this disclosure. A stem-like TCF1 high population can be maintained, and these can be called precursors of exhaustion or PEXcells. Like naive cells, these cells give rise to functional, effector-like cells, often called intermediate cells or TINT cells. These intermediate cells (TINT), upon chronic stimulation, rapidly give rise to exhausted cells (TEX), which have a loss of function. These three subsets can be distinguished by differential expression of TCF1, which is a marker of sternness, and TIM3, which is an inhibitory receptor that is uprcgulatcd in exhausted cells. Importantly, exhaustion is detrimental for tumor control, and therefore the more TCF1+cells a patient has the better their clinical responsiveness.

[0059] FIG. 2B shows a diagram illustrating exhausted cells are epigenetically fixed. Exhaustion can only be reversed early, and terminally exhausted cells may be epigenetically fixed in an irreversible state.

[0060] FIG. 2C shows a diagram and experimental results showing chronic stimulation drives stable repression of Tcf7 upon rest. The ex vivo model system accurately recapitulated the exhausted state. Increased stimulation time led to increased exhaustion differentiation as evidenced by the increase in Tcf7- YFP T IM3+cells. Tcf7 negative cells were sorted from each time point, and it was observed that the Tcf7 reactivation potential decreased with increasing time on stimulation.

[0061] FIG. 2D shows experimental results showing exhaustion differentiation drives stable repression of Tcf7 upon rest in an ex vivo chronic stimulation model. Cells stimulated for 6 days to generate exhausted cells and intermediate vs exhausted populations were sorted and recultured in the absence of stimulation. Intermediate population wasobserved to still have the potential to reactivate Tc / 7 with 4 days of rest compared to the exhausted cells which had diminished Tcf7 reactivation potential.

[0062] FIG. 2E shows epigenetic lockdown of TcJ7 reactivation potential in exhausted T cell population. Experimental results show stable silencing of Tcf7 is present in vivo in a B16-melanoma model. It was found that the TEX population had diminished reactivation potential, and this result was stable over time with rest. As such, the exhausted state is stable even in the absence of stimulation, which can indicate that Tcf7 is epigenetically locked down in these cells.

[0063] FIG. 2F shows an experimental setup for a Cas9 knockout (KO) screen to identify regulators controlling the initiation and maintenance of Tcf7 silencing, according to aspects of the disclosure. To address a question about the mechanisms governing the transition between the plastic TINT state and the epigenetically fixed TEX state, a CRISPR-Cas9 KO screen with 118 different gene targets was designed, including transcription factors (e.g., TcfT) and chromatin modifying enzymes. Cells from Cas9 mice crossed into the TC17 reporter were used; these cells were infected with a library of guide RNA at 1 day after activation to assay the initial silencing phase, and at 6 days after activation to measure the maintenance phase. For both screens, Tcf7 low and high bins were sorted to sequence and see which gRNA were enriched in each bin.

[0064] FIG. 2G shows an experimental design for sgRNA screen to identify genes responsible for promoting and for opposing Tcf7 silencing, according to aspects of the disclosure.

[0065] FIG. 2H shows experimental results from a sgRNA screen showing that early perturbation reveals multiple transcription factor (TF) regulators are responsible for initiation of Tc / 7 silencing. The results show that knocking out Tcf7 decreases Tcj7, indicating that the screen is working as intended, and in addition, transcription factors including Myc and Foxol decrease Tcf7 levels when knocked out, and other transcription factors including Prdml and Tbx21 increase Tcf7 expression when knocked out. Unexpectedly, many of the hits of this stage of the screen were transcription factors, not chromatin regulators, except histone deacetylase 1.

[0066] FIG.21 shows an experimental setup for sgRNA screen to identify which genes are responsible for stable maintenance of Tcf7 silencing, according to aspects of the disclosure. With the maintenance screen, TEX cells were generated, and after 6 days the library was transduced into the cells with the plot being the same as in the previous screen.

[0067] FIG. 2J shows experimental results from the maintenance screen, which reveals distinct regulators that maintain Tcf7 silencing in TEX. According to the results, transcription factors including Prdlm and Tbx21 are locking in the Tc / 7 off state, and Tc / 7 and Foxol knockouts are decreasing Tcf7 levels. The maintenance screen unexpectedly demonstrated more chromatin modifying enzymes in the results.

[0068] FIG. 2K shows a diagram illustrating polycomb repressive complexes (PRC) 1 and 2 that maintain Tcf7 silencing, according to aspects of the disclosure.

[0069] FIG.2L shows experimental results indicating that PRC 1 / 2 are important for maintenance, but not initiation, of silencing, according to aspects of the disclosure. A 2D plot of initial silencing versus maintenance (left) shows that the polycomb hits are anticorrelated between the two screens. To validate this anticorrelation, an inhibitor of a subunit of PRC2 drug was tested at the early silencing phase, and it was observed that inhibiting PRC2 decreased Tc / 7 levels, consistent with this anticorrelation effect.

[0070] FIG. 2M shows flow cytometry histograms showing that PRC 1 / 2 KO restores TCF1 high state in maintenance phase. CRISPR Cas9 was used to knock out individual PRC1 and PRC2 components in 6-day activated Tc / 7 silent cells, and it was observed that many cells were stuck in the TEX phase; however, with PRC1 component knockouts, increased Tc / 7 reactivation was observed, and this effect was also observed with knockout of PRC2 components. It was then asked whether PRC1 and PRC2 were working cooperatively.

[0071] FIG. 2N shows experimental results showing that PRC 1 / 2 cooperate to lock down the TEX state, according to aspects of the disclosure. Small molecule drug inhibitors of PRC1 (PRCli) and PRC2 (PRC2i) were contacted to TEX cells, and it was observed that the inhibitors were able to rescue cells from the TEX state, individually, and when combined, this effect was unexpectedly even more pronounced (quantified at right).

[0072] FIG.20 shows experimental results showing that joint PRC 1 / 2 inhibition can restore sternness in tumor-derived TEX cells, according to aspects of the disclosure. The B 16 melanoma model was used for collection and sorting of TEX cells at day 8 of the tumor. It was observed that there was minimal Tcf7 reactivation after 4 days of rest. With PRC2 inhibition, some increased reactivation was observed, but this effect was not seen with PRC1 inhibition; however, by combining PRC1 and PRC2 inhibition, the most reactivation potential was observed in the tumor derived cells.

[0073] FIG. 3A shows an overview of example experiments for the generation of improved TIL therapies, according to aspects of the disclosure. One can develop an epigenetic reprogramming strategy to revert terminally exhausted TILs to a stem-like state for enhanced persistence and antitumor function. This can be done in a mouse TIL model and in tumor-infiltrating lymphocytes (TILs) from human patients.

[0074] FIG. 3B shows a diagram illustrating Polycomb Repressive Complexes lock down Tcf7 silencing and uphold loss of sternness in terminally exhausted T cells, according to aspects of the disclosure. (A) A CRISPR / Cas9 screen implicates subunits of PRC1 and PRC2 in stable maintenance of Tcf7 silencing in exhausted T cells. (B) TCR and cytokine signals modulate Polycomb silencing of Tcf7 and other sternness regulators. (C) Terminally exhausted (Tc / 7‘TIM3+) T cells were sorted, cultured under epigenetic reprogramming conditions (+ / -PRC inhibition, no TCR stim, IL- 15), then analyzed by flow cytometry. Histograms show that reprogramming reverses stable silencing of TCF-1 and FoxOl, central transcriptional regulators of T cell sternness and self- renewal.

[0075] FIG.4A shows a diagram showing example approaches for elucidation of the mechanism of Polycomb-mediated Tcf7 silencing and commitment to terminal exhaustion, according to aspects of the disclosure; example approaches for determination of how this mechanism is modulated by cytokine-induced transcription factors; and example approaches, according to some aspects of the disclosure, to test whether inhibiting Polycomb restores sternness and antitumor function to exhausted TILs.

[0076] FIG. 4B shows a diagram of example approaches for elucidation of the mechanism by which PRC1 / 2 upholds Tcf7 silencing and loss of self-renewal, according to aspects of the disclosure. (A) One can distinguish two models: PRC2 read-write, followed by cPRCl chromatin compaction upholds silencing (Model 1; FIG. 4B (A)); alternatively, PRC2 / vPRCl read-write, coupled to H2AK119ubl -mediated repression upholds silencing (Model 2; FIG.4B (A). Table of gain and loss-of-function perturbations used to test these hypotheses (FIG.4B (B)). FIG.4B (C) shows experimental workflow.FIG. 4B (D) shows IP-MS data using FLAG-tagged EED (Embryonic Ectoderm Development (EED) a crucial core component of Polycomb Repressive Complex 2 (PRC2)) in hematopoietic stem cells, establishing feasibility for proposed proteomics work to detect Polycomb subunit composition. Scatterplot shows p- value vs fold increase for enrichment in Flag-EED cells relative to untagged cells. Table lists individual associated proteins. FIG. 4B (E) shows a SCEPTRE super-resolution image to visualizethe Tc / 7 promoter and H3K27me3 (red) in hematopoietic progenitors. Images show example single cell, with Tc / 7 locus boundaries circled.

[0077] FIG. 4C shows experimental results from an example minimal in vitro system that recapitulates differentiation of CD8+T cells into a terminally exhausted state, according to aspects of the disclosure. FIG. 4C(A) shows the experimental set up where CD8 T cells are isolated from 7c / 7‘YFP mice, cultured as indicated and analyzed by flow cytometry, scRNA-seq or sorting and reculture. FIGS. 4C(B) -4C(D) show flow plots showing that cells enter an exhausted state (TEx:7c 7+Tim3 ) upon 6 days of stimulation FIG. 4C(B), up-regulate the exhaustion-associated transcription factor Tox FIG. 4C(C) and show decreased cytokine production upon re-stimulation FIG. 4C(D). FIG. 4C(E) shows scRNA-seq UMAP dimensionality reduction plots (left) and condition heatmaps (right) show activated exhaustion programs at the transcriptional level. FIG.4C(F) shows TEX (7C 7TIM3+) or TINT (rc / 7Tim3 ) cells arising after 6 days of culture were sorted (left), re-cultured with rest (IL-2 only) after 4 days (right). Histograms show that terminally exhausted cells (TEX), in contrast to non-exhausted cells (TINT), show greatly reduced Tcf7 reactivation potential upon rest.

[0078] FIG. 4D shows experimental results showing that Polycomb Repressive Complexes (PRC) 1 and 2 maintain stable Tcf7 silencing during terminal T cell exhaustion, according to aspects of the disclosure; FIG. 4D(A) shows a CR1SPR / Cas9 screen workflow. CD8 T cells from 7c / 7YFP / Cas9 mice were cultured. 6 days later, TEX were sorted, transduced with a gRNA library targeting 118 TFs and chromatin regulators, then re-cultured with rest for 4 days. Transduced Tcf7+and Tcf7~ populations were then sorted and sequenced to obtain the gRNA enrichment in these two populations. FIG. 4D(B) is a scatterplot showing two replicates, revealing genes that up-regulate or silence Tcf7 expression. PRC1 / 2 are key mediators of Tcf7 silencing. FIGS. 4D(C)-4D(E) show individual PRC1 / 2 hits validated in TEX by Cas9-RNP nucleofection (FIG.4D(C)) or drug inhibition (FIG. 4D(D)) using the same experiment design. Inhibition of PRC1 (by Cbx4 KO; or Ringla inhibition with 10 pM PRT4165) or PRC2 (by Ezh2 KO or inhibition with 1 pM Taz) increases fraction of precursor (7c / 7+Tim3 ) cells FIGS. 4D(C)-4D(D), with combined drug inhibition of PRC1 / PRC2 synergistically enhancing Tcf7 activation FIG.4D (D, right).

[0079] FIG.4D(E) shows that when Ezh2 inhibitor (1 pM Taz) is added prior to exhausted state entry, it has the opposite effect, enhancing Tcf7 silencing. The Polycombsystem also underlies exhausted state stability in adoptive T cells from a mouse tumor model. FIG.4D(F-G) shows an experimental set up to test whether Polycomb mechanisms also operate in tumor-infiltrating T cells, 7c / 7YFP OT-I CD8 T cells were transferred into mice with B16-Ova tumors, sorted TEX within tumors after 8 days, then re-cultured them with + / -Taz (1 pM) ((FIG. 4D(F)). Flow plots show exhausted cells after isolation (left) and after 8 days of rest (right), showing that Ezh2 inhibition enhances Tcf7 re-activation ((FIG.4D(G)).

[0080] FIG.4E shows experimental results showing that Tcf7 and other memory genes acquire stable Polycomb domains during terminal T cell exhaustion in vitro, according to aspects of the disclosure. CD8+T cells were stimulated with aCD3 / CD28, IL2 and IFNa for 6 days. Tcf7~TIM3+cells were then sorted and rested for 8 days. Samples were removed at the indicated days for CUT&RUN analysis. d2,6 samples were cultured for 1 day in rest, then sorted for intermediate (7c / 7Tim3 ) and terminal (7c / 7 TIM3+) subpopulations. FIG. 4E(A) shows Genome-browser tracks show H3K27me3 and H2AK119ubl distributions at the Tcf7 locus, both for the samples, as well for precursor (PDTTIM3 ) and terminally exhausted (PD1+TIM3+) OT-I T cells populations in B16-Ova tumors at Day 14 (from 24). T cells in the in vitro system establish a large Polycomb (H3K27me3 / H2AK119ubl) domain at Tcf7 similar to that observed for B16 TILs. FIG.4E(B) shows line charts show normalized H3K27me3 / H2AKl 19ubl counts at Tcf7 locus (shading in FIG. 4E(A)) across time. FIG.4E(C) shows a Heatmap showing z-scores for H3K27me3 / H2AK119ubl levels over time for two groups of genes obtained from hierarchical clustering: one that stably gaining (top) or losing (bottom) both modifications (top). Tcf7 and other memory genes (e.g. Bach2, Id3, Lefl) acquire both histone modifications and stably maintain their levels after stimulation removal.

[0081] FIG. 4F shows experimental results showing that Polycomb-mediated lock down of Tcf7 silencing is reinforced by IL-2 but antagonized by IL- 15, according to aspects of the disclosure. ((FIGS.4F(A)-4F(Q). P14 CD8+T cells were exhausted in vitro for 6 days, sorted for TEX (Tc / 71ow, Tim3 high) phenotype, and rested with or without the Ezh2 inhibitor Tazemetostat (Ezh2i), in IL-2 or IL- 15. FIG. 4F(A) shows representative histograms of 7c / 7YFP expression after rest in indicated condition. FIG. 4F(B) shows representative flow cytometry plots showing differences in Tc / 77Slamf6+cells after 7 days of rest. FIG. 4F(C) shows a summary bar chart of FIG. 4F(A), three technical replicates per condition. FIG. 4F(D) shows representative histograms of Foxol levels in CD8+T-cells stimulated in vitro for 6 days, then rested in IL- 15 with or without Ezh2i for 4 or 8 days. FIG.4F(E) shows P14 CD8+T-cells were stimulated in vitro for 6 days, then rested in IL- 15 with or without Ezh2i for 4 or 8 days; representative flow cytometry plots showing differences in Tcf7+CD62L+cells. Thus, rest in combination with PRC inhibition and IL-15 broadly restores sternness in previously exhausted T-cells.

[0082] FIG. 4G shows an example experimental setup for testing Polycomb inhibition as a strategy for restoring sternness and antitumor function to exhausted T cells and TILs for adoptive cell therapy, according to aspects of the disclosure. Experiments can use a mouse neoantigen TIL therapy model (A) or human TCR-transduced T cells model (B).

