Leveraging TCF1 and LEF1 to enhance stemness in car t cells

Abstract

The present disclosure is directed to compositions and methods for modulating TCF1 or LEF1 activity to enhance CAR-T cell therapies. In one aspect, there are provide TCF1 fusions or LEF1 fusions to β-catenin that can be used to re-engineer CAR-T cells to suppress inhibitory receptors, increase memory-associated markers, increase cell size and metabolic activity, and augment cytokine secretion and cell killing. This approach has the potential to dramatically improve the function of CAR-T cells.

Description

[0001] LEVERAGING TCF1 AND LEF1 TO ENHANCE STEMNESS IN CAR T CELLS PRIORITY CLAIM

[0002] This application claims benefit of priority to U. S. Provisional Application Serial No.

[0003] 63 / 716,093, filed November 4, 2024, the entire contents of which are hereby incorporated by reference.

[0004] REFERENCE TO A SEQUENCE LISTING

[0005] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on October 23, 2025, is named CHOPP0086WO.xml and is 25,942 bytes in size.

[0006] BACKGROUND

[0007] 1. Field of the Disclosure

[0008] The present disclosure relates generally to the fields of molecular biology, oncology, and cancer immunotherapy. More particularly, the disclosure relates to compositions and methods for modulating TCF1 activity and LEF1 activity to enhance CAR-T cell therapies.

[0009] 2. Background

[0010] Cancer immunotherapy is a class of cancer therapeutics that leverages the human immune system to treat cancer. T cells play an integral role in responses to immunotherapy due to their ability to recognize and kill tumor cells and orchestrate the recruitment of other immune cells. Chimeric antigen receptor (CAR) T cell therapy, whereby a patient’s T cells are engineered outside of the body to express a CAR that enables tumor killing, has demonstrated remarkable clinical responses in patients with aggressive hematologic malignancies and represents a major advancement in the treatment of cancer. Yet less than 50% of patients exhibit durable remission at 12 months1and CAR-T cells for solid tumors have been largely ineffective. A major barrier to progress for CAR-T cell therapy and cancer immunotherapy, more broadly, is poor T cell fitness (i.e., T cell dysfunction), which results in diminished antitumor responses2Methods to enhance CAR-T cell fitness have resulted in encouraging preclinical data but have not translated into meaningful improvements in clinical responses, indicating a knowledge gap in the mechanisms by which human CAR-T cells lose or durably maintain function. Hence, there is a critical unmet need to define mechanisms that limit CAR- T cell fitness and develop translational approaches that enhance fitness and CAR-T therapeutic efficacy in patients. SUMMARY

[0011] Thus, in accordance with the present disclosure, there is provided a recombinant fusion protein comprising (a) a P-catenin protein or fragment that comprises, consists, or consists essentially of a putative transactivation domain; (b) (i) a TCF1 protein or fragment that binds to the truncated P-catenin protein and retains TCF1 -driven T cell memory phenotype and / or sternness; (ii) a LEF1 protein or fragment that binds to the truncated P-catenin protein and retains LEF1 -driven T cell memory phenotype and / or sternness; and optionally (c) a destabilizing domain (DD) attached to said TCF1 / LEF1 protein or fragment, wherein the order of elements starting from the amino-terminus is (b) to (a) or (c) to (b) to (a).

[0012] The P-catenin fragment may lack the armadillo repeats, the TCF1 fragment may comprise, consist, or consist essentially of the HMG box domain, the P-catenin binding domain and the C-terminal transactivation, the LEF1 fragment may comprise, consist, or consist essentially of the HMG box domain, the P-catenin binding domain and the C-terminal transactivation, and / or the DD may be an E. coli DHFR degron or FK BP12. One or both of the truncated P-catenin protein and DD may be attached to said TCF1 protein or fragment with a linker, such as [Glycine-Serine linker (Gly-Ser), (GGGGS)N linker, or XTEN linker. The recombinant fusion protein may comprise, consist essentially of or consist of any one of SEQ ID NOS: 1-8, 10, 11, 12, 14, or 15. Also provided is a polynucleotide encoding the recombinant fusion protein as described herein. The polynucleotide may further comprise a promoter that facilitates expression of the encoded recombinant fusion protein.

[0013] Further provided is an engineered cell or non-engineered cell (a) that expresses the recombinant fusion protein as described herein; and / or (b) that comprises the polynucleotide as described herein under the control of a promoter active in said engineered cell. The cell may express the recombinant fusion protein either transiently, constitutively, or inducibly. The coding region for the recombinant fusion protein may be fused to a motif which modulates expression levels or enhances intracellular degradation. The cell may be a T cell or a tumor infiltrating lymphocyte. The T cell may be one that maintains functionality under conditions in which unmodified T cells display exhaustion. The T cell may comprise a nucleic acid encoding a recombinant receptor. The recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The recombinant receptor may be specific for a tumor antigen. The recombinant fusion protein and the engineered receptor may be encoded by separate nucleic acids or encoded by a single nucleic acid. The recombinant fusion protein and the engineered receptor may be expressed under different promoters. The cell may be obtained from a sample from a subject, such as a tumor sample, a lymph node or blood. The engineered cell may be expanded ex vivo.

[0014] In another embodiment, there is provided a pharmaceutical composition comprising the recombinant fusion protein as described here, the polynucleotide as described herein, or the engineered cell as described herein, and a pharmaceutically acceptable buffer, diluent or excipient.

[0015] In a further embodiment, there is provided a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of a cell as described herein. The cell may be allogeneic or autologous to the patient. The cell may be administered systemically. The method may further comprise administering a second anticancer therapy to the patient, such as surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. The cell may be a T cell or a tumor infiltrating lymphocyte. The T cell may be a CAR-T cell. The cancer may be a solid tumor. Administering may reduce the number of cancerous cells in the patient, reduce and / or eliminate the tumor burden in the patient, exhibit enhanced cancer treatment compared to administration of unmodified T cells, and / or result in lower expression of inhibitory receptors in the subject in comparison to administration of unmodified T cells.

[0016] A method of inducing a naive T cell expression signature, increasing CAR T persistence, increasing CAR T proliferation, and / or reinvigorating exhausted CAR T cells, the method comprising transforming a T cell / CAR T cell with a recombinant fusion protein comprising (a) a P-catenin protein or fragment that comprises, consists, or consists essentially of a putative transactivation domain; (b)(i) a TCF1 protein or fragment that binds to the truncated P-catenin protein and retains TCFl-driven T cell memory phenotype and / or sternness; (ii) a LEF1 protein or fragment that binds to the truncated P-catenin protein and retains LEF1 -driven T cell memory phenotype and / or sternness; and optionally (c) a destabilizing domain (DD) attached to said TCF1 / LEF1 protein or fragment, wherein the order of elements starting from the aminoterminus is (b) to (a) or (c) to (b) to (a).

[0017] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0018] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIGS. 1A-C. Increasing 0-catenin levels to promote P-catenin / TCFl interaction.

[0021] (FIG. 1 A) Schematic depicting amino acid changes made to achieve constitutive expression of P-catenin. (FIG. IB) Flow cytometric analysis of CCR7 and IL-7Ra in CD19.28^ CAR T cells overexpressing tNGFR (control) or the stabilized P-catenin variant (sP-cat). CCR7 and IL-7Ra are canonical memory markers and putative TCF1 targets. An increase in both markers is observed upon overexpressing P-catenin in CAR T cells, suggesting the exogenous P-catenin is interacting with endogenous TCF1 to increase expression of its targets. (FIG. 1C) In vitro expansion curve of tNGFR- and sP-cat-CD19.28^ CAR T cells. While the expected phenotype is observed in FIG. IB, sP-cat-CD19.28^ cells do not expand similarly to tNGFR control cells, which is unfavorable for manufacturing and supports the use of a more specific approach to leverage the P-catenin / TCFl interaction.