[0083] FIG. 4H shows an example of modeling of neoantigen TIL therapy production and testing in mice using B16 melanoma. FIG.4H (A) shows an experimental workflow. Pmel-1 TCR transgenic mice were inoculated with B16F10 melanoma cells. After 2-3 weeks, TILs or splenic T cells were isolated from established tumors, expanded in vitro to generate infusion products, then adoptively transferred into the mice implanted with gplOOKVP-expressing B 16 cells. In all conditions, a gplOOKVPvaccinia virus vaccine was co-administcrcd during adoptive transfer. FIGS. 4H(B)-4H(C) show flow plots and bar charts show % of stem-like (CD39 CD69 ) populations in TIL versus splenic T cell infusion products. TIL products showed lower percentages of stem-like T cell populations compared to their splenic counterparts. FIG. 4H(D) shows growth curves for Bl 6-gplOOKVP tumors, showing that TIL infusion products show significantly weaker tumor control compared to infusion products generated from splenic T cells.

[0084] FIG. 41 shows example experimental results showing that TCR-engineered human T cells mount cytotoxic responses against a cancer cell line expressing the cognate neoantigen. Peripheral blood cells were transduced with R175H TCR, sorted for the indicated T cell subsets and cocultured in vitro with fluorescent TYK-nu cells, an ovarian cancer cell line that expresses p53R175H. Curves show tumor cell area over time upon co-culture with different TCR-transduced T cell subsets.

[0085] FIG. 5 shows TCF7 reactivation in terminally exhausted cells rested for 4 days. Terminally exhausted (Tcf7-, TIM3+) mouse primary CD8+ T-cells were generated in vitro, sorted, and rested for 4 days in: Ezh2 inhibitor (luM Tazemetostat) or vehicle control (0.1% DMSO); Paired with: IL-15 (50 ng / mL), IL-7 (5 ng / mL) or a combination of IL-15 (50ng / mL) and IL-7 (5 ng / mL).DETA1LED DESCRIPTION

[0086] In various aspects, the disclosure provides methods for reversal of exhaustion phenotypes and restoration of stem-like phenotypes of T-cells, particularly in vitro, such that the rejuvenated T-cells exhibit greater anti-tumor potency for TIL treatments, and the disclosed methods can also be useful for generating CAR T cells for CAR T cell treatments. In some embodiments, the method comprises resting the T-cell in absence of T-cell receptor (TCR) stimulation (including, for example, absence of downstream signaling such as anti-CD3 stimulation). In some embodiments, the method further comprises inhibiting at least a portion of polycomb repressive complex 1 and / or polycomb repressive complex 2 (PRC 1 / 2) in the T-cell and contacting the T-cell with at least one cytokine. In an embodiment the exhaustion phenotype may be an intermediate exhaustion phenotype. In an embodiment, the exhaustion phenotype is a terminal exhaustion phenotype.

[0087] In an aspect, the disclosure provides a method of reversing an exhaustion phenotype and restoring a stem-like phenotype of a T-cell comprises resting the T-cell in absence of T-cell receptor (TCR) stimulation (including, for example, absence of downstream signaling such as anti-CD3 stimulation), inhibiting at least a portion of polycomb repressive complex 1 and / or polycomb repressive complex 2 (PRC 1 / 2) in the T-cell, and contacting the T-cell with at least one cytokine. In some embodiments, the at least one cytokine comprises interleukin- 15 (IL- 15) (z.e., IL- 15 protein or a biologically active portion thereof), interleukin-7 (IL-7) (z'.e?., IL- 15 protein or a biologically active portion thereof), or a combination thereof.

[0088] In at least some instances, an exhaustion phenotype can be characterized, at least in part, by the absence or decreased potency of an anti-tumor activity of a T-cell (e.g., by a functional characterization), and / or the presence of one or more markers indicative of the exhaustion phenotype e.g., by a structural characterization). In at least some instances, the exhaustion phenotype is a terminal exhaustion phenotype that is characterized at least in part by the downregulation of transcription factor 7 (TCF7), which encodes TCF1 protein in the T-cell (e.g., relative to a reference or non-exhausted T cell). Furthermore, in at least some instances, the stem-like phenotype is characterized at least in part by upregulation of TCF7 / TCF1 in the T-cell relative to the exhaustion phenotype (e.g., relative to a reference or non-stem-like T-cell).

[0089] In an embodiment the exhaustion phenotype may be an intermediate exhaustion phenotype. In some aspects, the exhaustion phenotype is at least partially characterized by loss or downregulation of TCF1 and TIM3 (e.g., relative to a reference or non-exhausted T cell). In an embodiment, the exhaustion phenotype is a terminal exhaustion phenotype. In some aspects, the exhaustion phenotype is at least partially characterized by loss or downregulation of TCF1 and upregulation or an increased expression of TIM3 (e.g., relative to a reference or non-exhausted T cell).

[0090] In embodiments, the portion of PRC 1 / 2 that is inhibited by the method comprises a protein component of polycomb repressive complex 2 (PRC2) e.g., a protein or polypeptide that constitutes at least a portion of the PRC2 complex). In example embodiments, the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof. However, one or more other protein components of PRC2 can be inhibited as part of a method of the disclosure, without departing from the scope and spirit of the disclosure.

[0091] In some embodiments, the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cell with an agent capable of inhibiting a component of the polycomb repressive complex 2 (PRC2) (e.g., a protein or polypeptide that constitutes at least a portion of the PRC 2 complex). In embodiments, the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of EZH2. In example embodiments, the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide), CPI- 1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

[0092] In embodiments, the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with an RNA interference (RNAi) inhibitor of EZH2. Inembodiments, the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

[0093] In embodiments, the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises knocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique. While any genome editing technique can be implemented, in example embodiments, the nuclear genomic editing technique comprises a clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) genomic editing technique.

[0094] In embodiments, the portion of PRC 1 / 2 comprises a protein component of polycomb repressive complex 1 (PRC1) (e.g., a protein or polypeptide that constitutes at least a portion of the PRC1 complex). In embodiments, the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), RING1 A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof. However, one or more other protein components of PRC 1 can be inhibited as part of a method of the disclosure, without departing from the scope and spirit of the disclosure.

[0095] In some embodiments, the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with an agent capable of inhibiting a component of the polycomb repressive complex 1 (PRC 1) (e.g., a protein or polypeptide that constitutes at least a portion of the PRC 1 complex). In embodiments, the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of RING1 A. In embodiments, the small molecule inhibitor of RING1 A comprises PRT4165 (2-(3-pyridinylmethylene)-lH-indene-l,3(2H)-dione).

[0096] In embodiments, the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique. While any genome editing technique can be implemented, in exemplary embodiments, the nuclear genomic editing technique comprises a CRISPR-Cas9 genomic editing technique.

[0097] In embodiments, the method further comprises contacting the T-cell with interleukin-2 (IL-2), which can be before, after, or concomitantly with contacting the T-cell with IL- 15.

[0098] Adoptive cell therapies harnessing patient-derived tumor infiltrating lymphocytes (TILs) hold tremendous promise for curative treatment of advanced stage cancers. T cells have unique abilities to recognize and eliminate cancer cells and are now being intensely investigated as therapeutic agents against diverse solid cancers. Compared to other T cell therapies, TILs have two advantages: First, T cells in TIL products target multiple tumor antigens and are polyclonal, making them potentially less susceptible to antigen escape compared to antigen receptor-engineered T cells. Second, as TILs are selected to target tumor-specific antigens, they are less likely to destroy healthy tissues compared to engineered T cells. Indeed, adoptive cell therapy (ACT) using TILs has shown promise against multiple types of solid cancers and has gained FDA approval for treatment of metastatic melanoma refractory to immune checkpoint inhibitors. However, despite their promise, TIL therapies remain largely ineffective against most solid tumors, as TILs with neoantigen specificity often exist in an exhausted state with limited antitumor efficacy.

[0099] In various aspects, the disclosure provides approaches for the improvement or enhancement of TIL therapies. TIL therapy includes types of adoptive cell transfer (ACT) immunotherapy in which the therapy uses a patient’s own tumor-infiltrating lymphocytes (TILs), which are isolated from a resected growing tumor, expanded to large numbers in the laboratory (e.g., optionally using interleukin-2 (IL- 2), a T cell growth factor), and then reinfused into the patient under specialized conditions where they target tumor cells, (see, e.g., Steven A. Rosenberg, Lymphocytes as a living drug for cancer. Science. 385,25-26(2024). DOLlO.l 126 / science.adpl 1 0).

[0100] In an aspect, the disclosure provides a method for preparation of a TIL therapy for a subject, the method comprising: isolating a T-cell from a tumor of the subject and, after isolating the T-cell from the tumor of the subject and before administering the T-cell to the subject, reversing an exhaustion phenotype of the T-cell and restoring a stemlike phenotype of the T-cell. The method of reversing the exhaustion phenotype and restoring the stem-like phenotype can comprise resting the T-cell in absence of TCR stimulation (including, for example, absence of downstream signaling such as anti-CD3 stimulation), inhibiting at least a portion of PRC1 / 2 in the T-cell, and contacting the T-cellwith at least one cytokine, where the cytokine is IL-15, IL-7, or a combination thereof (z'.e., a protein or a biologically active portion thereof).

[0101] In another aspect, the disclosure provides a method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject the TIL therapy of the present disclosure; optionally wherein the cancer comprises melanoma, renal cell carcinoma, or both. The administering to the subject can be by way of infusion or another suitable administration route, with timing and dosages administered determined by a person having ordinary skill in the art. In embodiments, the method further comprises administering to the subject one or more other anti-cancer therapies, treatments, or agents, such as IL-2 (e.g., in an effective amount or as part of a combination therapy). Administration of IL-2 to the subject can be before, after, or concomitantly with contacting the T-cell with the at least one or more cytokine.

[0102] T cell exhaustion has been increasingly incriminated as a cause of T cell dysfunction in CAR T cells. Tonic antigen-independent signaling, due to scFv aggregation, commonly occurs in T cells expressing CARs and can induce rapid exhaustion (Long AH, Haso WM, Shem JF, et al. 4- IBB co-stimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med.2015;21(6):581-590. doi:10.1038 / nm.3838; herein incorporated by reference in its entirety). Integration of the CD28 endodomain into second generation CAR T cell receptors enhances expansion, but also predisposes CAR T cells to exhaustion, both in the setting of Ionically signaling receptors and in CD19-28z CAR T cells exposed to high tumor burdens. Increased frequency of T cells bearing exhaustion characteristics contained within CD19-BBz CAR grafts were recently demonstrated to distinguish nonresponding from responding patients treated for CLL3. A broad base of data from diverse studies implicates intrinsic T cell dysfunction due to T cell exhaustion as a major factor limiting the efficacy of CAR T cell therapeutics and raises the prospect that engineering exhaustion-resistant CAR T cells could substantially improve clinical outcomes.

[0103] In another aspect, the disclosure provides a method for preparation of a chimeric antigen receptor (CAR) T-cell therapy. The method comprises: resting a CAR T-cell in absence of TCR stimulation, inhibiting at least a portion of PRC1 / 2 in the CAR T-celL and contacting the CAR T-cell with at least one cytokine, where the cytokine is IL- 15 (i.e., IL- 15 protein or a biologically active portion thereof), IL-7 (i.e., IL-7 protein or abiologically active portion thereof), or a combination thereof. The CAR T-cell therapy comprises the CAR T-cell, for example, as a composition (e.g., pharmaceutical composition). In another aspect, the disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject the CAR T-cell (e.g., administering to the subject a pharmaceutical composition comprising the CAR T-cell).

[0104] In yet another aspect, the disclosure provides a kit comprising one or more agents, e.g., a PRC1 / 2 inhibitor, an IL-15 protein, an TL-7 protein, or a combination thereof, or a biologically active portions thereof, or the like, in combination with an instructional material directing a user in a use of the kit in a method of the disclosure. The kit can be configured for reversal of exhaustion and restoration of sternness in T-cells isolated from a cancer patient for use in a TIL therapy, for preparation of CAR T cells for use in a CAR T cell therapy, or the like. Generally, the kit can be provided for scale-up and / or facilitation of a procedure for rejuvenating T cells for use in T cell therapies.

[0105] In another aspect, the disclosure provides a composition comprising isolated T cells modified ex vivo to reverse an exhaustion phenotype in the isolated T cells. In some embodiments, the modification comprises resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the isolated T cells with an inhibitor effective in inhibiting at least a portion of polycomb Repressive Complex 1 and / or polycomb repressive complex 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

[0106] In some aspects, the isolated T cells are further modified to express a recombinant receptor. In an embodiment, the recombinant receptor is an engineered T cell receptor (TCR). In an embodiment, the recombinant receptor is specific for a tumor antigen.

[0107] The isolated T cells contemplated by this disclosure include but are not limited to native, naturally occurring T cells, autologous T cells, or T cells with specificity for and activity against a tumor. In some embodiments, the native, naturally occurring T cells are obtained from resected tumors, and wherein the T cells are expanded ex vivo prior to the modification. In some embodiments, the T cells with specificity for and activity against a tumor are peripheral blood derived-T cells genetically modified to express a receptor that recognizes and responds to tumor.

[0108] The isolated T cells disclosed herein may be selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, gamma delta T cells, a combination of CD4+ and CD8 T+ cells, memory T cells, cytokine-induced killer cells, and combinations thereof. In an embodiment, the isolated T cells are a combination of CD4+ and CD8+ cells.

[0109] The methods and compositions disclosed herein are effective in reversing the exhaustion phenotype of a T cell. The methods and compositions as disclosed herein are useful for treating a disease or pathological condition in a subject. In some embodiments, the method comprising administering to the subject an effective amount of one or more of the compositions disclosed herein. Diseases or the pathological conditions treatable by the compositions and methods disclosed herein include but are not limited to a tumor or cancer, optionally a solid tumor or hematopoietic malignancy, bacterial and / or parasitic infections.

[0110] In some embodiments, the methods of treatment disclosed herein may be combined with one or more other therapeutic agents. Exemplary therapeutic agents that can be combined include but are not limited to anticancer agents and / or one or more chemotherapeutic agents, anti-bacterial or anti-parasitic agent.

[0111] Also provided herein are kits comprising the compositions of the disclosure.

[0112] The disclosure also provides a method for treating a cancer in a subject in need thereof. In some embodiments, the method comprises administering to the subject the TIL therapy disclosed herein or any one or more of the compositions disclosed herein. In some embodiments, the cancer comprises melanoma, renal cell carcinoma, or both. In some embodiments, further comprises administering to the subject an effective amount of IL-2.

[0113] Methods for preparation of a chimeric antigen receptor (CAR) T-cell therapy are also disclosed herein. In some embodiments, the method comprises resting CAR T-cells in absence of TCR stimulation; inhibiting at least a portion of PRC1 and / or PRC2 in the CAR T-cell; and contacting the CAR T-cells with at least one cytokine. In some embodiments, the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof. In an embodiment, the CAR T-cell therapy comprises the CAR T-cell.

[0114] Aspects of the disclosure provide a method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cells disclosed herein.

[0115] In yet another aspect, the disclosure provides an in vitro method of reversing T cell exhaustion comprising modifying isolated T cells. In some embodiments, the modification comprising resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the T cells with an inhibitor effective in inhibiting at least a portion of polycomb repressive complexes 1 and / or 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

[0116] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.Terminology

[0117] Unless stated otherwise, experimental hypotheses or forward-looking models are not intended to be binding on the applicant or exhaustive of the range of possible experimental hypotheses or forward-looking models, but rather arc intended to be illustrative, non-limiting examples for aiding those in the art in the understanding and practice of elements of the disclosure.

[0118] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0119] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0120] Unless the context clearly requires otherwise, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

[0121] Unless the context clearly requires otherwise, the phrase “consisting of’ excludes any element, step, or ingredient not specified.

[0122] If an element is described or claimed herein such that it “comprises” a feature, that description or claim also includes embodiments wherein the element “consists essentially of’ and embodiments wherein the element “consists of’ the feature, unless something else is specifically stated to the contrary.

[0123] Unless otherwise stated or the context clearly requires otherwise, methods of the disclosure can be performed, in whole or in part, in any order of steps, including steps that are performed subsequently, in parallel, and in combination. Tn addition, methods can be performed, in whole or in part, by humans optionally assisted by one or more machines such as one or more computational devices or systems (e.g., computer(s)). In at least some instances, methods can be performed by one or more humans with little or no substantive assistance by one or more machines. In at least some other instances, methods can be performed by one or more humans with substantive assistance by one or more machines, and in at least some instances, one or more machines can perform methods autonomously or semi-autonomously.

[0124] Unless otherwise stated or required by the context, an agent (e.g., small molecule inhibitor, protein, etc.) of the disclosure can be provided or formulated as an clement of a composition for its use in a method of the disclosure. As known in art, compositions can be water or oil-based, and can include carriers, salts, lipids, surfactants, pH buffers, and the like to configure the composition to be suitable for its intended purpose (e.g., acceptance for biological, pharmacological, or medical applications).

[0125] A composition of the disclosure can be formulated as a pharmaceutical composition. As used herein, “pharmaceutical composition” can be a cell therapy or medication and refers to a composition that includes at least one pharmacologically active cell or compound in combination with a buffer, a pharmaceutical excipient, a surfactant, a salt, a solvate, a lipid, a phospholipid, a carrier, a diluent, or any combination thereof that preserves and configures the at least one pharmacologically active cell or compound for storage, transport, and pharmacologically activity upon administering the pharmaceutical composition to a subject.

[0126] As used herein, “administering” refers to the physical introduction of a composition comprising an agent, such as a therapeutic cell or agent, to a subject, using any of the various methods and delivery systems known to those skilled in the art. Example routes of administration for antibodies include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example by injectionor infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0127] As used herein, the terms “antagonize”, “inhibit”, and “block” are used interchangeably and encompass both partial and complete inhibition / blocking of a structure or function of a given target molecule, pathway, or biological activity. As a non-limiting example, the inhibition / blocking can be by at least about 50%, for example, at least about 60%, 70%, 80%, 90%, 95%, or 99%, or 100%.

[0128] As used herein, an “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein.

[0129] The disclosure also provides kits comprising at least one agent capable of reversing T cell exhaustion and instructions for use.

[0130] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of immunotherapeutic agents, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and / or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant immunotherapeutic agents (e.g., modified T cells and / or supporting materials). As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising an immunotherapeuticcomposition for a particular use, while a second container contains a second agent (e.g., a chemotherapeutic agent). Indeed, any delivery system comprising two or more separate containers that each contains a sub portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0131] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of one or more elements of a kit of the disclosure for carrying out a testing method, a screening method, a treatment method, or other method of the disclosure, including methods for alleviation of one or more diseases or disorders as described herein or as known in the art. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit can, for example, be affixed to a container which contains an identified compound or can be shipped together with a container which contains the identified compound. Alternatively, the instractional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively.

[0132] As used herein the terms “disease” and “pathologic condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g., humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such a deviation can manifest as a state, signs, and / or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions.

[0133] A disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infective agent (e.g., a virus or bacteria)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and / or climate), may be responsive to an inherent or latent defect in the organism (e.g., genetic anomalies) or to combinations of these and other factors.

[0134] The terms “host,” “subject,” or “patient” are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., humans, mice, rats, monkeys, horses, cows, pigs, dogs, cats, and the like). In the context of the invention, the term “subject” generally refers to an individual who will be administered or who has been administered one or more compositions of the present invention (e.g., modified or engineered (e.g., genetically) T cells described herein).

[0135] “T cell exhaustion” refers to loss of T cell function, which may occur as a result of an infection (e.g., a chronic infection) or a disease. Exhaustion may arise due to chronic antigen exposure, immunosuppressive microenvironments, or persistent inflammatory signaling. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, apoptosis, and reduced cytokine secretion. Accordingly, the terms “ameliorate T cell exhaustion,” “inhibit T cell exhaustion,” “reverse T cell exhaustion” and the like refer to a condition of restored functionality of T cells and restoration of a stem-like phenotype characterized by one or more of the following: decreased expression and / or level of one or more of PD-1, TIM-3, and LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased antigen-induced cytokine (e.g., IL-2) production and / or secretion; enhanced killing capacity; increased recognition of tumor targets with low surface antigen; enhanced proliferation in response to antigen.

[0136] The term “exhaustion phenotype” refers to a phenotype of a T-cell that is characterized at least in part by the T-cell exhibiting a decreased function relative to that of a reference T-cell. Non-limiting examples of decreased function relative to that of a reference T-cell include loss of potential for long-term self-renewal, loss of potential for forming memory, loss of differentiation potential, loss of proliferation, reduced ability to produce effector cytokines, and reduced cytotoxicity.

[0137] As used herein, the term “exhausted” or “exhaustion”, unless otherwise stated, generally refers to effector T cells with a reduced capacity to secrete cytokines (e.g. IL-2), a reduced capacity to proliferation, and increased expression of inhibitory receptors (e.g. PD-1, Tim-3, Lag-3). Other examples of markers of T-cell exhaustion include, but are not necessarily limited to, downregulation of transcription factor 7 (TCF7) / TCF1, FOXO1, Sell (also known as CD62L) in the T-cell. Thus exhaustion-pathways may include without limitations (a) inhibitory receptor signaling, including but not limited to, PD-1, LAG-3,TIM-3, TIGIT, .CTLA-4, and related pathways; (b) transcriptional regulation, including but not limited to, transcription factors such as TOX, NR4A family members, BAIT’, 1RF4, NFAT, and combinations thereof; (c) epigenetic regulation, including but not limited, to chromatin remodeling, histone modification, DNA methylation, and non-coding RNA regulation; (d) metabolic pathways, including but not limited to, mitochondrial function, glycolysis, oxidative phosphorylation, fatty acid metabolism, and metabolism and redox balance; and (d) cytokine and growth factor signaling, including and not limited to, IL-2, IL-7, IL-15, IL-21, interferons, and associated downstream pathways.

[0138] Exhaustion is characterized by progressive loss of T cell effector functions, wherein under certain conditions (i.e., persistent exposure to antigens), T cells become incapable of elaborating effector-related activities including the production of effector and memory T cell populations. Exhausted T-cell responses have been observed under various circumstances including, but not limited to, lymphocytic choriomeningitis virus (LCMV) infection, polyoma virus infection, adenovirus infection, Friend leukemia virus infection, mouse hepatitis virus infection, human immunodeficiency virus (HIV) infection, hepatitis B vims (HBV) infection, hepatitis C vims (HCV) infection, and have also been reported in subjects with malignancies.

[0139] Exhausted T cells suitable for the methods and compositions disclosed herein may include CD4+ T cells, CD8+ T cells, regulatory T cells, memory T cells, effector T cells, or genetically modified T cells, including chimeric antigen receptor (CAR) T cells or T cell receptor (TCR) engineered cells.

[0140] Table

[0141] Agents useful in the disclosed methods may include, without limitation: small molecules, polypeptides or peptides, nucleic acids (DNA, RNA, modified RNA), gene-editing constructs (e.g., CRISPR systems), viral or non-viral delivery systems, and nanoparticle or lipid formulations. Agents disclosed herein may act by inhibiting, activating, repressing, enhancing, or otherwise modulating exhaustion associated molecules and / or pathways.

[0142] In certain embodiments, T cells are isolated from a subject, treated ex vivo under conditions sufficient to reverse exhaustion or exhaustion phenotype, and optionally administered back to the subject.

[0143] Reversal of exhaustion phenotype and restoration of sternness may be assessed by one or more of: increased cytokine secretion (e.g., IL-2, IFN-g), increased expression of TCF7, CD62L, Slamf6, and / or IL-7Ra, enhanced cytotoxic activity and proliferation, enhanced persistence and survival following transfer. These markers may be assessed by one or more methods known in the art, including but not limited to flow cytometry.

[0144] The disclosed methods may be used to treat diseases associated with T cell exhaustion, including but not limited to, cancer, chronic viral infection, persistent bacterial or parasitic infection, and immune dysregulation disorders.

[0145] The disclosed methods may be used alone or in combination with other therapies. In certain embodiments, the disclosed methods are combined with immune checkpoint inhibitors, vaccines, cytokine therapies, adoptive cell therapies, radiation, chemotherapy, or targeted therapies.

[0146] As used herein, the terms “cancer” and “tumor” refer to a tissue or growth comprising cells that have lost the ability to control growth and proliferation. Cancer and tumor cells generally are characterized by a loss of contact inhibition, may be invasive, and may display the ability to metastasize (e.g., they have lost the ability to adhere to other cells / tissues). The present invention is not limited by the type of cancer or the type of treatment (e.g., prophylactically and / or therapeutically treated). Indeed, a variety of cancers may be treated with compositions and methods described herein including, but not limited to, brain cancer or other cancers of the central nervous system, melanomas, lymphomas, bone cancer, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, sarcomas, carcinomas, and / or a combination thereof.

[0147] An “effective amount” refers to an amount of a pharmaceutical composition, anticancer agent, or other drug effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result (e.g., relief of some or all symptoms of the disease being treated).

[0148] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the rate of tumor growth (e.g., reduces and / or eliminates the tumor burden in the patient), decreases tumor mass, decreases the number of metastases, decreases tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0149] As used herein, the terms “administration” and “administering” refer to the act of giving a composition to a subject. Exemplary routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intraperitoneally, intratumorally, etc.), topically, and the like. In one embodiment, administration of T cells of the invention is via intravenous infusion.

[0150] As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) (e.g., modified immune cells and one or more other agents — e.g., anti-cancer agents) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. In some embodiments, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0151] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or other immunologic reactions) when administered to a subject.

[0152] As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[0153] As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., nontransformed cells), and any other cell population maintained in vitro.

[0154] As used herein, the term “tumor-infiltrating lymphocyte (TIL) therapy” is inclusive and includes a cell therapy in which some or all of the T-cells isolated from a patient expresses TIM3, Lag3, CD39, CD69, or any combination thereof, and / or has a relatively lower expression of TCF1, FoxOl, Sell / CD69, Slamf6 / Lyl08, or anycombination thereof, compared to a reference T cell isolated from the patient or another subject.

[0155] A “small molecule” or “small organic molecule” is one that has a molecular weight below about 500 Daltons.

[0156] An “interfering RNA” “RNAi” is RNA of 10 to 50 nucleotides in length which reduces expression of a target gene, wherein portions of the strand are sufficiently complementary (e.g., having at least 80% identity to the target gene). The method of RNA interference refers to the target- specific suppression of gene expression (i.e., “gene silencing”), occurring at a post-transcriptional level (e.g., translation), and includes all posttranscriptional and transcriptional mechanisms of RNA mediated inhibition of gene expression, such as those described in P.D. Zamore, Science 296:1265 (2002) and Hannan and Rossi, Nature 431:371-378 (2004). As used herein, RNAi can be in the form of small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA). Such RNAi molecules are often a double stranded RNA complexes that may be expressed in the form of separate complementary or partially complementary RNA strands. Methods are well known in the art for designing double- stranded RNA complexes. For example, the design and synthesis of suitable shRNA and siRNA may be found in Sandy ct al., BioTechniques 39:215-224 (2005).

[0157] A “small interfering RNA” or siRNA is a double stranded RNA (dsRNA) duplex of 10 to 50 nucleotides in length which reduces expression of a target gene, wherein portions of the first strand is sufficiently complementary (e.g., having at least 80% identity to the target gene). siRNAs are designed specifically to avoid the anti-viral response characterized by elevated interferon synthesis, nonspecific protein synthesis inhibition and RNA degradation that often results in suicide or death of the cell associated with the use of RNAi in mammalian cells. Paddison et al., Proc Natl Acad Sci USA 99 (3): 1443-8. (2002).

[0158] The term “hairpin” refers to a looping RNA structure of 7-20 nucleotides. A “short hairpin RNA” or shRNA is a single stranded RNA 10 to 50 nucleotides in length characterized by a hairpin turn which reduces expression of a target gene, wherein portions of the RNA strand are sufficiently complementary (e.g., having at least 80% identity to the target gene). The term “stem-loop” refers to a pairing between two regions of the same molecule base-pair to form a double helix that ends in a short, unpaired loop, giving a lollipop- shaped structure.

[0159] A “microRNA” or “miRNA” (previously known as stRNA) is a single stranded RNA of about 10 to 70 nucleotides in length that are initially transcribed as pre-miRNA characterized by a “stem-loop” structure, which are subsequently processed into mature miRNA after further processing through the RNA-induced silencing complex (RISC).

[0160] Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0161] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0162] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0163] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0164] All of the references cited herein are incorporated by reference. Aspects of the disclosure can be modified, if necessary, to employ the systems, functions, and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0165] It will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modificationscan be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as stated by the claims.EXAMPLES

[0166] Example 1. Restoring Sternness in Tumor-Infillrating Lymphocytes by Epigenetic Reprogramming

[0167] Relevance to Cancer Immunology.

[0168] Adoptive cell therapies harnessing patient-derived tumor infiltrating lymphocytes (TILs) hold tremendous promise for curative treatment of advanced stage cancers. T cells have unique abilities to recognize and eliminate cancer cells and are now being intensely investigated as therapeutic agents against diverse solid cancers. Compared to other T cell therapies, TILs have two advantages: First, T cells in TIL products target multiple tumor antigens and are polyclonal, making them potentially less susceptible to antigen escape compared to antigen receptor-engineered T cells. Second, as TILs are selected to target tumor-specific antigens, they are less likely to destroy healthy tissues compared to engineered T cells. Indeed, adoptive cell therapy (ACT) using TILs has shown promise against multiple types of solid cancers and has gained FDA approval for treatment of metastatic melanoma refractory to immune checkpoint inhibitors. However, despite their promise, TIL therapies remain largely ineffective against most solid tumors, as TILs with neoantigen specificity often exist in an exhausted state with limited antitumor efficacy.

[0169] Most human epithelial cancer types harbor TILs that recognize neoantigens expressed in their native tumors. However, the vast majority of neoantigentargeting TILs tend to exist in a terminally exhausted state with limited persistence and efficacy. While this terminally exhausted state likely reflects chronic tumor antigen engagement, a key driver of exhaustion, it would limit the efficacy of these neoantigenspecific TILs after re-infusion. Surprisingly, the presence of stem-like neoantigen-targeting T cells in TIL infusion products determines the efficacy of TIL therapies. While the absence of stem-like TILs predicted poor outcomes, even a small fraction of stem-like neoantigen targeting TILs in the infusion product improved clinical responses and drove cancer regression. These findings underscore the importance of stem-like phenotypes in TIL products for therapeutic efficacy. To fully realize the therapeutic potential of TIL therapies, there is a need for approaches to reverse terminal exhaustion and revive self-renewal potential in exhausted neoantigen-specific TILs.

[0170] The switch into a terminally exhausted state can include an antigen-driven differentiation process involving global changes in the T cell’s state, however, other factors not related to antigens can be involved. While initial changes due to antigen stimulation are reversible, the transition to a terminally exhausted state is considered irreversible, such that it persists even when initiating signals are gone. Because of this epigenetic stability, exhausted TILs persist in this state after isolation and during ex vivo TIL production, and remain limited in their antitumor efficacy upon reinfusion. Therefore, to fully restore TIL performance, one may need to epigenetically reprogram terminally exhausted T cells into a stem-like state with long-term effector capabilities. However, to date, there are no interventions that can reverse exhaustion and restore self-renewal capabilities to terminally exhausted T cells. While many strategies to counter exhaustion have been investigated, all such prior approaches aim to block or delay exhaustion, and none is capable of reversing an exhausted state once it is established.

[0171] This example enables one to implement functional T cell immunology, and models for murine T cells and neoantigen-targeting human T cells, to epigenetically revive exhausted TIL subsets that are enriched with antitumor T cells to improve cell therapy (FIG. 3A).

[0172] An epigenetic mechanism upholding commitment of T cells to a terminally exhausted state is elucidated herein. To study mechanisms upholding T cell exhaustion, an in vitro system that recapitulates the antigen-driven entry of T cells into terminally exhausted state, defined by silencing of the transcription factor TCF-1 (encoded by TcJ ) and up-regulation of TIM-3 (FIG. 1A), was developed. Importantly, Tcf7 silencing is stably maintained after antigen removal, pointing to epigenetic mechanisms underlying this stability. Indeed, by CRISPR / Cas9 screening using this system (FIG. IB), it was found that the Polycomb Repressive Complexes (PRC)l and PRC2 together lock down Tcf7 silencing and loss of sternness in terminally exhausted T cells (FIG. 3B (A)).By chromatin profiling and by analysis of TILs from B16 melanoma tumors, it was found that the same mechanism locks down Tcf7 silencing and loss of sternness in CD8+TILs.