[0022] FIGS. 2A-C. Designing a chimeric protein, TCFlfusion, that specifically enforces the P-catenin / TCF1 interaction. (FIG. 2A) Schematics depicting the design of TCFlftlslonand the variants created to achieve specific P-catenin / TCFl interaction. TCFlfuslon-vl is comprised of full length P-catenin (fip-catenin), a glycine-serine linker, and truncated TCF1 (tTCFl). In tTCFl, amino acids 1-59 of TCF1 isoform 4L were removed to shorten the transgene while retaining the region thought to be necessary for P-catenin binding. TCFlftlslon-v2 is comprised of fip-catenin, a glycine-serine linker, and TCF1 isoform 4L (flTCFl). TCFl^^-vS is comprised of the transactivation domain of P-catenin (tp-cat), a glycine-serine linker, and flTCFl. TCFl^H-vd is comprised of the transactivation domain of P-catenin (tp-cat), a glycine-serine linker, and tTCFl. (FIG. 2B) Flow cytometric analysis of P-catenin and TCF1 inT cells overexpressing tNGFR (control), TCFl, or TCFl^^variants. All TCFlfuslonvariants result in increased levels of P-catenin and TCF1, supporting that the chimeric proteins are being properly expressed. (FIG. 2C) In vitro expansion curve of tNGFR- and

[0023]

[0024] CAR T cells. As opposed to overexpressing P-catenin (FIG. 1C), overexpressing TCFlft,slondoes not impair CAR T cell expansion.

[0025] FIGS. 3A-E. TCFlfusionis functional and increases a memory phenotype in CAR T cells. (FIG. 3A) Western blot for chromatin-bound P-catenin in untransduced T cells and T cells overexpressing tNGFR, P-catenin, TCF1, TCFlfuslon_v3, or TCFl^81011-V4. Since P-catenin does not have a DNA binding domain, it is not expected to be detected in this assay. Detection of chromatin-bound P-catenin only in T(T’1|U8I°" -v3 and TCT’I|USI°" -v4 cells supports that P-catenin was successfully tethered to TCF1, the chimeric proteins are properly folding, and that TCFlfuslonis functioning as a transcription factor. Additionally, the TCF111181011proteins are detected at their expected molecular weights. (FIG. 3B) Relative luciferase activity in TCF1 reporter assay where 293GP cells transfected with either TOPFlash (Addgene Plasmid#! 2456) or FOPFlash (Addgene Plasmid#! 2457), Renilla luciferase-Pol III (Addgene Plasmid# 37380), and either tNGFR, TCF1, TCFlfusion-v2, TCFlfusion-v3, or TCF11=1181011-v4. TCF1 alone induces ~20-fold luciferase activity compared to tNGFR. However, TCF1 activity increases when it is tethered to P-catenin as TCI;111181011-v2, TC 111181011-v3. and TCFl11181™1-v4 induce -1000-fold, ~400-fold, and ~350-fold more luciferase activity compared to tNGFR, respectively. Data are mean ± s.e.m. of 3 technical replicates. The TCF / LEF binding sites from TOPFlash were cloned into a lentiviral backbone to control a minimal promoter regulating GFP expression to perform TCF1 reporter assay in primary human T cells. (FIG. 3C) Flow cytometric analysis of the mean fluorescence intensity (MFI) of GFP in the CAR+NGFR+ population of tNGFR-, TCF1-, or TCFlfusion-CD19. BB^ CAR T cells (n=2 donors). Similarto the observations of FIG.

[0026] 3B, all variants of TCFl^81011drive greater expression of GFP compared to TCF1 alone in human T cells. (FIG. 3D) Relative mRNA expression of AXIN2 in NGFR+ tNGFR-, TCF1-, or TCFlfuslon-CD19.28 CAR T cells. Data are mean ± s.e.m. of 3 technical replicates. AXIN2 is a negative regulator of Wnt signaling, where it functions to block the P-catenin / TCFl interaction. Expression of AXIN2 increases in response to Wnt signaling. A significant increase in AXIN2 expression is observed when TCFlfll81on-v2, TCFlfuslon-v3, or TCFlfuslon-v4 is overexpressed. This supports that the P-catenin / TCFl interaction observed in the presence of a Wnt signal is being recapitulated synthetically with TCFlfllslon. (FIG. 3E) Flow cytometric analysis of putative TCF1 -targets IL-7Ra and CCR7 in the CD8 CAR+NGFR+ population of tNGFR- or TCFlfuslon-CD19. BB^ CAR T cells. Expression of both targets is increased when TCFlfl,slonis overexpressed, regardless of variant form. This observation is indicative of increased TCF1 activity leading to a more memory-like CAR T cell. Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 3B, FIG. 3D).

[0027] FIGS.4A-C. Constitutive TCFlfusionexpression impairs CAR T function. (FIG. 4A) Cytotoxicity of tNGFR-, TCF1-, TCFlft,sion-CD19. BBg CAR T cells against Nalm6. GD2 leukemia seeded at a 1:2 (left) or 1:16 (right) effector-to-target (E: T) ratio in vitro. At the 1:2 E: T, overexpression of TCFlfuslonhas minimal effects on CAR T function. However, while tNGFR-CAR T cells maintain tumor control at a 1: 16 E: T, TCFlfuslon-CAR T cells allow tumor outgrowth suggesting impaired anti-tumor function. (FIG. 4B) IFNy (left) and IL-2 (right) secretion from tNGFR-, TCF1-, TCFlfusion-CD19. BB^ CAR T cells challenged with Nalm6. GD2 leukemia at a 1:1 effector-to-target ratio. Data are mean ± s.e.m. of 3 technical replicates. Overexpression of TCFlfuslonresults in significantly less IFN-y secretion, supporting the notion that TCFIfuslon-CAR T cells have impaired anti-tumor function. The effects on IL-2 secretion vary depending on TCF1fusionvariant. (FIG. 4C) Nalm6-bearing NSG mice were treated with a subcurative dose of 0.6x106NGFR+ tNGFR-, TCF1-, TCFlfasion-CD19.28 cells six days after engraftment (left). Quantification of tumor bioluminescence (middle) and survival (right) with 5 mice per condition. Mice treated with TCFlfilslon-v2-CAR T cells had greater tumor outgrowth and decreased survival. However, mice treated with TCFl^^-vS-and TCFl^^-v -CAR T cells performed similarly to those treated with control tNGFR-CAR T cells. Statistical comparisons performed using ANOVA with Dunnctt’s multiple comparisons test (FIG. 4B) and log-rank Mantel-Cox test (FIG. 4C, right). NS, not significant.

[0028] FIGS.5A-C. Designing an inducible TCFl / p-catenin expression in human T cells through DD-TCFlfusion. (FIG. 5A) Schematics depicting the design of DD-TCF1lll',l<,nprotein. DD-TCFlfuslon-v2 is comprised of an N-terminal ecDHFR sequence, full length P-catenin (fip-catenin), a glycine-serine linker, and full-length TCF1 (flTCFl). DD-TCFlfuslon-v3 is comprised of an N-terminal ecDHFR sequence, truncated P-catenin (tp-catenin), a glycineserine linker, and full-length TCF1 (flTCFl). The ecDHFR domain is an unstructured, unstabilized short amino acid sequence derived from E. coli dihydrofolate reductase that tags DD-TCFlfuslonfor degradation by the proteasome. The ecDHFR domain in DD-TCFlfuslonis stabilized by exposure to a small-molecule ligand, trimethoprim (TMP). Addition of TMP allows for transient and dose-dependent induction of DD- TCFlfuslonvariants. (FIG. 5B) Flow cytometric analysis of TCF1, P-catenin, IL7RA and CCR7 expression in human CD8+ T cells overexpressing tNGFR (control, black) and DD-TCFl^81011variants treated increasing doses of TMP (representative histogram of n=2 donors). Culturing DD-TCFlfuslonvariants with increasing concentrations of TMP results in increased expression of TCF1 and P-catenin as well as TCF1 targets, IL7RA and CCR7. (FIG. 5C) Representative biaxial plot of CD45RA and CD62L of human CD8+ T cells overexpressing DD-TCFlfuslonvariants treated increasing doses of TMP (representative flow plot of n=2 donors). Increasing concentrations of TMP results in increasing frequency of TSCM (CD45RA+CD62L+) cells. FIGS. 6A-E. DD-TCFlfusion function? (FIG. 6A) Primary human T cells were activated and then transduced with CAR and DD-TCFlft,slonconstructs. At D7, post-activation, tNGFR+ cells were selected. TMP was added to induce TCI;I|USI°" expression at D8 post activation until DI 5, when the TMP was washed out and CAR-T cells were co-cultured with Nalm6. GD2 tumor cells at different Effector: Target (E: T) ratios (1:2, 1:4, 1:8, 1:16) for 7 days. Immunophenotyping by flow cytometry was done at indicated times. (FIG. 6B) Flow cytometric analysis of TCF1 and -catenin in human CD8+ T cells overexpressing DD-TCF1fusionvariants cuitured in either no TMP for luM TMP for 24, 48 and 72H. (FIG. 6C) Flow cytometric analysis of TCF1 and P-catenin in human CD8+ T cells overexpressing tNGFR (control, black) and DD-TCF 111181011variants after washout for 24, 48 and 72H upon being cultured with luM TMP for 5 days. TCFl^1011protein levels return to baseline after 72H after washout, indicating that TCF111181011levels can be controlled transiently by removing TMP from culture media. (FIG. 6D) T cell expansion curves of CAR T cells cultured with Nalm6. GD2 cells at 1:2 E: T ratio. Upon clearance of tumor around 62H, CART cells “primed” with DD-TCFlLlslonwere able to expand at a greater fold change than control tNGFR cells, indicating that CFl6*51011may play a role in T cell expansion after clearance of tumor. (FIG.