[0173] Signaling and gene regulatory linkages modulating Polycomb silencing of TcJ7 and self- renewal gene loci are also elucidated. As PRC1 / PRC2 do not have inherent sequence specificity in DNA binding, they depend on trans- factors to specify their genomic targets. Indeed, T cell receptor (TCR) stimulation and high doses ofIL-2 reinforce Polycomb silencing of Tcf7 and other sternness-associated genes (e.g., FoxOl), whereasIL-15 and IL-7, singly or in combination, antagonize their silencing and synergize with Polycomb perturbations to promote restoration of sternness gene programs in terminally exhausted cells (FIG. 3B (B)). These findings highlight the relevance of signaling context when designing molecular interventions to revive exhausted T cells, which are accounted for below.

[0174] The disclosure contemplates and provides an epigenetic reprogramming strategy to restore sternness and antitumor efficacy to exhausted TTLs, where cells are subject to PRC1 / PRC2 inhibition with IL- 15 and without TCR stimulation (FIGs 3A, 3B (B)). Indeed, as described herein, these conditions not only efficiently reverse TCF-1 silencing, but also reawaken sternness programs more broadly (FIG. 3B(C)); thus, this disclosure provides novel evidence that terminally exhausted T cells can be reverted to a stem-like / precursor state. Similar reversal of exhaustion and restoration of sternness was observed terminally exhausted mouse primary CD8+ T (TCF7-, TIM3+) cells subject to PRC1 / PRC2 inhibition with IL-L5, IL-7, or a combination of IL-15 and IL-7 and without TCR stimulation (FIG. 5).

[0175] Based on these unexpected results, it was concluded that epigenetic reprogramming via cytokine-modulated Polycomb modulation may revert TILs into a stem-like state, restoring their self-renewal, long-term cytotoxic function, and ability to induce tumor regression (FIG. 3A). One can evaluate the effects of epigenetic reprogramming on TILs from a neoantigen mouse melanoma model. One can also evaluate whether epigenetic reprogramming can restore stem-like states in exhausted TILs, and whether it can boost their proliferative potential and ability to drive tumor control upon reinfusion into tumor-bearing mice. One can also evaluate the effects of epigenetic reprogramming on TIL infusion products from metastatic melanoma or colorectal cancer patients. The ability of epigenetic reprogramming to restore Tcf7 expression and a stemlike state in these human TILs can be evaluated. One can also determine whether this sternness restoration occurs for TILs with tumor neoantigen specificity, which can be important for this intervention to be usable for boosting TIL therapeutic efficacy. Finally, one can evaluate whether epigenetically reprogrammed human TILs have superior proliferative potential and antitumor capabilities, using both in vitro restimulation assays and in vivo patient-derived xenograft mouse models to evaluate TIL product efficacy.

[0176] As an element of this embodiment, one can evaluate epigenetic reprogramming as a strategy for restoring TIL sternness and antitumor performance in amouse melanoma model. A mouse neoantigen model for TIL therapy can be implemented; this model utilizes the established B16 melanoma line, which recapitulates terminal exhaustion of CD8+T cells and enables functional testing of self-renewing TCF1+subsets after adoptive transfer. In this model, tumor cells are engineered to express a mutated peptide from the melanoma differentiation antigen Pmel, or gplOO (mut:KVPRNQDWL vs wt:EGSRNQDWL); this mutant peptide is recognized by cognate Pmel-1 TCR CD8+T cells more strongly than its wildtype counterpart, enabling modeling of neoantigen response. To obtain exhausted TILs, one can implant Bl 6F10 cells into mice with the Pmel-1 TCR and a 7c / 7YFP reporter, then isolate exhausted (7c / 7’TIM3+) CD8+TILs from established tumors 2-3 weeks later. This procedure yields large numbers of TILs (~106 / mouse) for facile downstream analysis or transfer. One can then expand these exhausted PmeLl TILs and culture them with or without epigenetic reprogramming. Epigenetic reprogramming may comprise resting the TILs (i.e., no TCR stimulation or no anti-CD3 / CD28 antibody), and contacting the TILs with either a PRC1 inhibitor, a PRC2 inhibitor or both, and at least one of IL- 15, IL-7 or a combination of IL- 15 and IL-7.

[0177] One can then evaluate resultant effects of epigenetic reprogramming on T cell persistence and antitumor activity through secondary transfer of these cells into mice bearing B16KVP tumors. To gain insights into the resultant effects of reprogramming on T cell states, one can also analyze cells using either flow cytometry or single cell transcriptomic and chromatin accessibility analysis (joint RNA / ATAC-seq). These experiments enable to evaluate use of epigenetic reprogramming to restore antitumor function to exhausted TILs in the context of an established mouse model.

[0178] As another element of this embodiment, one can evaluate epigenetic reprogramming as a strategy for restoring function in exhausted human TILs. The landscape of experimentally defined human tumor neoantigens has been significantly expanded by the Surgery Branch, National Cancer Institute (NCLSB) as a part of in vitro expanded clinical TIL administered to metastatic cancer patients as ACT (NIH clinical protocol 10-C-0166). For this element, one can leverage NCLSB ’s unique repository of neoantigen-specific dysfunctional human antitumor TILs identified as a part of clinical protocols, to evaluate if they can be epigenetically reprogrammed to a stem-like state for adoptive cell transfer (ACT) in humans. To this end, one can identify up to 20 in vitro expanded TIL populations obtained from metastatic melanoma and colon cancer patients with defined neoantigen-specificities. One can stimulate these TILs to expand them, thensubject them to epigenetic reprogramming (PRC1 inhibitor, PRC2 inhibitors, or a combination thereof; IL- 15, IL-7, or a combination thereof; and no TCR stimulation), to generate TIL infusion products that can be scaled for clinical use.

[0179] One can similarly determine the effects of epigenetic reprogramming on the phenotype, antigen specificity, expansion potential and antitumor function of human neoantigen CD8+TILs. To uncover if terminally exhausted human antitumor TILs have been truly reprogrammed to a stem-like state, one can measure phenotypic changes by flow cytometry analyses of neo-antigen-tetramer gated TILs. One can also perform single cell RNA sequencing and TCR sequencing (scRNA-scTCR) of treated TILs to track frequencies of neoantigen-TCR clones expressing stem-like TILs, optionally using one or more previously established methods. If revival of sternness in neoantigen-specific TILs is observed, one can optimize conditions with respect to dose and duration of Polycomb inhibition, to maximize TIL expansion and enhance clinical scalability for human TIL-ACT administration. Finally, one can evaluate the persistence and antitumor function of reprogrammed neoantigen TILs in vitro, by co-culture with neoantigen-expressing autologous antigen presenting cells and, if available, in vivo, using autologous patient-derived tumor xenograft models in established immunodeficient mice models for ACT. Together, these preclinical experiments can enable the utility and scale of epigenetic reprogramming of human neoantigen-TIL to stem-like states as a scalable, deployable cell therapy strategy for metastatic cancer patients.

[0180] These elements establish approaches for epigenetic reprogramming as a strategy for enhancing TIL therapies against metastatic cancers, particularly for patients without stem-like T cells in their TIL infusion products. Reprogramming can be tested clinically for melanoma and colon cancer patients and can also be applicable towards other solid cancers, especially those where neoantigen-specific TILs are lower in frequency and / or more exhausted and harder to expand for TIL manufacturing. Finally, as exhaustion limits T cell antitumor function across different immunotherapy modalities, these approaches also have relevance in the broader field of cancer immunology.

[0181] Example 2. Reversing T cell Exhaustion

[0182] Stem-like precursor T cells self-renew to sustain cytotoxic responses against cancer. However, upon chronic stimulation in the tumor microenvironment, these cells relinquish self-renewal capabilities and commit to an exhausted state with limited antitumor functions and persistence. Despite much work, it has remained unclear how Tcells commit to a terminal exhausted state after tumor engagement. Stem-like precursor T cells are defined by the expression of memory and self-renewal genes, including the key memory transcription factor TCF1 (encoded by Tcj7). Upon tumor stimulation, cells silence TCF1 and other memory genes and up-regulate genes controlling effector function and exhaustion. Initial regulatory changes due to tumor stimulation are reversible; however, the eventual transition to an exhausted TCFF state is irreversible, such that this state is epigenetically stable, persisting when stimulatory signals are withdrawn.

[0183] Mechanisms upholding the commitment of T cells to a terminal exhausted state have remained elusive, because it is challenging to interrogate its molecular basis using prior approaches. The development of in vitro systems for T cell exhaustion due to chronic stimulation has opened doors to mechanistic studies. In particular, by facilitating CRISPR / Cas9 screens, these systems enable to systematically identify underlying genes and pathways. However, while powerful, none of these prior systems have recapitulated the differentiation of T cells to a terminal exhausted state. As such, they only enable to uncover genes initiating exhaustion and not those maintaining the epigenetic stability of the exhausted state after entry.

[0184] The Poly comb system, including Poly comb Repressive Complex (PRC)l and PRC2, are involved in the maintenance of differentiated cell identity across mammalian development.

[0185] To stably maintain cell type-specific gene expression programs in differentiated cells, PRC1 and PRC2 work together to enforce silencing of stem cell genes or genes associated with other lineages. To do so, they establish Polycomb chromatin domains at target genomic loci consisting of two repressive histone modifications -monoubiquitylation of histone-2A at Lysl l9 (H2AK119ubl), written by PRC1, and methylation of histone-3 at Lys27 (H3K27me3), written by PRC2. Once established, these Polycomb domains persist when initial signals are absent and are heritable over cell division, a property that could be utilized by cells to stably maintain their identity after lineage commitment. Indeed, during acute infection, PRC2 locks down silencing of memory genes during CD8+T cell terminal effector differentiation; this silencing is mediated in part by the deposition of H3K27me3 at these loci. However, during chronic stimulation in a tumor setting, roles for PRC2 in T cell terminal exhaustion differentiation have previously remained unclear: while H3K27me3 levels increase at Tcf7 and other memory gene loci during the course of T cell exhaustion in tumors, Ezh2 deletion orinhibition can also enhance effector activity and even exacerbate dysfunction. It is also unclear what roles, if any, PRC1 plays in terminal exhaustion, and whether PRC1 cooperates with PRC2 to enforce gene silencing during T cell differentiation.

[0186] Using a novel in vitro system to study terminal T cell exhaustion, this disclosure elucidates a role for PRC1 and PRC2 in locking down silencing of TcJ7 and other sternness genes in terminally exhausted T cells. To overcome limitations of prior systems, the disclosure provides a novel in vitro differentiation system that uniquely recapitulates the irreversible commitment of T cells into a TCF1'TIM3+exhausted state (FIGS. 2A-2C). By performing a series of CRISPR / Cas9 knockout screens using this system, and through complementary studies of T cells from a tumor model, it is disclosed that PRC1 and PRC2 are dispensable for initial Tcf7 silencing but are needed for the stable maintenance of Tcf7 silencing in terminally exhausted cells, and also for upholding silencing of self-renewal genes more broadly (FIG.4D).

[0187] Building on these findings, one can elucidate the molecular mechanism by which PRC1 / PRC2 lock down Tcf7 silencing and terminal exhaustion and examine how this lockdown mechanism is modulated by signaling pathways and trans-factors acting at silenced memory gene loci (FIG. 4A). Embodiments of the present disclosure also contemplate Polycomb targeting as a therapeutic strategy for boosting the efficacy of tumor infiltrating lymphocyte (TIL) therapies against cancer. Adoptive cell therapies harnessing patient-derived TILs have shown remarkable efficacy against a range of solid cancers and recently gained FDA approval for use in late-stage melanoma patients refractory to immune checkpoint inhibitors. However, despite these breakthroughs, TIL therapies remain ineffective in -70% of patients.

[0188] The presence ofTCFl+stem-like, tumor neoantigen-specific T cells in TIL infusion products is highly predictive of clinical efficacy.

[0189] Remarkably, even when present as a small fraction of the infusion product, these stem-like sub-populations can mediate cancer regression and clinical responses. Together, these findings underscore the importance of having TIL expansion protocols that give rise to infusion products with stem-like, neoantigen-specific populations for enhanced anti-tumor efficacy. Polycomb inhibition can enhance the production of stemlike TILs in infusion products, as it can reverse terminally exhausted states during expansion. In different human tumor types, TILs bearing tumor antigen-specific TCRs tend to show a terminally exhausted phenotype prior to expansion. This exhausted phenotype,possibly arising from tumor engagement and stimulation, highlights the need for culture conditions that not only block or slow down exhaustion, but also restore self-renewal capabilities and sternness to exhausted cells during cell therapy. As Polycomb inhibition not only reverses Tcf7 silencing but also re-activates memory programs more broadly, this approach can restore sternness to terminally exhausted T cells and TILs and improve their long-term antitumor functions. This can be evaluated (FIG.4A), first in a neo-antigen TIL therapy mouse model, then in human T cells, using an in vitro system to generate terminally exhausted cells for analysis as described herein.

[0190] Broader impact.

[0191] Aspects of the disclosure also relate to mechanisms by which the Polycomb system upholds commitment of T cells to a terminally differentiated state. The present disclosure not only informs efforts to target the Polycomb system to restore antitumor T cell self-renewal and longevity but also yields fundamental insights into how epigenetic mechanisms maintain cell identity in differentiated cells, with relevance for many mammalian systems. The tumor studies disclosed herein reveal the efficacy of Polycomb inhibition as a strategy for restoring sternness to exhausted antitumor T cells. Thus, the present disclosure provides improved and efficacious TIL therapies that can more broadly lead to better treatments for cancers where T cell exhaustion is a barrier to efficacy.

[0192] In vitro system for deep mechanistic analysis of T cell terminal exhaustion.

[0193] Prior mouse models of cancer and chronic infection are powerful but limited in their ability to support mechanistic studies requiring large numbers of cells and facile manipulation / observation. A number of in vitro models of exhaustion have been developed previously, but none recapitulate the irreversible transition of T cells into a terminal state. By recapitulating terminal T cell state entry using minimal, defined signals, the disclosed in vitro system has facilitated the genetic perturbation and mechanistic determination of the Polycomb lockdown state. The present disclosure contemplates utilizing the foregoing powerful system and expand for chromatin profiling, proteomics, and imaging approaches, including as described herein.

[0194] Exhaustion reversal ' / de-differentiation as an approach for boosting T cell function.

[0195] Prior efforts to counter T cell exhaustion to boost efficacy have mainly focused on blocking or delaying exhaustion differentiation. As described herein, by targeting mechanisms underlying the maintenance of the differentiated state, aspects of thedisclosure enable reversing differentiation and restoring sternness to exhausted T cells. This approach is relevant and applicable in TIL therapies as many T cells already exist in an exhausted state in the primary tumor.

[0196] Modeling of neoantigen TIL therapies in a melanoma mouse model.

[0197] A unique mouse model for TIL therapies, in which neoantigen-specific T cells are harvested from primary melanomas, expanded ex vivo, then re-transferred into secondary tumor-bearing hosts, can be implemented (FIG. 3A). This model enables evaluation of the effects of different ex vivo treatments or conditions on expansion and infusion product generation.

[0198] An element of this embodiment enables one to elucidate a mechanism by which Polycomb Repressive Complexes lock down Tcf7 silencing and terminal exhaustion. For PRC1 and PRC2 to stably maintain silencing of Tcf7 and other memory genes in terminally exhausted cells, histone modifications written by these enzyme complexes can have two properties: first, they propagate over DNA replication at silent gene loci; second, they repress transcriptional activity at these loci. Two candidate models have emerged to explain how Polycomb maintains heritable gene silencing at target genes (FIG. 4B (A)).In the first model (Model I), silent states arc maintained through ‘read-write’ positive feedback in histone H3K27me3 deposition. Here, H3K27me3 binds PRC2 allosterically via the EED subunit to catalyze H3K27me3 deposition at nearby nucleosomes. Such readwrite feedback enables H3K27me3 marks to become replenished after dilution by DNA replication and spread out from nucleation sites to establish large silencing domains. To instruct gene silencing, H3K27me3 recruits a subset of PRC1 complexes - termed canonical (c)PRCl - that can then drive chromatin compaction to block transcriptional activity.

[0199] Canonical PRC1 binds H3K27me3-marked chromatin via their chromobox (CBX) subunits. Chromatin-bound cPRCl can self-associate (via Phc45-47 or CBX subunits) to form larger assemblies to drive chromatin compaction and / or limit access to the transcriptional machinery. In the second model (Model 2; FIG.4B (B)), silent state maintenance involves not only H3K27me3, but also read- write positive feedback in H2AK119ubl deposition and / or crosstalk between H2AK119ubl and H3K27me3. Another subset of PRC 1 complexes, named variant (v)PRCl, bind H2AK119ubl through their RYBP subunit and catalyze H2AK119ubl deposition at neighboring nucleosomes, enabling propagation of silencing domains over cell division. H2AK119ubl can furtherrecruit PRC2 complexes containing the JARID2 subunit and stimulate its methyltransferase activity, enabling positive crosstalk between these two modifications. High H2AK119ubl levels due to vPRCl activity then instruct transcriptional silencing, by directly blocking RNA polymerase recruitment or activity.

[0200] Based on the example data, stable TcJ7 silencing can involve read-write feedback in PRC2-mediated H3K27 trimethylation, coupled to canonical PRC 1 -driven chromatin compaction (z.e., Model 1; FIG. 4B (A)). The disclosed CRISPR / Cas9 screen revealed that core PRC1 and PRC2 subunits are important for locking down TcJ7 silencing. However, it additionally revealed that canonical PRC1 subunits (PCGF2 / 4, Phc3 and Cbx4) had a stronger impact on Tcf7 silencing when knocked out compared to variant PRC1 subunits, consistent with Model 1 ; FIG.4B (A). One can test two predictions of this model: First, read-write activities of PRC2, but not that of vPRCl, underlie maintenance of Polycomb domain at Tcf7 and other loci (FIG. 4B (A)). One can test this prediction by perturbing PRC2 and vPRCl subunits involved in read-write positive feedback and measuring resultant effects on H3K27me3 / H2AK119ubl levels at Tcf7 and other memory gene loci by CUT&RUN chromatin profiling. Second, cPRCl complexes are present in terminally exhausted cells and uphold Tcf7 silencing by driving chromatin compaction (FIG. 4B (B)). To test these, one can measure the subunit composition of PRC1 family members in terminally exhausted T cells using immunoprecipitation followed by mass spectrometry (IP-MS). One can then perturb cPRCl subunits regulating compaction and analyze resultant effects on Tcf7 levels and locus compaction state by using a chromatin imaging method such as a method named SCEPTRE (for Single Cell Evaluation of Post Translational Epigenetic encoding), to visualize locus conformation and compaction state at the single-cell, single-locus level. Finally, to determine whether similar cPRCl -mediated compaction mechanisms maintain Tcf7 silencing in vivo, one can repeat cPRCl perturbation experiments on TILs isolated from B 16 tumors (FIG.4B (C)).

[0201] As described herein, an in vitro assay recapitulates CD8 T cell commitment into a terminal exhausted state. The disclosure provides an in vitro T cell exhaustion assay (FIG. 4C (A)). In this assay, CD8 T cells are stimulated by aCD3, IL-2, and Type I interferon (IFNa), which is present in many ‘hot’ tumors where exhaustion occurs. Upon chronic TCR stimulation (~6d), cells transition from a precursor (Tc / 7+Tim3 ) to an exhausted state (7c 7‘TIM3+) (FIG. 4C(B)), as observed in tumors. Cells acquire other hallmarks of exhaustion, including reduced cytokine secretion, upregulation of PD- 1and the exhaustion regulator Tox, as well as dysfunction-associated transcriptomic states (FIG. 4C (C-E)). When rested from stimulation early (d2), cells regain a memory phenotype. However, after exhausted state entry (d6), cells no longer re-activate Tc / 7 and exit the exhausted state (7c / 7’TIM3+) upon rest (FIG. 4C(F)). Thus, T cells enter an epigenetically stable exhausted state that persists after antigen removal.

[0202] CRISPR / Cas9 knockout screen implicates the Polycomb system in maintenance ofTcf7 silencing.

[0203] To identify genes maintaining Tcf7 silencing in exhausted cells, a pooled Cas9 deletion screen was performed, knocking out 118 transcription factors (TFs) and chromatin regulators in sorted terminally exhausted (7c / 7 TIM3+) cells, and assaying for Tcf7 re-expression through re-sorting and sequencing of Tcf7+I- populations (FIG.4D (A)).This screen design differs with earlier screens that identify genes initiating exhaustion (FIG.4D (B)). Knockout of pro-memory genes (MYB, FOXO1) reinforced Tcf7 silencing, whereas knockout of effector genes (Prdml, Tbx21, Stat4) disrupted Tcf7 silencing (FIG.4D (B)). Strikingly, knockout of both PRC1 (Cbx4 and Ringla) and PRC2 (Ezh2, EED, Rbbp4) subunits disrupted Tcf7 silencing (FIG. 4D (B)), implicating PRC1 / PRC2 in locking down Tcf7 silencing. In validation assays, Cbx4 or Ezh2 knockout not only reversed Tcf7 silencing but also reversed Tim3 up-regulation, indicating a broader role for Polycomb in upholding the terminally exhausted (7c / 7TIM3+) state (FIG. 4D (C)). Joint PRC1 and PRC2 inhibition synergistically increased Tcf7 re-activation, suggesting functional cooperation (FIG. 4D (D)). Finally, strikingly, when Ezh2 was inhibited early during activation, it instead accelerated Tcf7 silencing, highlighting its context-dependent action and underscoring the relevance of probing its functions specifically in terminally exhausted cells.

[0204] The Polycomb system also locks down the exhausted state in TILs.

[0205] To test whether Polycomb mechanisms also uphold terminal exhaustion in T cells differentiating inside tumors, CD8 T cells were adoptively transferred from Tcf7~ YFP I OT-I TCR-transgenic mice into recipient mice bearing B16-Ova tumors, and exhausted (7e 7 TIM3+) cells sorted after 8d, then rested with or without the Ezh2 inhibitor Taz (FIG. 4D (F)). It was observed that Ezh2 inhibition increased Tcf7 re-expression in tumor infiltrating cells (FIG. 4D (G)), indicating that the exhaustion lockdown mechanisms identified in the CRISPR / Cas9 screen are also operational in antitumor T cells in vivo.

[0206] PRC1 / PRC2 establish stable Polycomb chromatin domains at TcJ7 and other memory gene loci. To test if PRC1 / PRC2 deposits H3K27me3 and H2AK119ubl at Tcf7 and other loci, and whether these modifications are epigenetically stable, CUT&RUN profiling of H3K27me3 and H2AK119ubl was performed in CD8 T cells before stimulation (d0), during stimulation (d3 and d7), and after stimulation withdrawal (dl4). Indeed, the Tcf7 locus gained a large (~100kb) Polycomb domain with both H3K27me3 and H2AK119ubl after silencing (d3 and d7), that persisted for a week after removal of stimulation (dl4) (FIG.4E (A)). Stable Polycomb domains were also established at other memory loci (e.g., Bach2, Lefl, Klf2), and were removed from effector gene loci (e.g., Prdml and Id2), indicating broader establishment of an epigenetically stable exhausted state.

[0207] It was asked whether PRC2 and PRC1 read- write activities impact Poly comb domain stability in exhausted T cells. A potential model (Model 1 ; FIG.4B (A)) posits that Polycomb domain stability is primarily upheld by PRC2 read-write abilities, not by vPRC 1 read- write abilities or vPRCl / PRC2 crosstalk. A prediction of this model is that disrupting the read- write abilities of PRC2 - but not that of vPRCl - can reverse Tcf7 silencing and disrupt the repressive H3K27mc3 / H2AK119ubl chromatin domains at Tc / 7 and other silenced memory gene loci. To test this prediction, one can perturb potential H3K27me3 and H2AK119ubl read-write activities in exhausted CD8 I cells, either by CRISPR / Cas9 deletion of PRC read- write subunits (EED, Ezh2 for PRC2; Ringl, RYBP for PRC1), or through transduction of dominant negative subunits that do not disrupt complex formation, but are unable to bind these modifications (EED and RYBP) (Model 2; FIG.4B (B)). One can then read out effects of these perturbations on 7c 7YFP silencing, as measured by flow cytometry, as well as on the maintenance of H3K27me3 / H2AK119ubl domains by chromatin profiling (FIG.4B (C)).

[0208] Design and generation of retroviral constructs.

[0209] For CRISPR / Cas9 knockout, one can perform retroviral transduction of gRNAs into terminally exhausted T cells from Cas9-expressing (Hl 1 Cas9) mice. To do so, one can generate murine-stem cell virus (MSCV)-mCherry retroviral constructs containing a U6 promoter driving expression of gRNAs targeting Ezh2, EED, Ringl and Cbx4 (SEQ ID NOs: 1-8). The MSCV (Murine Stem Cell Virus) retroviral system is commonly used for over-expression in stem cells and hematopoietic cells, featuring bicistronic expression with mCherry to identify transduced cells. These constructs are commercially available.For each gene, one can test 3 gRNAs identified from the CRISPR / Cas screen to have the strongest magnitude effects on Tcf7 silencing maintenance. To disrupt the ability of PRC2 to read H3K27me3 without disrupting complex formation, one can also use MSCV-mCherry retroviral constructs to over-express EED with alanine mutations at H3K27me3 contact residues (Eed-3A: F97A, Y148A, Y365A); these mutations abrogate EED chromatin binding and exhibit dominant negative effects in a developmental context. As a negative control, one can also clone and test wildtype EED (EED-wt) (Mus musculus NCBT Gene ID: 13626; Homo sapiens NCBI Gene ID: 8726). Similarly, to specifically disrupt vPRC2 reading of H2AK119ubl, one can generate MSCV-mCherry construct to overexpress dominant-negative RYBP with mutations that abrogate H2AK119ubl binding (T31A, F32A), along with its wildtype (Mus musculus NCBI Gene ID: 56353; Homo sapiens NCBI Gene ID: 23429) counterpart. All constructs can be made using standard restriction-enzyme cloning methods and can be utilized to generate mouse ecotropic retrovirus using standard packaging procedures in a HEK293T cell line.

[0210] In vitro T cell differentiation assays.

[0211] Using the in vitro differentiation system (FIG. 4C), one can generate terminal cxhaustcd / cffcctor (7c 7TIM3+) CD8+T cells, transduce with the retroviral constructs as described above, then measure resultant effects on the maintenance of Polycomb domains and gene silencing at Tcf7 and other memory loci. CD8+T cells can be isolated from 7c / 7YFP / + mice or 7c / 7YFP / +; HllCas9 / + / mice. Cells can then be stimulated continuously for 6 days with aCD3 / CD28 and IFNa to induce Tc / 7 silencing and entry into the terminal (7c / 7 TIM3+) state. After 6 days, one can retrovirally transduce T cells to introduce PRC2 perturbations, then purify transduced, terminal cells (mCherry+7c / 7’YFP-TIM3+) using fluorescence-activated cell sorting. One can then reculture sorted cells in the absence of stimulation for an additional 4-8 days, then measure how these transduced perturbations affect Polycomb repressive chromatin domains over time, both at Tc / 7 and more global at other gene loci.

[0212] CUT&RUN Chromatin profiling.

[0213] One can isolate CD8+T cells at successive time points after reculture (dO, d4 and d8), then measure genome-wide distributions for H3K27me3 and H2AK119ubl in these cells using the chromatin profiling method CUT&RUN82. In brief, for CUT&RUN, sorted, re-cultured populations can be permeabilized with mild detergents, then treated in situ with an anti-H3K27me3 or an anti-H2AKl 19ubl antibody, and a secondary MNase-conjugated antibody. Released DNA fragments can then be mixed in with E. coli spike-in normalization controls, which can enable quantification of the effects of PRC perturbations on histone modification levels. DNA can then be PCR-amplified with adapters for sequencing on an Illumina platform. One can then map sequenced reads to the mouse genome using Bowtie 2, then segment Polycomb domains based on initial H3K27me3 marking on TcJ7 and other memory gene loci of interest prior to stimulation withdrawal. One can then quantify the levels and width of H3K27me3 and H2AK119ubl distributions on these gene loci.

[0214] It can then be asked whether cPRCl complexes are present in terminally exhausted cells and do they uphold silencing of Tcf7 and other memory genes by driving chromatin compaction. To address these questions, one can first isolate terminally exhausted T cells generated in vitro (FIG.4C), then identify the PRC1 subunit composition by immunoprecipitation with epitope tagged-PRCl subunits followed by mass spectrometry (IP-MS) (FIG. 4B (D)). To determine if cPRCl subunits maintain Tcf7 locus compaction and silencing, one can knockout cPRCl subunits involved in compaction (Cbx4, Phc3), then measure effects on the Tcf7 locus conformation and localized cPRCl using SCEPTRE (for Single Cell Evaluation of Post TRanslational Epigenetic encoding), a super-resolution chromatin imaging assay, that enables concurrent visualization of multiple genomic loci using DNA-F1SH and immunolabeled proteins. In preliminary experiments, SCEPTRE was utilized to measure Tcj7 and its associated chromatin states in single cells (FIG.4B (E)). One can also use flow cytometry to measure 7c / 7YFP and other memory markers (FoxOl, CD62L, Slamf6).

[0215] Immunoprecipitation-mass spectrometry (IP-MS) of PRC 1 subunits.

[0216] For proteomics of PRC1, one can generate murine-stem cell virus (MSCV)-IRES-mCherry retroviral constructs driving expression of Flag-Ringl and Flag-Cbx4 and their untagged controls. CD8+T cells can be isolated from 7c / 7YFP / + mice, stimulated and differentiated continuously for 6 days to induce Tc / 7 silencing and entry into the terminal (7c 7TIM3+) state. After 6 days, one can retrovirally transduce T cells with Flag-Ringl, Flag-Cbx4, or untagged -Ringl -Cbx4 control. Using fluorescence-activated cell sorting, one can sort mCherry+7c / 7 YFP-TIM3+cells and fractionate nuclei to remove cytoplasm extract. The nuclei can then be disrupted with MNase digestion or sonication to release chromatin-bound proteins and the proteins associated with soluble or chromatin-bound Ringl or Cbx4 can be affinity purified using a FLAG- IP-MS approach.The PRC 1 -associated polypeptides can then be identified by mass spectrometry (MS) and including an untagged control can be implemented to assist in narrowing unique polypeptides associated with PRC 1. These proteomics experiments can be repeated at least four independent times to support a reproducible and statistically significant candidate list of proteins.

[0217] Retroviral constructs for cPRCl perturbation.

[0218] Canonical Polycomb Repressive Complex 1 (cPRCl) associates via sterile-alpha- motif (SAM) domains of Polyhomeotic (PHC1, PHC2, or PHC3) subunits; and can also undergo phase separation via interactions between intrinsically disordered domains of CBX subunits. Thus, to disrupt cPRCl interactions, one can generate MSCV-mCherry vectors expressing gRNAs targeting Cbx4 and Phc3, the two subunits showing the greatest Tcf7 silencing reversal in the Cas9 screen. One can also target the core subunit Ringl as a positive control for the disruption of all PRC1 complexes. To selectively disrupt Phc3 oligomerization, one can generate MSCV-mCherry constructs overexpressing PHC3 with three mutations (L9 1A, L965A, L969A) that disrupt PRC1 interactions and clustering. To disrupt the Cbx4 interactions via positively charged intrinsically disordered regions, one can generate MSCVmChcrry constructs for Cbx4 containing 10 mutations that reduce positive charge in the intrinsically disordered region (lOx R,K,H —>■ A; 283-451); for both subunits, one can express wildtype proteins as negative controls.