[0029] 6E) Tumor fluorescence curves of CAR T cells cultured with Nalm6. GD2 cells at a 1:16 E: T ratio. Priming of CAR T cells with DD- TCFIlu'’l°11was able to rescue T cell control of tumors and led to similar ability to control tumor growth as compared to tNGFR control under T cell stress conditions.

[0030] FIGS. 7A-G. Designing LEFlfusionand validating its effects on CAR T cell phenotype and function. (FIG. 7A) Schematics depicting the design of LEFlfuslonand the variants created to achieve specific -catenin / LEFl interaction. LEFlftl81on-vl is comprised of H -catenin, a glycine-serine linker, and LEF1 isoform 1 (flLEFl). LEFlfuslon-v2 is comprised of fip-catenin, a glycine-serine linker, and flLEFl. (FIG. 7B) Flow cytometric analysis of -catenin and LEF1 in T cells overexpressing tNGFR (control), LEF1, or LEF111181011variants. Both LEFluslonvariants result in increased levels of P-catenin and LEF1, supporting that the chimeric proteins are being properly expressed. (FIG. 7C) Relative luciferase activity in LEF1 reporter assay where 293GP cells transfected with either TOPFlash (Addgene Plasmid# 12456) or FOPFlash (Addgene Plasmid#! 2457), Renilla luciferase-Pol III (Addgene Plasmid# 37380), and either tNGFR, LEF1, or ITT'Ilusion-v2, LEF1 alone induces ~10-fold luciferase activity compared to tNGFR. However, LEF1 activity increases when it is tethered to P-catenin as LEFlfuslon-v2 induces ~200-fold more luciferase activity compared to tNGFR. (FIG. 7D) Flow cytometric analysis of GFP MFI in the CAR+NGFR+ population of tNGFR-, LEF1-, or LEFlfuslon-CD19. BB CAR T cells (n=2 donors). Both variants of L\EFlfuslondrive greater expression of GFP compared to LEF1 alone in human T cells. (FIG. 7E) Relative mRNA expression of AXIN2 in NGFR+ tNGFR-, LEF1-, or

[0031]

[0032] T cells. Data are mean ± s.e.m. of 3 technical replicates. A significant increase in AXIN2 expression is observed when EEFIlusion-vl is overexpressed. As observed with TCFlfuslon, the increase of AXIN2 suggests the specific modulation of this axis with LEF1lusion. (FIG. 7F) Flow cytometric analysis of putative TCF1 / LEF1 -targets IL-7Ra and CCR7 in the CD8 CAR+NGFR+ population of tNGFR- or TCFI^^-CDIQ. BB^ CAR T cells. Expression of both targets is increased when LEF1lusionis overexpressed, regardless of variant form. This observation is indicative of increased LEF1 activity leading to a more memory-like CAR T cell. (FIG. 7G) Nalm6-bearing NSG mice were treated with a subcurative dose of 0.6xl06NGFR+ tNGFR-, LEF1-, LEFlfuslon-CD19.28 cells six days after engraftment (left). Quantification of tumor bioluminescence (middle) and survival (right) with 5 mice per condition. Similar to TCF1lllsl°"-v2-CAR T cells (FIG. 4C), mice treated with LEFl^^-vl-CAR T cells had decreased survival. However, mice treated with LEFlfuslon-v2-CAR T cells performed similarly to those treated with control tNGFR-CAR T cells. Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple comparisons test (FIG. 7C, FIG. 7E) and log-rank Mantel-Cox test (FIG. 7G, right).

[0033] FIGS.8A-C. TCFlfusion promotes a stem- and naive-like phenotype. Bulk RNA-seq in tNGFR+CD8+ CD19.28^ CAR T cells overexpressing tNGFR, TCF1, or TCFlfusion (n=3 donors). (FIG. 8A), RNA-seq PCA. TCFlfusion CAR T cells cluster separately from tNGFR or TCF1-CAR T cells, suggesting TCFlfusion overexpression is driving transcriptional changes different from TCF1 alone. (FIG. 8B) Volcano plot of differentially expressed genes in TCFlfusion versus tNGFR (Bonferroni-adjusted P < 0.05 with absolute log 2- transformed fold change (abs(log2(fold change)) > 0.5). TCFlfusion cells upregulate many genes related to T cell sternness and persistence. TCFlfusion cells also downregulate many genes related to terminal differentiation and exhaustion that counteract T cell sternness and persistence. (FIG.

[0034] 8C) Gene set variation analysis (GSVA) using a naive T cell signature, with isolated CD8+ naive T cells as a positive control. TCFlfusion CAR T cells are enriched in this naive T cell signature, suggesting TCFlfusion is promoting a naive-like phenotype that is not achieved with TCF1 overexpression. Statistical comparisons were performed using repeated-measures oneway ANOVA with Dunnett’s test.

[0035] FIGS. 9A-C. TCFlfusion increases CAR T persistence in a tumor re-challenge model. (FIG. 9A) Nalm6. GD2-bearing NSG mice were treated with a curative dose of 2.5xl06 tNGFR+ tNGFR, TCF1, or TCFl*"118'011CD19.28^ cells ten days after tumor engraftment. Upon tumor clearance, mice were rechallenged with lOxlO6Nalm6. GD2 (CD19+, n=3) or Nalm6. HER2 (CD19-, n=2) on day 35 post initial CAR T engraftment (left). Quantification of circulating human CD45+ T cells (i.e., CAR T) throughout the experiment (n=5, right). An increase in CAR T cells is observed after tumor rechallenge only in mice treated with TCFlfusion CAR T cells. (FIG. 9B) Quantification of human CD45+ T cells in spleen and bone marrow on day 35. Mean ± s.e.m. of n = 5 mice per group from one donor. (FIG. 9C) Quantification of human CD45+ T cells in spleen and bone marrow on day 70. Mean ± s.e.m. of n = 3 mice per group from one donor. Statistical comparisons were performed using oneway ANOVA with Dunnett’s test. On both day 35 and day 70, there are more T cells found in the spleen and bone marrow of mice treated with TCFlfusion CAR T cells.

[0036] FIG. 10. Transient TCFlfusion expression increases CAR T proliferation upon tumor challenge. TCFlfusion expression is controlled by the degron domain, where TCFlfusion is expressed (ON) in the presence of TMP. To test how transient TCFlfusion expression affects proliferation, TMP was added during CD 19.28^ CAR T manufacturing from days 8-15 and removed on day 15 (PRIMED). CAR T cells were stained with proliferation dye Cell Trace Violet (CTV) prior to tumor challenge. CAR T cells were co-cultured with Nalm6. GD2 tumor cells at a 2:1 E: T and proliferation was measured six days post-challenge. Data show flow cytometric analysis of CTV in TCF1± DD (left) and TCFlfusion-v2 ± DD (right) CAR T cells. Data from representative donor (n=2 donors). Primed DD-TCFlfusion-v2 CAR T cells had increased proliferation compared to constitutive TCFlfusion-v2 and tNGFR cells, consistent with the notion that transient TCFlfusion expression enhances persistence. Importantly, this effect is not observed with transient overexpression of wild-type TCF1.