[0219] In vitro T cell differentiation and analysis.

[0220] One can generate sorted terminal (7c / 7 TIM3+) T cells as above, then transduce with the cPRC 1 perturbation constructs described above. One can then re-culture them at successive time points (dO, d4 and d8) after rest from stimulation, then analyze the effects of these perturbations on Tcf7 compaction state and expression using SCEPTRE and flow cytometry respectively, as below.

[0221] Supe r-resolution chromatin imaging.

[0222] SCEPTRE uses expansion microscopy (EXM) to achieve imaging at ~70 nm resolution, sufficient to resolve the large Polycomb domains (>100 kb) formed at the Tcf7 locus. Using SCEPTRE, one can analyze either resting CD8+T cells prior to activation, or terminally-exhausted T cells subject to different durations of rest (dO and d4), generated as described above. Following the SCEPTRE protocol, one can immunolabel using fluorescent antibodies targeting either core PRC1 subunits (Ringla / b) or the cPRCl subunits (PCGF4 or Cbx4). To visualize the Tcf7 locus and measure its degree ofcompaction, one can perform multi-color DNA-FISH to label the Tcf7 promoter and a -lOOkb upstream region within the same Polycomb domain. Through automated image analysis, one can quantify, for each perturbation condition, the degree of Tcf7 compaction, defined as the distance between the promoter and upstream region, along with the amount of cPRCl enrichment at each locus.

[0223] Flow cytometry.

[0224] One can determine whether the cPRCl perturbations described above are sufficient to reverse the silencing of Tcf7' YFP and other memory gene loci. One can use flow cytometry to assay the re-expression of 7c 7 YFP or other memory markers.

[0225] It can be asked whether PRC2 / cPRCl mechanisms also uphold silencing of Tcf7 and memory genes in TILs undergoing terminal exhaustion in vivo. To address this question, one can generate (7c / 7’TIM3+) T cells using the B16-Ova model (FIG. 4B (C)).One can inoculate congenic (CD45.2+) C57 / BL6 recipient mice with B 16-Ova tumor cells, then adoptively transfer CD8 T cells from OT-I;7c / 7 YFP mice into tumor-bearing mice after 7 days. After 7 days, one can then harvest tumor tissues, sort terminally exhausted (7c / 7 YFP-TIM3+) T cell populations within these tissues, retrovirally transduce them to perturb cPRCl -mediated compaction, then assay resultant effects on silencing at Tcf7 and other memory genes (FoxOl, CD62L and Slamf6) using flow cytometry.

[0226] If Model 1 ; FIG. 4B (A) is correct, one can observe Polycomb domains (H3K27me3 / H2AK119ubl) at Tcf7 and other genes to be reduced by PRC2 perturbations but not by vPRCl perturbations. One can further observe that cPRCl complexes can be present and that their perturbation can disrupt TcJ7 locus compaction and silencing, together with silencing of memory gene programs more broadly. Though one can favor Model 1 ; FIG. 4B (A) based on preliminary data, it remains possible that Model 2; FIG.4B (B), involving variant PRC1 feedback and H2AK119Ub transcriptional repression may also play a role in silencing maintenance. If this is the case, one can evaluate this model using a similar perturbation approach as those laid out above. In any case, this work can illuminate questions of how Polycomb extinguishes self-renewal and enforces terminal differentiation. These insights can also restore persistence and self-renewal to CD8+T cells for therapeutic benefit.

[0227] The mutant PRC1 and PRC2 subunits generated, though previously validated, may be too weak to exert a dominant negative effect in the system. If this is the case, one can instead use these mutants in a knockout / rescue setting, where one can useCRISPR / Cas9 to knockout the endogenous gene, then retrovirally transduce either wildtype or mutant versions of the same gene to determine whether they can rescue loss-of-function phenotypes. Additionally, functional redundancy between paralogs may diminish observable functional phenotypes; in this case, one can use CRISPR / Cas9 to concurrently knockout multiple genes. One can achieve concurrently targeted multiple loci by nucleofection of multiple Cas9-RNPs, if necessary.

[0228] One can also determine how Polycomb lockdown of terminal exhaustion is differentially impacted by IL-2 and IL- 15 and their downstream transcription factors. The ability of the Polycomb system to stably maintain silencing of target genes depends on the levels and activity of TFs acting at these gene loci and, consequently, the signaling pathways upstream of these TFs. While some TFs can enhance Polycomb recruitment or function at their silent loci to enhance silencing stability, some TFs could antagonize Polycomb function at silenced genes by modulating the chromatin states or transcriptional activity at these loci. To understand the degree to which sternness is irreversibly lost upon terminal exhaustion - and can be restored with appropriate intervention - it can be important to elucidate this interplay between Polycomb silencing and signaling pathway / TF activity at Tcf7 and other memory regulatory genes in terminally exhausted cells. Consistent with this idea, data shows that interleukin (IL)-2 and IL- 15 exert opposing effects on Polycomb-mediated lockdown of Tcf7 silencing. IL-2 drives proliferation differentiation of CD8 T cells after antigen encounter and activation, whereas IL- 15 supports the survival and maintenance of memory T cell populations after antigen clearance. Strikingly, it was found that IL-2 prevents and IL- 15 enhances re-expression of PRC2-silenced Tcf7 and other memory markers upon Polycomb inhibition. Based on these findings, it can be hypothesized that IL-2 and IL-15 differentially modulate Polycomb silencing of memory and self-renewal genes in terminally exhausted cells by up-regulating distinct downstream TFs. IL-2 can turn on Prdml (BLIMP-1), a TF that drives terminal effector cell differentiation and represses Tcf7 expression. On the other hand, IL- 15 can turn on Foxol, a TF that mediates T cell memory and sternness and induces Tc 7 expression. These TFs, amongst others, could mediate the opposing effects of IL-2 and IL-15 on Polycomb silencing at Tcf7 and other memory genes. Consistently, Prdml and Foxol were identified from the Cas9 screen as factors promoting / opposing Tcf7 silencing respectively (FIG.4D (B)).

[0229] One can further test this hypothesis in two stages: first, one can test whether 1L2- and IL- 15 differentially impact Polycomb silencing of memory gene programs in terminally exhausted cells. To do so, one can measure transcriptomic states of exhausted T cells recultured with both cytokines, both with and without PRC2 inhibition. Single-cell (sc)RNA sequencing can be used as re-activation of Polycomb silenced genes often show cell-to-cell heterogeneity not well captured by bulk-averaged measurements. Second, one can identify the TFs responsible for modulating the effects of IL-2 or IL-15 on Polycomb silencing of Tcf7 and other self-renewal genes. To do so, one can perturb candidate TFs in terminally exhausted cells, then measure how these perturbations impact the genes re-expressed upon PRC2 inhibition. To identify candidate TFs, one can identify TFs from scRNA-seq data that are differentially up-regulated in response to IL-2 and IL-15 and show evidence for direct binding to Poly comb- silenced memory genes. Finally, one can test whether these TFs also impact the stability of Polycomb domains of terminally exhausted T cells in vivo. These experiments can involve isolation and analysis of TILs from tumor-bearing mice, using B 16 melanoma as a model.

[0230] Experimental data suggests IL-2 and IL- 15 can differentially impact the stability of Polycomb-mcdiatcd silencing of Tcf7. Here, to determine whether IL-2 and IL-15 affect Poly comb-mediated Tcf7 silencing in terminally exhausted cells, terminally exhausted (Tcf7 'T1M3+) CD8+T cells were generated from naive and memory T cells from 7c / 7YFP mice using the in vitro assay. Exhausted cells were then recultured under resting conditions with either IL-15 or IL-2, and with or without the Ezh2 inhibitor Taz. Because IL-2 has dose-dependent effects on survival versus differentiation, cells were rested at low and high IL-2 doses (10 versus 100 U / mL). It was found that while Ezh2 inhibition moderately destabilized Tcf7 silencing in low IL-2 doses, its destabilizing effects on Tcf7 silencing were greatly enhanced in IL-15 (-75% 7c / 7’YFP+cells versus 30% in low IL-2 at Day 7, FIG.4F (A-C)). In contrast, high IL-2 doses abrogated TcJ7 re-expression upon Ezh2 inhibition (-1% with or without Taz, FIG. 4F (A, C)). Thus, IL-2 and IL-15 differentially modulate Polycomb-mediated Tc 7 silencing in terminally exhausted cells, with IL- 15 antagonizing and IL- 2 reinforcing silencing. The antagonistic effects of IL- 15 on Polycomb lockdown of Tcf7 silencing may also extend to other memory genes silenced during terminal exhaustion. When terminally exhausted T cells were rested in IL- 15, they also showed enhanced re -expression of the memory regulators FoxOl and CD62L uponEzh2 inhibition (FIGS. 4F(D)-4F(E)). Thus, cytokines may broadly modulate Polycomb silencing of self-renewal genes in terminally exhausted cells.

[0231] It can be asked whether IL-2 and IL- 15 differentially modulate Polycomb silencing of memory gene programs in terminally exhausted cells on a more global level. A hypothesis can predict that IL- 15 antagonizes Poly comb silencing at memory gene loci, whereas high IL-2 reinforces Polycomb silencing at these loci and may instead antagonize Polycomb silencing at effector genes. To test this prediction, one can re-culture terminally exhausted CD8+T cells under resting conditions in the presence of either IL- 15 or IL-2, subject them to PRC2 inhibition, then measure effects on transcriptomes using scRNA-seq.

[0232] In vitro differentiation assays.

[0233] As above, CD8+T cells can be isolated from 7c 7YFP mice, then stimulated in vitro using aCD3 / aCD28 antibodies, IL-2, and IFNa. After 6 days of stimulation, during which the majority of cells differentiate, one can use fluorescence-activated cell sorting (FACS) to isolate Tc / 7TIM3+cells. One can then re-culture these cells under resting conditions (no aCD3 / aCD28 or IFNa) with either low IL-2 (10 U / mL), high IL-2 (100 U / mL), or IL-15 (50 ng / mL). Re-cultured T cells can additionally be treated with cither 1 pM Tazcmctostat (Taz) to inhibit Ezh2 activity, or DMSO vehicle controls. After 4 days, one can harvest cells for scRNA-seq.

[0234] scRNA seq and analysis.

[0235] One can use the 10X Genomics platform. Here, one can prepare singlecell libraries multiplexed with hashtag oligonucleotides and sequence libraries using an Illumina Next-Seq. One can then use Monocle3 to perform differential gene expression analysis to uncover genes up-regulated by Taz in either IL- 15 or with high doses of IL-2. Specific candidate TFs identified can be further validated by intracellular staining flow experiments.

[0236] It can be asked which TFs act downstream of IL- 15 and IL-2 to modulate Polycomb silencing of target genes in terminally exhausted cells. To answer this question, one can first identify candidate TFs from scRNAseq data in two days: first, one can perform a TF binding motif analysis of genes differentially activated upon Polycomb inhibition in the presence of either IL-2 and / or IL-15. To do so, one can use the analysis package SCENIC to identify candidate TFs with enriched binding to these gene loci. Second, one can identify specific TFs that are differentially enriched in the presence of IL- 15 and / or IL-2 without Ezh2 inhibition. Through loss and gain-of-function perturbation analyses, onecan test whether these identified candidates mediate, at least in part, the impact of the IL-2 or IL-15 on Polycomb silencing of Tcf7 and other gene loci. One can perturb terminally exhausted cells generated using the in vitro exhaustion system, reading out the effects of these perturbations on the maintenance of Tcf7 silencing as well as other memory loci.

[0237] Design and generation of retroviral constructs.

[0238] As above, for CRISPR / Cas9 knock out of candidate TFs, one can generate murine-stem cell virus (MSCV)-mCherry retroviral const ructs expressing gRNAs targeting candidate TFs identified above. One can identify and test candidate TFs induced in response to IL-2 versus IL-15 (Prdml versus FoxOl), though one can also identify other candidates as well. As positive controls, one can knock out second messengers of IL-2 / IL-15 signaling (e.g., Stat5a, Stat5b, Erk). One can also clone MSCVmCherry retroviral constructs to over-express full-length TFs. All constructs can be made using standard restriction enzyme cloning methods and can be utilized to generate mouse ecotropic retrovirus using standard packaging procedures in a HEK293T cell line.

[0239] Analysis of terminally exhausted cells generated ex vivo.

[0240] As above, one can generate exhausted CD8+T cells using the in vitro exhaustion system, then sort terminally exhausted (7c / 7 TIM3+) cells emerging after 6 days of stimulation. One can then retrovirally transduce sorted cells with constructs described above, re-culture these cells under resting conditions (no aCD3 / aCD28 or IFNa) with 1L-2 (both low or high) or IL- 15, then treat cells with either 1 pM Taz to inhibit Ezh2 activity, or DMSO controls. One can then utilize flow cytometry to measure resultant effects on silencing reversal of TcJ7 or other memory gene loci.

[0241] It can be asked whether IL-2 and IL- 15 and their downstream TFs also modulate Polycomb silencing in CD8 T cells undergoing terminal exhaustion in vivo. To address this question, one can use, generate, and sort terminally exhausted (7c / 7YFP-TIM3+) OT-I CD8 TILs from B16-Ova melanoma model, as above. One can incubate them with IL-2 and IL- 15, transduce them with retroviral constructs to perturb TF activity, then re-culture them in IL-2 / IL-15, with or without Taz. After 4-8 days, one can measure the effects of Ezh2 inhibition on expression of Tcf7 and other memory markers (FoxOl, Slamf6, and CD62L) using flow cytometry.

[0242] IL- 15 can more broadly antagonize Polycomb-mediated silencing of other genes besides Tcf7, thereby antagonizing commitment to a terminally exhausted state. On the other hand, one can observe that high IL-2 can reinforce the silencing of Tcf7 andmemory programs, thus upholding commitment to a terminal state. One can further identify TFs activated by IL- 2 or IL- 15 that are functionally responsible for the modulatory effects of these cytokines on Polycomb silencing in exhausted cells. This knowledge can elucidate how trans- factors work with the Polycomb machinery to uphold terminal state stability, informing strategies for reversing exhaustion differentiation and enhancing T cell selfrenewal to improve the persistence of antitumor T cells for therapeutic purposes.

[0243] Transcriptomic data of IL-2 / IL-15 and Taz treated T cells may not reveal clear TF candidates for testing. This may be because trans-regulators downstream of cytokine signaling may not be induced via transcriptional changes or, alternatively, because of limitations of using TF binding site analysis to identify candidates. In this case, one can perform a CRLSPR / Cas9 screen to more broadly interrogate TFs expressed in exhausted cells, to identify those responsible for IL- 15 (IL-2) dependent antagonism (reinforcement) of Polycomb-mediated silencing lockdown.

[0244] IL-7 can also antagonize Polycomb-mediated silencing of genes including Tcf7, thereby antagonizing commitment to a terminally exhausted state. One can culture terminally exhausted T-cells at rest from TCR stimulation, in Tazemetostat or another inhibitor of a Polycomb complex protein, and IL-7 in place of, or alongside, IL- 15, measuring the effects of this combination on expression of Tcf7 or other genes relevant for T-cell sternness and memory (FIG. 5).