[0037] FIGS. 11A-B. Transient TCFlfusion expression reinvigorates exhausted CAR T cells. HA.28^ CAR T cells target the antigen, GD2, and exhibit tonic CAR signaling that induces T cell exhaustion. To test whether different durations of TCFlfusion expression can reinvigorate exhausted CAR T cells, TMP was added during HA.28^ CAR T manufacturing from days 9-14 and removed on day 14 (PRIMED). Conditions where TMP was always present (ALWAYS ON) or absent (ALWAYS OFF) were included as controls. (FIG. HA) IFNy secretion from CAR T cells challenged with 143B tumor cells at a 1:1 effector-to-target ratio. PRIMED cells secrete greater amounts of IFNy compared to other DD-TCFlfusion conditions, suggesting transient TCFlfusion expression provides a functional benefit. In a separate healthy donor, TMP was added during CAR T manufacturing from days 7-11 and removed on day 11 (PRIMED). (FIG. 11B) IFNy secretion from CAR T cells challenged with 143B at a 1:1 effector-to-target ratio. PRIMED cells secrete greater amounts of IFNy compared to other DD-TCFlfusion conditions, supporting the notion that transient TCFlfusion expression reinvigorates exhausted CAR T cells. Data are mean ± s.e.m. of 3 technical replicates from a representative donor (n=2 donors). DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0038] As discussed above, there is an urgent need to improve the performance of CAR-T cell therapies, in particular with respect to their tendency to lose long term persistence in a clinical setting. However, CAR-T cells with a memory-like phenotype and high expression of TCF7 (encodes TCF1) have enhanced persistence that could, in theory, translate into longer action in vivo and greater efficacy.

[0039] TCF1 and LEF1 are transcription factors critical for T cell differentiation, specifically driving self-renewal in memory T cells in mice. The inventors have shown that overexpressing TCF1 alone does not promote CAR-T memory or enhance function (Figure 1, Appendix A). The inventors’ results suggest that TCF1 / LEF1 activity is altered in engineered human T cells and highlight a gap in knowledge for this vital transcription factor. TCF1 / LEF1 activity can be regulated by P-catenin as it displaces repressors and recruits activators of transcription when bound to TCF1. Based on the data presented here, the inventors propose that leveraging the β-catenin-TCF1 / Lef1 interaction will drive the development of memory-like CAR-T cells and improve their function through the activation of a TCF1 / LEF1 -mediated transcription profile.

[0040] These and other aspects of the disclosure are described in detail below.

[0041] I. TCF1

[0042] A. TCF1 / TCF7

[0043] Transcription factor 7 is the gene that in humans encodes for the TCF1 protein. It is a member of the TCF / LEF family (T cell factor / lymphoid enhancer factor family), a group of genes that encode transcription factors which bind to DNA through a SOX-like high mobility group domain. They are involved in the Wnt signaling pathway, particularly during embryonic and stem-cell development, but also have been found to play a role in cancer and diabetes. TCF / LEF factors recruit the coactivator beta-catenin to enhancer elements of genes they target. They can also recruit members of the Groucho family of corepressors.

[0044] The TCF7 gene is expressed predominantly in T-cells and plays a critical role in natural killer cell and innate lymphoid cell development. TCF1 protein forms a complex with beta-catenin and activates transcription through a Wnt / beta-catenin signaling pathway. Mice with a knockout of this gene are viable and fertile, but display a block in T-lymphocyte differentiation. Alternative splicing results in multiple transcript variants. Naturally-occurring isoforms lacking the N-terminal beta-catenin interaction domain may act as dominant negative regulators of Wnt signaling. B. LEF1

[0045] Lymphoid enhancer-binding factor 1 (LEF1) is a protein that in humans is encoded by the LEF1 gene. It is a member of T cell factor / lymphoid enhancer factor (TCF / LEF) family. Lymphoid enhancer-binding factor- 1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. It binds to a functionally important site in the T-cell receptor-alpha (TCRA) enhancer and confers maximal enhancer activity. LEF1 belongs to a family of regulatory proteins that share homology with high mobility group protein- 1 (HMG1). LEF1 is highly overexpressed and associated with disease progression and poor prognosis in B-cell chronic lymphocytic leukemia and other kinds of malignancies like colorectal cancer. It is also a promising potential drug target. Lymphoid enhancer-binding factor 1 has been shown to interact with ALX4, AML-1, Catenin beta-1 / β-catenin / CTNNB1 (including transgenically), EP300, MIFF, PIAS4, SMAD2, and SMAD3.

[0046] C. Nucleic Acids Encoding TCF1 / LEF1 and Expression Vectors Expressing the Same

[0047] Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product, e.g., and TCR1 / LEF1, in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0048] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0049] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0050] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde- 3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

[0051] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0052] The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ P, P-interferon, interleukin- 2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, p-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, oc-fetoprotein, t-globin, P-globin, c-fos, c-HA-ra.v, insulin, neural cell adhesion molecule (NCAM), ai-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B vims, human immunodeficiency vims, cytomegalovirus (CMV), and gibbon ape leukemia vims.

[0053] In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor vims), P-interferon, adenovims 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, a-2-macroglobulin, vimentin, MHC class I gene H-2Kb, HSP70, proliferin, tumor necrosis factor, and / or thyroid stimulating hormone a gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rl)x, poly(rc), E1A, phorbol ester (TP A), interferon, Newcastle Disease Vims, A23187, IL-6, semm, interferon, SV40 large T antigen, PMA, and / or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein.

[0054] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. D. Delivery of Expression Vectors

[0055] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0056] “Adenovirus expression vector’’ is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0057] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double- stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0058] Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5’ -tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0059] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the E3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.

[0060] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0061] Adenoviruses may be replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0062] As stated above, the typical vector according to the present disclosure is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, or in the E4 region where a helper cell line or helper virus complements the E4 defect.

[0063] Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109- 1012plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

[0064] Adenovirus vectors have been used in eukaryotic gene expression and vaccine development. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.

[0065] Retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double- stranded DNA in infected cells by a process of reversetranscription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5’ and 3’ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.

[0066] In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.

[0067] One approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors. A different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor are used. The antibodies are coupled via the biotin components by using streptavidin.

[0068] There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.

[0069] A particular type of retrovirus is a lentivirus. The virus contains a reverse transcriptase molecule found to perform transcription of the viral genetic material upon entering the cell. Within the viral genome are RNA sequences that code for specific proteins that facilitate the incorporation of the viral sequences into the host cell genome. The "gag" gene codes for the structural components of the viral nucleocapsid proteins: the matrix (MA / pl7), the capsid (CA / p24) and the nucleocapsid (NC / p7) proteins. The "pol" domain codes for the reverse transcriptase and integrase enzymes. Lastly, the "env" domain of the viral genome encodes for the glycoproteins and envelope on the surface of the virus.

[0070] There are multiple steps involved in the infection and replication of a lentivirus in a host cell. In the first step the virus uses its surface glycoproteins for attachment to the outer surface of a cell. More specifically, lenti viruses attach to the CD4 glycoproteins on the surface of a host's target cell. The viral material is then injected into the host cell's cytoplasm. Within the cytoplasm, the viral reverse transcriptase enzyme performs reverse transcription of the viral RNA genome to create a viral DNA genome. The viral DNA is then sent into the nucleus of the host cell where it is incorporated into the host cell's genome with the help of the viral enzyme integrase. From now on, the host cell starts to transcribe the entire viral RNA and express the structural viral proteins, in particular those that form the viral capsid and the envelope. The lentiviral RNA and the viral proteins then assemble and the newly formed virions leave the host cell when enough are made.

[0071] One method of gene therapy involves modifying a virus to act as a vector to insert beneficial genes into cells. Unlike other retroviruses, which cannot penetrate the nuclear envelope and can therefore only act on cells while they are undergoing mitosis, lentiviruses can infect cells whether or not they are dividing. Many cell types, like neurons, do not divide in adult organisms, so lentiviral gene therapy is a good candidate for treating conditions that affect those cell types.

[0072] Some experimental applications of lentiviral vectors have been done in gene therapy in order to cure diseases like diabetes mellitus, murine hemophilia A, prostate cancer, chronic granulomatous disease, and vascular diseases. Therapy requires manipulation of the lentivirus genes and structure for delivery of specific genes to alter the course of the disease. Parts of the viral genome must be removed so that the virus can't replicate itself. It is replaced with a gene to permanently incorporate into the host cell’s genome using genetically modified virus. HIV-derived lentiviral vectors have been used for introducing libraries of complementary DNAs, short hairpin RNAs, and cis-regulatory elements into many targets, including embryonic stem cells.

[0073] Some specific retroviral / lentiviral vectors include, but are not limited to mouse stem cell virus-based splice-gag vector (MSGV) retroviral vector and EFla lentiviral vector. Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells. In embodiments, the AAV vector is replication-defective or conditionally replication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11 or any combination thereof.

[0074] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use. Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0075] In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate -precipitated plasmids results in expression of the transfected genes. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0076] In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0077] In some embodiments, the expression construct is delivered directly to the liver, skin, and / or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.