[0245] One can also further evaluate Polycomb inhibition as a strategy for longterm antitumor efficacy in terminally exhausted T cells and tumor infiltrating lymphocytes (TILs). In many tumors, TILs are already exhausted before ex vivo expansion. Thus, as Polycomb inhibition reverses silencing of Tcf7 and other memory genes, one can hypothesize that Polycomb inhibition can restore sternness in TILs and enhance their longterm antitumor efficacy. One can test this hypothesis in two settings: First, one can do so in the B 16 melanoma mouse model for adoptive cell therapy, using tumor cells expressing a mutated peptide from the melanoma antigen Pmel, or gplOO (B16KVP: mut:KVPRNQDWL vs wt:EGSRNQDWL) (FIG. 4G(A)). Specifically, this mutant peptide is recognized by PmeLI TCR CD8+T cells more strongly than its wildtype counterpart, enabling modeling of a neoantigen response. Also, B 16 is an established model for testing stem-like (TCF1+) T cell populations after adoptive transfer. Using this model, one can test whether Polycomb inhibition can restore sternness and antitumor functions to terminally exhausted PmeLI CD8+T cells (FIG. 4C). One can first test Polycombinhibition on in vz / ro-generated terminally exhausted (7c / 7 TIM3+) cells, as one can have extensively characterized them above. Next, one can test Polycomb inhibition on Pmel-1 CD8 TILs that have been isolated from primary B16 tumors, expanded, then re -transferred into tumor-bearing hosts. In both cases, one can test whether Polycomb inhibition enhances T cell self-renewal and anti-tumor activity in vivo. Second, as a first step towards potential translation, one can evaluate whether Polycomb inhibition can revive terminally exhausted human T cells (3B) (FIG. 4G (B)). To do so, one can leverage the in vitro system to generate terminally exhausted CD8+T-cells from human peripheral blood. One can also engineer T cells to express a neoantigen targeting TCR (p53R175H; HLA-A*02:01), to enable direct testing of T cell performance against the TYK-nu ovarian cancer cell line, a neoantigen TCR model for adoptive cell therapies (ACT). One can then subject exhausted TCR-expressing cells to Polycomb inhibition and evaluate their persistence and antitumor efficacy using in vitro assays involving co-culture with TYK-nu cells. For these experiments, one can implement a neo-antigen B16 TIL therapy model.

[0246] Modeling of neoantigen TIL therapy with B16KVP melanoma.

[0247] One can have established a workflow to isolate large numbers of PmeLI CD8 TILs from primary B 16KVP tumors for modeling generation of TIL infusion products and testing through subsequent transfer (FIG. 4H (A)). In this model, TIL products from primary tumors were mostly differentiated (-0.1%, CD39 CD69 ) (FIG.4H (B)), whereas infusion products from splenic T cells showed more stem-like phenotypes (-6% CD39~ CD69 ). Upon adoptive transfer into mice with secondary B16KVP tumors, TIL infusion products showed worse performance compared to those generated from peripheral T cells (FIG.4H (C)) One can note that mice were also administered a gplOOKVP vaccine during adoptive transfer, though similar differences can be observed without vaccination. These results establish the feasibility of modeling neoantigen TIL therapy production in B16 melanoma and reinforce the idea that the stem-like populations in TIL products enhance their efficacy.

[0248] Human T cells engineered with a neoantigen-specific TCR mount antitumor responses.

[0249] One can find that human T cells from peripheral blood transduced with the R175H TCR mount cytotoxic responses against the TYK-nu ovarian tumor cell line in co-culture assays (FIG. 41). One can apply this system in experiments below.

[0250] One can ask whether Ezh2 inhibition restores self-renewal and antitumor performance to exhausted T cells and TILs. One can test this hypothesis in two stages (FIG.4G (A)). First, one can test whether Polycomb inhibition can restore self-renewal and antitumor function to terminally exhausted Pmel-I CD8+T cells generated through the in vitro system. Second, one can test whether Polycomb inhibition restores sternness and function to exhausted Pmel-I TILs from primary B16 tumors. In both cases, one can adoptively transfer both neoantigen-specific T cell populations into B16KVP-bearing hosts, then evaluate their (A) sternness and (B) tumor control after adoptive transfer.

[0251] In vitro differentiation of terminally exhausted Pmel-I CD8+T cells and Polycomb inhibition.

[0252] One can generate terminally exhausted (7c / 7 YFP_TIM3+) CD8+T cells from CD45.1+ Tcf7' YFP; Pmel-I TCR transgenic mice, then purify them using FACS. Sorted cells can then be either used directly for adoptive transfer, or subject to Polycomb inhibition by re-culture in 1 pM Taz and 50 ng / mL IL- 15 without TCR stimulation for 4 days, during which a substantial fraction of these cells re-activate I'c / 7 and other memory loci (FIG.4F (A)). To evaluate Polycomb inhibition apart from other factors, one can also re-culture cells without Taz as a negative control. Both populations (+ / -Taz) can be analyzed with adoptive transfer experiments. Immediately prior to adoptive transfer, infusion products can be analyzed by flow cytometry to measure 7F / 7 YFP and sternness markers (CD62L, FoxOl).

[0253] Adoptive T cell transfer into B16KVP tumor-bearing mice.

[0254] Six- to eight- week-old adult female C57 / BL6 mice (CD45.2+) can be injected subcutaneously with 3.5 xlO5B16KVP melanoma cells. Tumor cells can be allowed to establish for 10 days, after which the tumor-bearing mice receive a total body irradiation dose of 6 Gy. One can then transfer exhausted (7c / 7TIM3+); or exhausted then rested in the absence or presence of 1 pM Taz cells into congenic (CD45.2) mice with established B16-gpl00KVP tumors, in each case transferring IxlO6T cells / mouse. To further effector function, one can dose recipients with 12 pg IL-2 / day for three consecutive days post-transfer, in accordance with TIL therapy workflows. One can then measure tumor growth for 60 days and analyze Pmel-1 T cells (CD45.1) in tumors and in lymph nodes at d7, 14, and 21 post-transfer using flow cytometry to assay both sternness (7e / 7’YFP, CD62L) and exhaustion markers (CD39, TIM3).

[0255] Expansion of Pmel-I CD8+TILs, Polycomb inhibition and adoptive transfer.

[0256] To generate TILs for expansion and transfer, one can subcutaneously inject Pmel-I TCR-transgenic mice with B16F10 tumor cells, which have low MHC expression and get established over 2-3 weeks (FIG.4H(A)). After establishment, one can harvest the tumors, isolate CD45+ immune cells, and expand tumor-infiltrating Pmel-1 TILs in vitro by co-culturing for 10 days with irradiated C57 / BL6 splenocytes pulsed with gplOO.KVP neoepitope (FIG. 4H(A)). The resulting PmeLl TIL infusion product is differentiated with terminally exhausted phenotypes (CD39+CD69+, FIG. 4H(B)). One can then directly transfer expanded Pmel TILs for adoptive transfer, or first rest cells (+ILL5) either without Polycomb inhibition or with inhibition (+Taz). In each case, one can transfer IxlO6T cells into tumor-bearing mice, then analyze tumor growth and donor T cell populations using flow cytometry, as above. Here, splenic Pmel CD8 T cells can be used as positive controls for infusion products with stem-like T cells.

[0257] It can be asked whether Polycomb inhibition reverses exhaustion and restores self-renewal in human T cells. To test this hypothesis, one can isolate CD8+T cells from human peripheral blood, transduce them with the R175H TCR, then differentiate them into a terminally exhausted state. One can then subject these cells to Polycomb inhibition and assay for restored expression of TCF1 and other memory regulators, as measured by flow cytometry, as well as persistent cytotoxic activity, measured by long-term co-culture with a p53175H-expressing cancer cell line (TYK-nu) (FIG.41).

[0258] Generation of TCR-lransduced, terminally exhausted human T cells and Polycomb inhibition.

[0259] Human peripheral blood from de-identified donors can be purchased at a local blood bank. Peripheral blood lymphocytes (PBL) can be activated using aCD3 / CD28 antibodies, then retrovirally transduced with a human TCR targeting TP53 R175H. The human TCR is chimerically fused to a murine TCR constant region enabling identification using an anti-murine-TCR constant antibody (clone H57). After transduction, one can differentiate them by stimulation with aCD3 / CD28 and IFNa. At successive time points (d4-dl4) after stimulation onset (6-10 days), one can analyze cells by flow cytometry to measure markers of precursor (TCF1, FoxOL Slamf6 and CD62L) and terminal states (TIM3, CD39). Based on this analysis, one can sort transduced, terminally exhausted cells (TCR+TIM3+CD39+Slamf6_) at a timepoint at which this population is prominent. Sorted,terminally exhausted R175H TCR CD8+cells can be used directly for adoptive transfer or rested (+IL-15) either in the absence or presence of 1 pM Taz. For rested cells, one can perform flow cytometry at successive time points (d4-dl0) to measure the activation of memory markers (TCF1, FoxOl, Slamf6 and Cd62L). Based on this analysis, one can then choose cells for adoptive transfer at a time point at which memory markers are re-expressed and stem-like phenotypes are restored in a significant (50%+) percentage of cells.

[0260] Evaluation of T cell performance using chronic tumor co-culture assays.

[0261] To evaluate the long-term tumor killing of neoantigen-expressing cells by the different T cell populations, one can take equal numbers (~104) of TCR-transduced T cells from each test population [exhausted, rested (+IL15) +Taz, rested (+15) -Taz], then coculture with TYK-nu cells at a 1:2 (tumor: T cell) ratio to assay serial T cell killing of neoantigen-expressing human tumors in vitro (FIG. 41). One can measure growth curves for tumor cell area over 3 days, as a readout of persistence in antitumor activity. One can then harvest T cells and assay them for expression of sternness (TCF1, FoxOl, Slamf6 and Cd62L) and exhaustion (TOX, CD39, TIM3) markers across different conditions.

[0262] One can observe that Polycomb inhibition can cause terminally exhausted Pmcl-1 CD8+T cells and TILs to rc-acquirc a stem-like phenotype and thus show enhanced persistence and ability to control B16KVP tumors. Likewise, one can observe Polycomb inhibition causes TCR-transduced, terminally exhausted (R175H TCR+) human CD8+T cells to re-acquire stem-like states and show improved persistence and cytotoxic activity. These outcomes can establish a rationale for translating the findings to human TIL expansion and manufacturing, with applicability to different tumor types.

[0263] Taz toxicity may offset benefits to T cell sternness and persistence. If so, one can perturb Polycomb subunits using Cas9 deletion. Human CD8 T cells may show different optimal culture conditions for terminal exhaustion compared to mouse T cells, though exhaustion-associated state changes also occur in human cells in culture conditions similar to the disclosed system (i.e., aCD3 / 28, IFN0). If the system sub-optimally generates exhausted human T cells, one can titrate stimulation and cytokines to optimize terminal exhausted cell production. Also, Polycomb may have distinct effects in human T cells due to species-specific control. If so, one can screen human T cells to identify underlying mechanisms.

[0264] Data Analysis and Statistical Considerations.

[0265] For mouse tumor studies, experiments can be conducted in a double blinded manner. To compare mean tumor sizes across T cell treatments to reach a statistical power of 80 at an alpha error rate of 0.05, one may need sample sizes of approximately 14-15 mice per group. To logistically achieve this, one can perform 3 repeats of in vivo experiments, using groups of 5 mice per condition. Two-way ANOVA can be used for statistical analysis of tumor growth between conditions, and the log-rank test can be performed for statistical analysis of mouse survival.

[0266] Sex as a biological variable: One can perform experiments on both matched male and female donor and recipient mice; if sex-specific differences in T cell function are seen, one can increase mouse numbers, then perform statistical testing and report on these sex-specific differences.Table 2. SequencesNON- LIMITING EMBODIMENTS

[0267] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting Embodiments contain elementsthat are modular and can be combined with each other in any number, order, or combination to form a new non-limiting Embodiment, which can itself be further combined with other non- limiting Embodiments.

[0268] Embodiment 1. A method for reversal of an exhaustion phenotype and restoration of a stem-like phenotype of a T-cell in vitro, the method comprising: resting the T-cell in absence of T-cell receptor (TCR) stimulation; inhibiting at least a portion of polycomb repressive complexes 1 & 2 (PRC1 / 2) in the T-cell; and contacting the T-cell with interleukin- 15 (IL- 15).

[0269] Embodiment 2. The method of Embodiment 1 or any other Embodiment, wherein the exhaustion phenotype is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the T-cell, and wherein the stem-like phenotype is characterized at least in part by upregulation of TCF7 in the T-cell relative to the exhaustion phenotype.

[0270] Embodiment 3. The method of any one of Embodiments 1-2 or any other Embodiment, wherein the portion of PRC1 / 2 comprises a protein component of poly comb repressive complex 2 (PRC2).

[0271] Embodiment 4. The method of any one of Embodiments 1 -3 or any other Embodiment, wherein the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SEI’ nuclear proto-oncogene (SE T), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

[0272] Embodiment 5. The method of Embodiment 4 or any other Embodiment, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of EZH2.

[0273] Embodiment 6. The method of Embodiment 5 or any other Embodiment, wherein the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl] -3-methyl- 1 - [( 1 S) - 1 -methylpropyl] -6- [6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide), CPI- 1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

[0274] Embodiment 7. The method of Embodiment 4 or any other Embodiment, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with an RNA interference (RNAi) inhibitor of EZH2.

[0275] Embodiment 8. The method of Embodiment 7 or any other Embodiment, wherein the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short haiipin RNA (shRNA), or any combination thereof.

[0276] Embodiment 9. The method of Embodiment 4 or any other Embodiment, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises knocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique.

[0277] Embodiment 10. The method of Embodiment 9 or any other Embodiment, wherein the nuclear genomic editing technique comprises a clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) genomic editing technique.

[0278] Embodiment 11. The method of any one of Embodiments 1 - 10 or any other Embodiment, wherein the portion of PRC1 / 2 comprises a protein component of polycomb repressive complex 1 (PRC1).

[0279] Embodiment 12. The method of Embodiment 11 or any other Embodiment, wherein the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), RTNG1 A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

[0280] Embodiment 13. The method of Embodiment 12 or any other Embodiment, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of RING1 A.

[0281] Embodiment 14. The method of Embodiment 13 or any other Embodiment, wherein the small molecule inhibitor of RING1A comprises PRT4165 (2-(3-pyridinylmethylene)- 1 H-indene- 1 ,3 (2H)-dione) .

[0282] Embodiment 15. The method of Embodiment 12 or any other Embodiment, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

[0283] Embodiment 16. The method of Embodiment 15 or any other Embodiment, wherein the nuclear genomic editing technique comprises a CRTSPR-Cas9 genomic editing technique.

[0284] Embodiment 17. The method of any one of Embodiments 1-16 or any other Embodiment, further comprising contacting the T-cell with interleukin-2 (IL-2).

[0285] Embodiment 18. A method for preparation of a tumor-infiltrating lymphocyte (TIL) therapy for a subject, the method comprising: isolating a T-cell from a tumor of the subject and, after isolating the T-cell from the tumor of the subject and before administering the T-cell to the subject, reversing an exhaustion phenotype of the T-cell and restoring a stem-like phenotype of the T-cell according to the method of any one of Embodiments 1-17 or any other Embodiment.

[0286] Embodiment 19. A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject the TIL therapy of Embodiment 18 or any other Embodiment; optionally wherein the cancer comprises melanoma, renal cell carcinoma, or both.

[0287] Embodiment 20. The method of Embodiment 19 or any other Embodiment, further comprising administering to the subject an effective amount of IL-2.

[0288] Embodiment 21. A method for preparation of a chimeric antigen receptor (CAR) T-cell therapy, the method comprising: resting a CAR T-cell in absence of TCR stimulation; inhibiting at least a portion of PRC 1 / 2 in the CAR T-cell; and contacting the CAR T-cell with IL- 15 according to the method of any one of Embodiments 1-17 or any other Embodiment; wherein the CAR T-cell therapy comprises the CAR T-cell.

[0289] Embodiment 22. A method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cell of Embodiment 21 or any other Embodiment.

[0290] Embodiment 23. A composition comprising isolated T cells modified ex vivo to reverse an exhaustion phenotype in the isolated T cells, wherein the modification comprises: resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation;contacting the isolated T cells with an inhibitor effective in inhibiting at least a portion of Polycomb Repressive Complexes 1 and / or 2 (PRC 1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

[0291] Embodiment 24. The composition of Embodiment 23, wherein the exhaustion phenotype in the isolated T cells is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the isolated T-cells relative to reference or a non-exhausted T cells.

[0292] Embodiment 25. The composition of Embodiment 23, wherein the modification is characterized at least in part by upregulation of TCF7 relative to the exhaustion phenotype levels of TCF7 in the isolated T cells before the modification.