[0078] In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000™ is widely used and commercially available.

[0079] In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG- 1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

[0080] Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific.

[0081] Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor- specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells.

[0082] IL Chimeric Antigen Receptors (CARs) and CAR-T Cells

[0083] Chimeric antigen receptor (CAR) molecules are recombinant fusion proteins and are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (ITAMs) present in their cytoplasmic tails in order to activate genetically modified immune effector cells for killing, proliferation, and cytokine production. Receptor constructs utilizing an antigen-binding moiety (for example, generated from single chain antibodies (scFv)) afford the additional advantage of being “universal” in that they bind native antigen on the target cell surface in an HLA-independent fashion.

[0084] Embodiments of the CARs described herein include nucleic acids encoding an antigenspecific CAR polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding domain. Optionally, a CAR can comprise a hinge domain positioned between the transmembrane domain and the antigen binding domain. A CAR may further comprise a signal peptide that directs expression of the CAR to the cell surface. One embodiment includes a chimeric antigen receptor comprising (i) an ectodomain comprising single chain antibody variable region that binds selectively to an antigen, wherein said antibody: (a) is an IgG antibody; (b) inhibits cancer cell growth; (c) induces cancer cell death, and has a flexible hinge attached at the C-terminus of said single chain antibody variable region; (ii) a transmembrane domain; and (iii) an endodomain, wherein said endodomain comprises a signal transduction function when said single-chain antibody variable region is engaged with an antigen. The transmembrane and endodomains may be derived from the same molecule. The endodomain may comprise a CD3-zeta domain or a high affinity FcsRl. The flexible hinge may be from CD8a or 1g. Still another embodiment comprises a cell expressing the chimeric antigen receptor as defined above. For example, a CAR may comprise a signal peptide from CD8. In one embodiment, the CAR comprises a single-chain variable fragment (scFv) targeting GPC2 (D3 binder; PCT Publn. WO2017 / 083296, which is incorporated by reference herein in its entirety) preceded by a CD8 leader sequence and followed by CD28 hinge / transmembrane / co-stimulatory domains, and a CD3 zeta co-stimulatory domain. A CAR may also be co-expressed with a membrane-bound cytokine to improve persistence. For example, a CAR may be co-expressed with membranebound IL- 15.

[0085] Depending on the arrangement of the domains of the CAR and the specific sequences used in the domains, immune effector cells expressing the CAR may have different levels activity against target cells. Different CAR sequences may be introduced into immune effector cells to generate engineered cells, the engineered cells selected for elevated SRC, and the selected cells tested for activity to identify the CAR constructs predicted to have the greatest therapeutic efficacy.

[0086] A chimeric antigen receptor can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. A nucleic acid sequence encoding the several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.). The resulting coding region can be inserted into an expression vector and used to transform a suitable expression host allogeneic or autologous immune effector cells, such as a T cell.

[0087] The chimeric construct may be introduced into immune effector cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U. S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression. Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lenti viral vector) can be used to introduce the chimeric construct into immune effector cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the immune effector cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0088] A. Antigen Binding Domains

[0089] An antigen binding domain may comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof. The antigen binding regions or domains may comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular mouse, human, or humanized monoclonal antibody. The fragment can also be any number of different antigen binding domains of an antigen-specific antibody. The fragment may be an antigenspecific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells. The prototypical CAR encodes a scFv comprising VH and VL domains derived from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains (e.g. costimulatory domains and signaling domains).

[0090] A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using Protein L since Protein L interacts with the variable region of kappa light chains. Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. For example, the linker may have a proline residue two residues after the VH C terminus and an abundance of arginines and prolines at other positions.

[0091] U. S. Patent No. 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation.

[0092] B. Hinge domains

[0093] A CAR polypeptide may include a hinge domain positioned between the antigen binding domain and the transmembrane domain. In some cases, a hinge domain may be included in CAR polypeptides to provide adequate distance between the antigen binding domain and the cell surface or to alleviate possible steric hindrance that could adversely affect antigen binding or effector function of CAR-modified immune effector cells. The hinge domain may comprise a sequence that binds to an Fc receptor, such as FcyR2a or FcyRla. For example, the hinge sequence may comprise an Fc domain from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD or IgE) that binds to an Fc receptor.

[0094] A CAR hinge domain may be derived from human immunoglobulin (Ig) constant region or a portion thereof including the Ig hinge, or from human CD8 a transmembrane domain and CD8a-hinge region. A CAR hinge domain may comprise a hinge-CTF-CHj region of antibody isotype IgG4. The hinge domain (and / or the CAR) may not comprise a wild type human IgG4 CH2 and CH3 sequence. Point mutations may be introduced in antibody heavy chain CH2 domain to reduce glycosylation and non-specific Fc gamma receptor binding of CAR-modified immune effector cells.

[0095] The hinge domain may comprise a sequence that is about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an IgG4 hinge domain, a CD8a hinge domain, a CD28 hinge domain, or an engineered hinge domain. C. Transmembrane Domains

[0096] The antigen- specific extracellular domain and the intracellular signaling-domain may be linked by a transmembrane domain. Polypeptide sequences that can be used as part of transmembrane domain include, without limitation, the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3^ domain, a cysteine mutated human CD3^ domain, or other transmembrane domains from other human transmembrane signaling proteins, such as CD 16, CD8, and erythropoietin receptor. For example, the transmembrane domain may comprise a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of those provided in U. S. Patent Publication No. 2014 / 0274909 (e.g., a CD8 and / or a CD28 transmembrane domain) or U. S. Patent No. 8,906,682 (e.g., a CD8a transmembrane domain), both incorporated herein by reference. Transmembrane regions may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In certain specific aspects, the transmembrane domain can be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a CD8a transmembrane domain or a CD28 transmembrane domain.

[0097] D. Intracellular Signaling Domains

[0098] The intracellular signaling domain of a CAR is responsible for activation of at least one of the normal effector functions of the immune cell engineered to express the CAR. The term “effector function” refers to a specialized function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Effector function in a naive, memory, or memory -type T cell includes antigendependent proliferation. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. The intracellular signaling domain may be derived from the intracellular signaling domain of a native receptor. Examples of such native receptors include the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules, such as CD3 beta or zeta and CD28, CD27, 4-1BB / CD137, ICOS / CD278, IL-2Rp / CD122, IL-2Rot / CD132, DAP10, DAP12, CD40, OX40 / CD134, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used.

[0099] While the entire intracellular signaling domain may be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term “intracellular signaling domain” is thus meant to include a truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal, upon CAR binding to a target. One or multiple cytoplasmic domains may be employed, as so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effect, for example the CD28 and 4- IBB can be combined in a CAR construct. In certain specific aspects, the intracellular signaling domain comprises a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a CD3 zeta intracellular domain, a CD28 intracellular domain, a CD 137 intracellular domain, or a domain comprising a CD28 intracellular domain fused to the 4- IBB intracellular domain. The CAR polypeptide may contain a CD28 or 4- IBB intracellular signaling domain fused to a CD3z intracellular signaling domain.

[0100] E. Immune Effector Cells

[0101] Immune effectors cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), natural killer (NK) cells, invariant NK cells, or NKT cells. Also provided herein are methods of producing and engineering the immune effector cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune effector cells may be used as immunotherapy, such as to target cancer cells.

[0102] The immune effector cells may be isolated from subjects, particularly human subjects. The immune effector cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, a subject who is undergoing therapy for a particular disease or condition, a subject who is a healthy volunteer or healthy donor, or from a blood bank. Immune effector cells can be collected, enriched, and / or purified from any tissue or organ in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. The isolated immune effector cells may be used directly, or they can be stored for a period of time, such as by freezing.

[0103] Tissues / organs from which the immune effector cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors. Immune effector cells isolated from cord blood may have enhanced inimunomodulation capacity, such as measured by CD4- or CD8-positive T cell suppression. The immune effector cells may be isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0104] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune effector cell activity. Thus, the cells will be autologous to the subject in need of therapy. Alternatively, the population of immune effector cells can be obtained from a donor, preferably an allogeneic donor. Allogeneic donor cells may or may not be human-leukocyte-antigen (HLA)-compatiblc. To be rendered subjectcompatible, allogeneic cells can be treated to reduce immunogenicity.

[0105] The immune effector cells may be T cells. The T cells may be derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. The T cells may be human T cells. The T cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. The cells may include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+cells, CD8+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, persistence capacities, antigenspecificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. For off-the-shelf technologies, the cells may be derived from pluripotent and / or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs).

[0106] Among the sub-types and subpopulations of T cells (e.g., CD4+and / or CD8+T cells) are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0107] One or more of the T cell populations may be enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).

[0108] T cells may be separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0109] CD8+T cells may be further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. Enrichment for central memory T (TCM) cells may be carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations.

[0110] The T cells may be autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL- 2). The cells are cultured until confluence (e.g., about 2xl06lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days.

[0111] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.

[0112] Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin- 15 (IL- 15), with IL-2 being preferred. The non-specific T-cell receptor stimulus can include around 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N. J.) and may involved anti-C3 / CD28 bead stimulation. Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 lU / mL IL-2 or IL-15, with IL-2 being preferred. The in vitro-induced T cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

[0113] The autologous T-cells can be modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells. Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL- 15, and IL- 12. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rded„ Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In particular aspects, modified autologous T-cells express the T-cell growth factor at high levels. T-cell growth factor coding sequences, such as that of IL-12, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression.

[0114] F. Engineering of Immune Effector Cells

[0115] The immune effectors cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells)) may be genetically engineered to express antigen receptors such as chimeric antigen receptors (CARs). For example, the host cells (e.g., autologous or allogeneic T-cells) may be modified to express a CAR having antigenic specificity for GPC2. Multiple CARs, such as to different antigens, may be added to a single cell type, such as T cells. In addition, in accordance with the present disclosure, expression constructs encoding TCR1 / LEF1 may also be introduced.

[0116] The cells may comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors or TCR1 / LEF1, and genetically engineered products of such nucleic acids. The nucleic acids may be heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. The nucleic acids may not be naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0117] In some aspects, the engineered immune effector cells are modified to decrease or eliminate the expression of one or more endogenous genes. For example, the engineered immune effector cells may be modified to knock down or knock out at least one immune checkpoint protein. The at least one immune checkpoint gene may be selected from the group consisting of: PD1, CTLA4, LAG3, TIM3, TIGIT, CD96, BTLA, KIRs, adenosine A2a receptor, Vista, IDO, FAS, SIRP alpha, GISH, SHP-1, FOXP3, LAIR1, PVRIG, PPP2CA, PPP2CB, PTPN6, PTPN22, CD160, CRTAM, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

[0118] As another example, HLA genes in the engineered immune effector cells may be modified in various ways. For example, the engineered immune effector cells may be engineered such that they do not express functional HLA-A on their surface. The HLA-A negative engineered immune effector cells may be derived from an HLA-homozygous individual. Alternatively, the engineered immune effector cells may be HLA-A homozygous. Further, the engineered immune effector cells, regardless of whether they are HLA-A negative or HLA-A homozygous, may be HLA-homozygous at HLA-B, HLA-C, and / or HLA-DRB1 alleles.

[0119] In some aspects, the engineered immune effector cells may be modified to knock down or knock out the expression of one or more T-cell receptor components. For example, in some aspects the cell lacks expression or has reduced expression of TCRa, TCRP, TCRa and TCR0, TCRy, TCR3, TCRy and TCR8, or any combination of the foregoing. Such can occur by any suitable manner, including by introducing zinc finger nucleases (ZFN), for example, targeting the constant region of one or more of the TCR receptor components.

[0120] G. Methods of Propagating Immune Effector Cells

[0121] In some cases, immune effector cells of the embodiments (e.g., T-cells) are co-cultured with activating and propagating cells (AaPCs), to aid in cell expansion. For example, antigen presenting cells (APCs) are useful in preparing therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U. S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U. S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. W02007 / 103009, each of which is incorporated by reference.

[0122] III. CAR-T Cell Based Therapies

[0123] A. Formulation and Administration

[0124] The present disclosure provides pharmaceutical compositions including engineered CAR-T cells. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0125] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0126] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0127] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0128] B. Cancers

[0129] Cancer results from the outgrowth of a clonal population of cells from tissue. The development of cancer, referred to as carcinogenesis, can be modeled and characterized in a number of ways. An association between the development of cancer and inflammation has long-been appreciated. The inflammatory response is involved in the host defense against microbial infection, and also drives tissue repair and regeneration. Considerable evidence points to a connection between inflammation and a risk of developing cancer, i.e., chronic inflammation can lead to dysplasia.

[0130] Cancer cells to which the methods of the present disclosure can be applied include but are not limited to cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma: adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor may comprise an osteosarcoma, angiosarcoma, rhabdosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, or leukemia.

[0131] In addition, the methods of the disclosure can be applied to a wide range of species, e.g., humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancers may also be recurrent, metastatic and / or multi-drug resistant, and the methods of the present disclosure may be particularly applied to such cancers so as to render them resectable, to prolong or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multi-drug resistant cancers. At a cellular level, this may translate into killing cancer cells, inhibiting cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0132] C. Combination Therapies

[0133] In the context of the present disclosure, it also is contemplated that engineered CAR-T cells described herein could be used similarly in conjunction with chemo- or radiotherapeutic intervention, or other treatments. It also may prove effective, in particular, to combine engineered CAR-T cells with other therapies.

[0134] To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present disclosure, one would generally contact a “target” cell with an engineered CAR-T cells according to the present disclosure and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the engineered CAR-T cells according to the present disclosure and the other agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the engineered CAR-T cells according to the present disclosure and the other includes the other agent.

[0135] Alternatively, the engineered CAR-T cell therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the engineered CAR-T cells are applied separately to the cell, one would generally ensure that a significant period of time did not expire between each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0136] It also is conceivable that more than one administration of either an engineered CAR-T cell or the other agent will be desired. Various combinations may be employed, where an engineered CAR-T cell according to the present disclosure therapy is “A” and the other therapy is “B”, as exemplified below:

[0137] A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B

[0138] Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell. Agents or factors suitable for cancer therapy include any chemical compound or treatment method that induces damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic” or “genotoxic agents,” may be used. This may be achieved by irradiating the localized tumor site; alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition. A combination therapy may also include surgery. Various modes of these therapies are discussed below.

[0139] 1. Chemotherapy

[0140] The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0141] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mclphalan, novembichin, phcncstcrinc, prcdnimustinc, trofosfamidc, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A uncialamycin and derivatives thereof; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0142] 2. Radiotherapy

[0143] Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0144] Radiation therapy used according to the present disclosure may include, but is not limited to, the use of y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors induce a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0145] Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.

[0146] Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility of side effects is reduced. A device called a multi-leaf collimator has been developed and may be used as an alternative to the metal blocks. The multi-leaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very important for conformal radiotherapy treatment and a special scanning machine may be used to check the position of internal organs at the beginning of each treatment.

[0147] High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area.

[0148] Although research studies have shown that conformal radiotherapy and intensity modulated radiotherapy may reduce the side effects of radiotherapy treatment, it is possible that shaping the treatment area so precisely could stop microscopic cancer cells just outside the treatment area being destroyed. This means that the risk of the cancer coming back in the future may be higher with these specialized radiotherapy techniques.

[0149] Scientists also are looking for ways to increase the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effect on cells undergoing radiation. Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation. Hyperthermia, the use of heat, is also being studied for its effectiveness in sensitizing tissue to radiation.

[0150] 3. Immunotherapy

[0151] In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e.. direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[0152] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, FLAP, estrogen receptor, laminin receptor, erb B and pl55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including cytokines such as IL-2, IL-4, IL-12, GM-CSF, y-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al., 2000). Moreover, antibodies against any of these compounds may be used to target the anti-cancer agents discussed herein.

[0153] Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U. S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy, e.g., interferons a, P, and y; IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998) gene therapy, e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U. S. Patents 5,830,880 and 5,846,945) and monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2, anti-pl85 (Pietras et al., 1998; Hanibuchi et al., 1998; U. S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the gene silencing therapies described herein.

[0154] In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or “vaccine" is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell etal., 1 93).

[0155] In adoptive immunotherapy, the patient’s circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al., 1988: 1989).

[0156] 4. Surgery

[0157] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present disclosure, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0158] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs’ surgery). It is further contemplated that the present disclosure may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0159] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0160] In some particular embodiments, after removal of the tumor, an adjuvant treatment with a agents of the present disclosure is believed to be particularly efficacious in reducing the reoccurrence of the tumor. Additionally, the compounds of the present disclosure can also be used in a neoadjuvant setting. It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating cancer. The skilled artisan is directed to “Remington’s Pharmaceutical Sciences” 15th Edition, Chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0161] IV. Examples

[0162] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0163] Example 1

[0164] Background. The concept of deep cellular reprogramming is relevant to CAR-T cell therapy. Recent studies indicate that the success of CAR-T and other T cell-based cancer immunotherapies is dependent on T cell fitness2, such as the ability to expand, persist, and durably maintain antitumor function. Currently, there is no consensus on how to best reprogram CAR-T cells with enhanced fitness.