[0293] Embodiment 26. The composition of any one of Embodiments 23-25, wherein the portion of PRC 1 / 2 comprises a protein component of Polycomb Repressive Complex 2 (PRC2).

[0294] Embodiment 27. The composition of Embodiment 26, wherein the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

[0295] Embodiment 28. The composition of Embodiment 27, wherein the inhibiting at least the portion of PRC1 / 2 in the T-cells comprises contacting the isolated T-cells with a small molecule inhibitor of EZH2.

[0296] Embodiment 30. The composition of Embodiment 28, wherein the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide), CPI-1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo- 1 H-pyridin-3-yl)methyl]-2-methyl- 1 -[(1R)- 1 - [1 -(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

[0297] Embodiment 31. The composition of any one of Embodiments 23-26, wherein the inhibiting at least the portion of PRC 1 / 2 in the isolated T-cells comprisesknocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique.

[0298] Embodiment 32. The composition of Embodiment 27, wherein the inhibiting at least the portion of PRC1 / 2 in the T-cell comprises contacting the isolated T-cells with an RNA interference (RNAi) inhibitor of EZH2.

[0299] Embodiment 33. The composition of claim 32, wherein the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

[0300] Embodiment 34. The composition of any one of claims 23-25, wherein the portion of PRC 1 / 2 comprises a protein component of Polycomb Repressive Complex 1 (PRC1).

[0301] Embodiment 35. The composition of claim 34, wherein the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), RING1A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), Polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

[0302] Embodiment 36. The composition of Embodiment 34 or Embodiment 35, wherein the inhibiting at least the portion of PRC1 / 2 in the isolated T-cell comprises contacting the isolated T-cells with a small molecule inhibitor of RING1 A.

[0303] Embodiment 37. The composition of Embodiment 36, wherein the inhibiting at least the portion of PRC1 / 2 in the isolated T-cell comprises contacting the isolated T-cells with a small molecule inhibitor of RING1 A.

[0304] Embodiment 38. The composition of Embodiment 37, wherein the small molecule inhibitor of RING1A comprises PRT4165 (2-(3-pyridinylmethylene)-lH-indene-l,3(2H)-dione).

[0305] Embodiment 39. The composition of Embodiment 34, wherein the inhibiting at least the portion of PRC1 / 2 in the isolated T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1 A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

[0306] Embodiment 40. The composition of Embodiment 23, wherein the isolated T cells are further modified to express a recombinant receptor.

[0307] Embodiment 41. The composition of Embodiment 40, wherein the recombinant receptor is an engineered T cell receptor (TCR).

[0308] Embodiment 42. The composition of Embodiment 40, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

[0309] Embodiment 43. The composition of Embodiment 40, wherein the recombinant receptor is specific for a tumor antigen.

[0310] Embodiment 44. The composition of Embodiment 23, wherein the isolated T cells are native, naturally occurring T cells, autologous T cells, or T cells with specificity for and activity against a tumor.

[0311] Embodiment 45. The composition of Embodiment 44, wherein the native, naturally occurring T cells are obtained from resected tumors, and wherein the T cells are expanded ex vivo prior to the modification.

[0312] Embodiment 46. The composition of Embodiment 44, wherein the T cells with specificity for and activity against a tumor are peripheral blood derived-T cells genetically modified to express a receptor that recognizes and responds to tumor.

[0313] Embodiment 47. The composition of Embodiment 23, wherein the isolated T cells arc selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, gamma delta T cells, a combination of CD4+ and CD8 T+ cells, memory T cells, cytokine-induced killer cells, and combinations thereof.

[0314] Embodiment 48. The composition of Embodiment 47, wherein the isolated T cells are a combination of CD4+ and CD8+ cells.

[0315] Embodiment 49. A composition according to any one of Embodiments 23-48, for use in a method of treating a disease or pathologic condition in a subject, the method comprising administering to the subject an effective amount of the composition.

[0316] Embodiment 50. The composition to Embodiment 49, wherein the disease or the pathologic condition is a tumor or cancer, optionally a solid tumor or hematopoietic malignancy.

[0317] Embodiment 51. The composition according to Embodiment 50, wherein the composition is used in combination with one or more of anticancer agents and / or one or more chemotherapeutic agents.

[0318] Embodiment 52. The composition according to Embodiment 49, wherein the disease or the pathologic condition is a bacterial and / or parasitic infection.

[0319] Embodiment 53. The composition according to Embodiment 52, the method further comprising administering to the subject one or more anti-bacterial or anti-parasitic agent.

[0320] Embodiment 54. A kit comprising the composition of claim 23 and instructions for use.

[0321] Embodiment 55. A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject the TIL therapy of Embodiment 22 or the composition of any one of Embodiments 23-48; optionally wherein the cancer comprises melanoma, renal cell carcinoma, or both.

[0322] Embodiment 56. The method of Embodiment 55, further comprising administering to the subject an effective amount of IL-2.

[0323] Embodiment 57. A method for preparation of a chimeric antigen receptor (CAR) T-cell therapy, the method comprising: resting CAR T-cells in absence of TCR stimulation; inhibiting at least a portion of PRC1 / 2 in the CAR T-cell: and contacting the CAR T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof; wherein the CAR T-cell therapy comprises the CAR T-cell.

[0324] Embodiment 58. A method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cells of Embodiment 57.

[0325] Embodiment 59. An in vitro method of reversing T cell exhaustion comprising modifying isolated T cells, the modification comprising: resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the T cells with an inhibitor effective in inhibiting at least a portion of polycomb repressive complexes 1 and / or 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for reversal of an exhaustion phenotype and restoration of a stem-like phenotype of a T-cell in vitro, the method comprising:resting the T-cell in absence of T-cell receptor (TCR) stimulation;inhibiting at least a portion of polycomb repressive complexes 1 and / or 2 (PRC 1 / 2) in the T-cell; andcontacting the T-cell with at least one cytokine, wherein the cytokine is interleukin-15 (IL-15), interleukin-7 (IL-7), or a combination thereof.

2. The method of claim 1 , wherein the exhaustion phenotype is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the T-cell, and wherein the stem-like phenotype is characterized at least in part by upregulation of TCF7 in the T-cell relative to the exhaustion phenotype.

3. The method of any one of claims 1-2, wherein the portion of PRC 1 / 2 comprises a protein component of polycomb repressive complex 2 (PRC2).

4. The method of any one of claims 1-3, wherein the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

5. The method of claim 4, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of EZH2.

6. The method of claim 5, wherein the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl] -3 - [ethy l(oxan-4-yl) amino] -2-methyl-5- [4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl] -3 -methyl- 1 - [( 1 S)- 1 -methylpropyl] -6- [6-( 1 -piperazinyl)-3 -pyridinyl] -lH-indole-4-carboxamide), CPI-1205 (also known as Lirametostat; N-[(4-methoxy-6-methyl-2-oxo- 1 H-pyridin-3-yl)methy l]-2-methyl- 1 - [( 1 R)- 1 - [ 1 -(2,2,2-trifluoroethyl)piperidin-4-ylJethylJindole-3-carboxamide), or any combination thereof.

7. The method of claim 4, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with an RNA interference (RNAi) inhibitor of EZH2.

8. The method of claim 7, wherein the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

9. The method of claim 4, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises knocking down or knocking out EZH2, SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique.

10. The method of claim 9, wherein the nuclear genomic editing technique comprises a clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) genomic editing technique.

11. The method of any one of claims 1-10, wherein the portion of PRC 1 / 2 comprises a protein component of Polycomb Repressive Complex 1 (PRC1).

12. The method of claim 11, wherein the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), R1NG1A (also known as R1NG1), ring finger protein 2 (also known as RING1B or RING2), polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

13. The method of claim 12, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cell with a small molecule inhibitor of RING 1 A.

14. The method of claim 13, wherein the small molecule inhibitor of RING 1 A comprises PRT4165 (2-(3-pyridinylmethylene)-lH-indene-l,3(2H)-dione).

15. The method of claim 12, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, RING1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

16. The method of claim 15, wherein the nuclear genomic editing technique comprises a CRISPR-Cas9 genomic editing technique.

17. The method of any one of claims 1-16, further comprising contacting the T-cell with interleukin-2 (IL-2).

18. The method of any one of claims 1-17, wherein the T cell is a tumor infiltrating lymphocytes (TIL) or genetically engineered T cell.

19. The method of claim 18, wherein the TIL is a CD8+T cell.

20. The method of claim 18, wherein the genetically engineered T cell is a TCR-modified T cell or CAR-T cell.

21. The method of any one of claims 1 -20, wherein the T cell is a tumor antigenreactive T cell.

22. A method for preparation of a tumor-infiltrating lymphocyte (TIL) therapy for a subject, the method comprising:isolating a T-cell from a tumor of the subject and, after isolating the T-cell from the tumor of the subject and before administering the T-cell to the subject, reversing an exhaustion phenotype of the T-cell and restoring a stem-like phenotype of the T-cell according to the method of any one of claims 1-17.

23. The method of claim 22, wherein the exhaustion phenotype is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the T-cell, and wherein the stem-like phenotype is characterized at least in part by upregulation of TCF7 in the T-cell relative to the exhaustion phenotype.-VO-24. A composition comprising isolated T cells modified ex vivo to reverse an exhaustion phenotype in the isolated T cells, wherein the modification comprises:resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the isolated T cells with an inhibitor effective in inhibiting at least a portion of Polycomb Repressive Complexes 1 and / or 2 (PRC 1 / 2) in the isolated T-cells; andincubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

25. The composition of claim 24, wherein the exhaustion phenotype in the isolated T cells is a terminal exhaustion phenotype characterized at least in part by downregulation of transcription factor 7 (TCF7) in the isolated T-cell.

26. The composition of claim 24, wherein the modification is characterized at least in part by upregulation of TCF7 relative to the exhaustion phenotype in the isolated T cells before the modification.

27. The composition of any one of claims 24-26, wherein the portion of PRC1 / 2 comprises a protein component of Polycomb Repressive Complex 2 (PRC2).

28. The composition of claim 27, wherein the protein component of PRC2 comprises enhancer of zeste homolog 2 (EZH2), SET nuclear proto-oncogene (SET), embryonic ectoderm development (EED), SUZ12 polycomb repressive complex 2 subunit (SUZ12), retinoblastoma binding protein 4 (RBBP4), retinoblastoma binding protein 7 (RBBP7), enhancer of zeste homolog 1 (EZH1), or any combination thereof.

29. The composition of claim 28, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cells comprises contacting the T-cell with a small molecule inhibitor of EZH2.

30. The composition of claim 28, wherein the small molecule inhibitor of EZH2 comprises tazemetostat (also known as EPZ-6438; N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methy 1] -3 - [ethyl(oxan-4-yl) amino] -2-methyl-5- [4-(morpholin-4-ylmethyl)phenyl]benzamide), GSK126 (N-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl] -3 -methyl- 1 - [( 1 S)- 1 -methylpropyl] -6- [6-( 1 -piperazinyl)-3 -pyridinyl] -lH-indole-4-carboxamide), CPI- 1205 (also known as Lirametostat: N-[(4-methoxy-6-methyl-2-oxo- lH-pyridin-3-yl)methyl]-2-methyl- 1 - [(1 R)- 1 - [1 -(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide), or any combination thereof.

31. The composition of any one of claims 24-26, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cells comprises knocking down or knocking out EZH2. SET, EED, SUZ12, RBBP4, RBBP7, or any combination thereof, by way of a nuclear genomic editing technique.

32. The composition of claim 28, wherein the inhibiting at least a portion of PRC 1 / 2 in the T-cell comprises contacting the T-cells with an RNA interference (RNAi) inhibitor of EZH2.

33. The composition of claim 32, wherein the RNAi inhibitor comprises a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or any combination thereof.

34. The composition of any one of claims 24-26, wherein the portion of PRC1 / 2 comprises a protein component of Polycomb Repressive Complex 1 (PRC1).

35. The composition of claim 34, wherein the protein component of PRC1 comprises chromobox 2 (CBX2), chromobox 4 (CBX4), chromobox 6 (CBX6), chromobox 7 (CBX7), chromobox 8 (CBX8), ring-finger protein 1 (RING1) and YY1 transcription factor (YY1) binding protein (RYBP), RING1A (also known as RING1), ring finger protein 2 (also known as RING1B or RING2), Polycomb group ring finger 1 (also known as PCGF or PCGF1), or any combination thereof.

36. The composition of claim 34 or claim 35, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cells with a small molecule inhibitor of RING1 A.

37. The composition of claim 36, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises contacting the T-cells with a small molecule inhibitor of RING1A.

38. The composition of claim 37, wherein the small molecule inhibitor of RING1A comprises PRT4165 (2-(3-pyridinylmethylene)-lH-indene-l,3(2H)-dione).

39. The composition of claim 34, wherein the inhibiting at least a portion of PRC1 / 2 in the T-cell comprises knocking down or knocking out CBX2, CBX4, CBX6, CBX7, CBX8, RYBP, RING1A, R1NG1B, PCGF, or any combination thereof, by way of a nuclear genomic editing technique.

40. The composition of claim 24, wherein the T cells are further modified to express a recombinant receptor.

41. The composition of claim 40, wherein the recombinant receptor is an engineered T cell receptor (TCR).

42. The composition of claim 40, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

43. The composition of claim 40, wherein the recombinant receptor is specific for a tumor antigen.

44. The composition of claim 24, wherein the isolated T cells are native, naturally occurring T cells, autologous T cells, or T cells with specificity for and activity against a tumor,45. The composition of claim 44, wherein the native, naturally occurring T cells are obtained from resected tumors, and wherein the T cells are expanded ex vivo prior to the modification.

46. The composition of claim 44, wherein the T cells with specificity for and activity against a tumor are peripheral blood derived-T cells genetically modified to express a receptor that recognizes and responds to tumor.

47. The composition of claim 24, wherein the isolated T cells are selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, gamma delta T cells, a combination of CD4+ and CD8 T+ cells, memory T cells, cytokine-induced killer cells, and combinations thereof.

48. The composition of claim 47, wherein the isolated T cells are a combination of CD4+ and CD8+ cells.

49. A composition according to any one of claims 24-48, for use in a method of treating a disease or pathologic condition in a subject, the method comprising administering to the subject an effective amount of the composition.

50. The composition for use according to claim 49, wherein the disease or the pathological condition is a tumor or cancer, optionally a solid tumor or hematopoietic malignancy.

51. The composition for use according to claim 50, the method further comprising administering to the subject one or more anticancer agents and / or one or more chemotherapeutic agents.

52. The composition for use according to claim 49, wherein the disease or the pathologic condition is a bacterial and / or parasitic infection.

53. The composition for use according to claim 52, the method further comprising administering to the subject one or more anti-bacterial or anti-parasitic agent.

54. A kit comprising the composition of claim 24 and instructions for use.

55. A method for treating a cancer in a subject in need thereof, the method comprising: administering to the subject the TIL therapy of claim 22 or the composition of any one of claims 24-48; optionally wherein the cancer comprises melanoma, renal cell carcinoma, or both.

56. The method of claim 55, further comprising administering to the subject an effective amount of IL-2.

57. A method for preparation of a chimeric antigen receptor (CAR) T-cell therapy, the method comprising:resting CAR T-cells in absence of TCR stimulation;inhibiting at least a portion of PRC 1 / 2 in the CAR T-cell; and contacting the CAR T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof;wherein the CAR T-cell therapy comprises the CAR T-cell.

58. A method for treating cancer in a subject in need thereof, the method comprising: administering to the subject the CAR T-cells of claim 57.

59. An in vitro method of reversing T cell exhaustion comprising modifying isolated T cells, the modification comprising:resting the isolated T-cells in absence of T-cell receptor (TCR) stimulation; contacting the T cells with an inhibitor effective in inhibiting at least a portion of polycomb repressive complexes 1 and / or 2 (PRC1 / 2) in the isolated T-cells; and incubating the isolated T-cells with at least one cytokine, wherein the cytokine is interleukin- 15 (IL- 15), interleukin-7 (IL-7), or a combination thereof.

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