[0165] TCR1 / LEF1 are transcription factors that regulate expression of memory-related genes and is associated with clinical CAR-T persistence. However, the definitive role of TCF1 or LEF1 has not been well characterized in human T cells. The activity of TCF1 / LEF1 is intertwined with its context-specific regulation, where an important regulatory mechanism to consider is the activation of TCF1 / LEF1 by its co-activator P-catenin. The inventors hypothesized that the P-catenin-TCFl / LEFl interaction is sufficient to promote a memory-like phenotype and enhance persistence in human CAR-T cells. To test this, they engineered chimeric proteins by fusing P-catenin to TCF1 or LEFT The results, shown in FIGS. 1-7 and Appendix A, indicate that TCF1 / LEF1 fusions are sufficient to promote a memory-like phenotype but attenuates CAR-T antitumor activity. SEQUENCES

[0166] TCFlfusion

[0167] • flTCFl (found in vl and v2) = entire sequence for TCF1 isoform 4L MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLV NESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAORLFPDKLPEPLEDGLKAPEC TSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSP HPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPG HPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEM RAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSAR DNYGKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 1) 269 AA from W02022040277A1 + 115 new AA = 384 AA total

[0168] 269 / 325 = -70% sequence identity to SEQ ID NO:02 of W02022040277A1

[0169] • tTCFl (found in v3 and v4) does not reflect a known isoform, instead it removes the annotated 0-catenin binding domain (amino acids 1-59) from isoform 4L EGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSG MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPT PAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPA AIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAK VIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNY GKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 2) 269 AA from patent + 56 new AA = 325 AA total

[0170] 269 / 325 = -83% sequence identity to SEQ ID NO:02 of W02022040277A1

[0171] • fl0-catenin (found in vl and v2) = entire sequence for full length 0-catenin (only known isoform) MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEE DVDTSQVLYEWEQGFSQSFTQEQVADIDGOYAMTRAQRVRAAMFPETLDEGMQIPS TQFDAAIIPTNVQRLAEPSOMLKIIAVVNLINYQDDAELATRAIPELTKLLNDEDQVV VNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLS HHREGLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGL QKMVALLNKTNVKFLAITTDCLOILAYGNQESKLIILASGGPOALVNIMRTYTYEKLL WTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAAT KQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALV RTVLRAGDREDITEPAICALRHLTSRHOEAEMAQNAVRLHYGLPVVVKLLHPPSHW PLIKATVGLIRNLALCPANHAPLREOGAIPRLVQLLVRAHODTORRTSMGGTQQQFV EGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCE LAODKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSV ELTSSLFRTEPMAWNETADLGLDIGAQGEPLGYRODDPSYRSFHSGGYGQDALGMD PMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL (SEQ ID NO: 3)

[0172] 87 AA from W02022040277A1 + 694 new AA= 781 AA total

[0173] 87 / 781 = -11% sequence identity to SEQ ID NO:05 of W02022040277A1 tfFcatenin (found in v3 and v4) does not differ from SEQ ID NO:05 found in W02022040277A1

[0174] FULL SEQUENCES

[0175] • Patent SEQ ID NO: 01 of W02022040277A1 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPT PAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPA AIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAK VIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNY GKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPAGGGGSGGGGSDLGL DIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDG LPDLGHAQDLMDGLPPGDSNQLAWFDTDL (SEQ ID NO: 4)

[0176] • VI flB-catenin-tTCFl MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEE DVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPS TQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVV VNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLS HHREGLLAIFKSGGIPALYKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGL OKMVALLNKTNVKFLAITTDCLOILAYGNOESKLIILASGGPOALVNIMRTYTYEKLL WTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAAT KOEGMEGLLGTLVOLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALV RTVLRAGDREDITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHW PLIKATVGLIRNLALCPANHAPLREOGAIPRLVOLLVRAHODTORRTSMGGTOOQFV EGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCE LAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSV ELTSSLFRTEPMAWNETADLGLDIGAOGEPLGYRQDDPSYRSFHSGGYGQDALGMD PMMEHEMGGHHPG AD YP VDGI UDI GII A Q D I, M DGI UPGDSNQI, A WFDTDEGGGGS GGGGSEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAP ECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHF NSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSG VPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYM KEMRAKVIAECTLKES AAINQILGRRWI I ALSREEQAKYYELARKERQLI IMQLYPG WSARDNYGKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 5)

[0177] Total AA = 1166

[0178] 418 / 1166 = -36% sequence identity to SEQ ID NO:01 of W02022040277A1

[0179] • V2 flB-catenin-flTCFl MATQADLMELDMAMEPDRKAAVSnWQQQSYLDSGIIISGATTTAPSLSGKGNPEEE DVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPS TQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVV VNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLS HHREGLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGL QKMVALLNKTNVKFLAITTDCLOILAYGNQESKLIILASGGPQALVNIMRTYTYEKLL WTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAAT KQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALV RTVLRAGDREDITEP AIC ALRI ILTSRI IQEAEMAQNAVRLI IYGLPVVVKLLI IPPSIIW PLIKATVGLIRNLALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFV EGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCE LAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSV ELTSSLFRTEPMAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMD PMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDLGGGGS GGGGSMPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAE LKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGL KAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLY EHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLG SGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLY MKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPG WSARDNYGKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 6)

[0180] Total AA = 1175

[0181] 366 / 1175 = -31% sequence identity to SEQ ID NO: 01 of W02022040277A1

[0182] • V3 tB-catenin-HTCFl MDLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGAD YPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDLGGGGSGGGGSMPQLDSGGGG AGGGDDLGAPDELLAFODEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGA GIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVY SAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQ KQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVP PSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLK ESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRS REKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 7)

[0183] Total AA = 482

[0184] 367 / 482 = -76% sequence identity to SEQ ID NO:01 of W02022040277A1

[0185] • V4 tB-catenin-tTCFl MDLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGAD YPVDGLPDLGHAODLMDGLPPGDSNQLAWFDTDLGGGGSGGGGSEGAAGGAGIPG VPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFN LLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQV HRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSG KQEEQPFDRNEKTQAESKAEKEAKKPTIKKPENAFMEYMKEMRAKVIAECTEKESA AINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREK HQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 8)

[0186] Total AA = 423

[0187] 367 / 423 = -87% sequence identity to SEQ ID NO:01 of W02022040277A1

[0188] amino acid sequence that differs from WQ2022040277A1 DD-TCFlfusion

[0189] • DD= destabilizing domain (ecDHFR) MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG RKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTH IDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 9) • DD-TCF1 MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG RKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTH IDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERRMPQLDSGGGGAG GGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGI PGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSA FNLLMHYPPPS GAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAP ADIS QKQ VHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPS GKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKES AAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSRE KHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 10)

[0190] • DD-flB-catenin-flTCFl MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG RKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTH IDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERRMATQADLMELD MAMEPDRKAAVSIIWQQQSYLDSGIIISGATTTAPSLSGKGNPEEEDVDTSQVLYEW EQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNV QRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVVVNKAAVMVHQ LSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAIFKS GGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKT NVKFEA1TTDCLQIEAYGNQESKEI1LASGGPQALVN1MRTYTYEKLLWTTSRVLKVE SVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAATKQEGMEGLLG TLVQLLGSDDINVVECAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDRED ITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRN LALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEG CTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAI EAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPM AWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGH HPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDLGGGGSGGGGSMPQLDS GGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGA AGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYK ETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAP ADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIP HPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIA ECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGK KKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 11) DD-tB-catenin-flTCFl MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG RKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTH ID AEVEGDTI IFPD YEPDDWES VFSEFI ID AD AQNSI IS YCFEILERRMDLGLDIGAQGE PLGYRQDDPS YRS FHS GGYGQD ALGMDPMMEHEMGGHHPGAD YPVDGLPDLGH A QDLMDGLPPGDSNQLAWFDTDLGGGGSGGGGSMPQLDSGGGGAGGGDDLGAPDE LLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGA RGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPS GAGQHPQPQPPEHKANQPPHGVPQLSEYEHFNSPHPTPAPADISQKQVHRPLQTPDLS GFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRN LKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRW HALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTETNW PRELKDGNGQESLSMSSSSSPA (SEQ ID NO: 12)

[0191] LEFlfusion

[0192] Domains:

[0193] • B-catenin sequences / domains from TCF1 fusion apply here as well

[0194] • F1LEF1 = LEF1 isoform 1 MPQLSGGGGGGGGDPELCATDEMIPFKDEGDPQKEKIFAEISHPEEEGDLADI KSSLVNESEIIPASNGHEVARQAQTSQEPYHDKAREHPDDGKHPDGGLYNKG PSYSSYSGYIMMPNMNNDPYMSNGSLSPPIPRTSNKVPVVQPSHAVHPLTPLI TYSDEHFSPGSHPSHIPSDVNSKQGMSRHPPAPDIPTFYPLSPGGVGQITPPLG WQGQPVYPITGGFRQPYPSSLSVDTSMSRFSHHMIPGPPGPHTTGIPHPAIVTP QVKQEHPHTDSDLMHVKPQHEQRKEQEPKRPHIKKPLNAFMLYMKEMRAN VVAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWS ARDNYGKKKKRKREKLQESASGTGPRMTAAYI (SEQ ID NO: 13)

[0195] FULL SEQUENCES:

[0196] • VI: flB-catenin-flLEFl MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEE DVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPS TQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVV VNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLS HHREGLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGL QKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIILASGGPQALVNIMRTYTYEKLL WTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAAT KQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALV RTVLRAGDREDITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHW PI IK ATVGI.1 K N I. Al. C PA N 11 A PI. REQGAIPRI. VQI, VR AHQDTQRRTSMGGTQQQFV EGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCE LAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSV ELTSSLFRTEPMAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMD PMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDLGGGGS GGGGSMPQLSGGGGGGGGDPELCATDEMIPFKDEGDPQKEKIFAEISIIPEEEGDLAD IKSSLVNESEIIPASNGHEVARQAQTSQEPYHDKAREHPDDGKHPDGGLYNKGPSYSS YSGYIMMPNMNNDPYMSNGSLSPPIPRTSNKVPVVQPSHAVHPLTPLITYSDEHFSPG SHPSHIPSDVNSKQGMSRHPPAPDIPTFYPLSPGGVGQITPPLGWQGQPVYPITGGFRQ PYPSSLSVDTSMSRFSHHMIPGPPGPHTTGIPHPAIVTPQVKQEHPHTDSDLMHVKPQ I IEQRKEQEPKRPI IIKKPLN AFMLYMKEMR AN V VAECTLKES AAINQILGRRWI IALS REEQAKYYELARKERQLHMQLYPGWSARDNYGKKKKRKREKLQESASGTGPRMT AAYI (SEQ ID NO: 14)

[0197] • V2: tB-catenin-flLEFl MDLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGAD YPVDGLPDLGIIAQDLMDGLPPGDSNQLAWFDTDLGGGGSGGGGSMPQLSGGGGG GGGDPELCATDEMIPFKDEGDPQKEKIFAEISHPEEEGDLADIKSSLVNESEIIPASNGH EVARQAQTSQEPYHDKAREHPDDGKHPDGGLYNKGPSYSSYSGYIMMPNMNNDPY MSNGSLSPPIPRTSNKVPVVQPSHAVHPLTPLITYSDEHFSPGSHPSHIPSDVNSKQGM SRHPPAPDIPTFYPLSPGGVGQITPPLGWQGQPVYPITGGFRQPYPSSLSVDTSMSRFS HHMIPGPPGPHTTGIPHPA1VTPQVKQEHPHTDSDLMHVKPQHEQRKEQEPKRPH1KK PLNAFMLYMKEMRANVVAECTLKESAAINQILGRRWHALSREEQAKYYELARKER QLHMQLYPGWSARDNYGKKKKRKREKLQESASGTGPRMTAAYI (SEQ ID NO: 16) All constructs have ribosomal skip sequence P2A and tNGFR at the end of the above sequences:

[0198] GSGATNFSEEKQAGDNEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGL YTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSM SAPCVEADDA VC RCA YGYYQDETTGRCEA CRVCEA GSGLVFSCQDKQNTVCEECPDGTY SDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEP EAPPEQDLIASTVAGVVTTVMGSSQPWTRGTTDNLIPVYCSILAAWVGLVAYIAFKR (SEQ ID NO: 15)

[0199] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. VI. References

[0200] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0201] 1. Pasquini MC, Hu Z-H, Curran K, Laetsch T, Locke F, Rouce R, et al. Real-world evidence of tisagenlecleucel for pediatric acute lymphoblastic leukemia and non-Hodgkin lymphoma. Blood Adv 2020; 4:5414—24.

[0202] 2. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD 19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 2018; 24:563-71.

[0203] 3. Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019; 576:293-300.

[0204] 4. Weber EW, Parker KR, Sotillo E, Lynn RC, Anbunathan H, Lattin J, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021; 372: doi.org / 10.1126 / science.abal786.

[0205] 5. Chen J, Ixipez-Moyado IF, Seo H, Lio C-WJ, Hempieman LJ, Sekiya T, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 2019; 567:530-4.

Claims

1. WHAT IS CLAIMED IS:

1. A recombinant fusion protein comprising:3.(a) a P-catenin protein or fragment that comprises, consists, or consists essentially of a putative transactivation domain;4.(b) (i) a TCF1 protein or fragment that binds to the truncated P-catenin protein and retains TCFl-driven T cell memory phenotype and / or sternness;5.(ii) a LEF1 protein or fragment that binds to the truncated P-catenin protein and retains LEF1 -driven T cell memory phenotype and / or sternness; and optionally (c) a destabilizing domain (DD) attached to said TCF1 / LEF1 protein or fragment,6.wherein the order of elements starting from the amino-terminus is (b) to (a) or (c) to (b) to (a).

2. The recombinant fusion protein of claim 1, wherein the P-catenin fragment lacks the armadillo repeats.

3. The recombinant fusion protein of claim 1, wherein the TCF1 fragment comprises, consists, or consists essentially of the HMG box domain, the P-catenin binding domain and the C-terminal transactivation.

4. The recombinant fusion protein of claim 1, wherein the LEF1 fragment comprises, consists, or consists essentially of the HMG box domain, the P-catenin binding domain and the C-terminal transactivation.

5. The recombinant fusion protein of claim 1, wherein the DD is an E. coli DHFR degron or FKBP12.

6. The recombinant fusion protein of claim 1, wherein one or both of the truncated P- catenin protein and DD are attached to said TCF1 protein or fragment with a linker, such as [Glycine-Serine linker (Gly-Ser), (GGGGS)N (SEQ ID NO: 17) linker, or XTEN linker.

7. The recombinant fusion protein of claim 1, wherein said recombinant fusion protein comprises any one of SEQ ID NOS: 1-8, 10, 11, 12, 14, or 15.

8. A polynucleotide encoding the recombinant fusion protein of any one of claims 1-7.

9. The polynucleotide of claim 8, further comprising a promoter that facilitates expression of the encoded recombinant fusion protein.

10. An engineered cell or non-engineered cell:15.(a) that expresses the recombinant fusion protein of any one of claims 1 -7; and / or (b) that comprises the polynucleotide of claim 9 under the control of a promoter active in said engineered cell.

11. The cell of claim 10, wherein the recombinant fusion protein is either transiently, constitutively, or inducibly expressed.

12. The cell of claim 10 or claim 11, wherein a coding region for the recombinant fusion protein is fused to a motif which modulates expression levels or enhances intracellular degradation.

13. The cell of any of claims 10-12, wherein the cell is a T cell or a tumor infiltrating lymphocyte.

14. The cell of claim 13, wherein the T cell is one that maintains functionality under conditions in which unmodified T cells display exhaustion.

15. The cell of claim 13 or claim 14, wherein the T cell comprises a nucleic acid encoding a recombinant receptor.

16. The cell of claim 15, wherein the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

17. The cell of claim 15 or claim 16, wherein the recombinant receptor is specific for a tumor antigen.

18. The cell of any of claims 15-17, wherein the recombinant fusion protein and the engineered receptor are encoded by separate nucleic acids.

19. The cell of any of claims 15-17. wherein the recombinant fusion protein and the engineered receptor are encoded by a single nucleic acid.

20. The cell of any of claims 15-19, wherein the recombinant fusion protein and the engineered receptor are expressed under different promoters.

21. The cell of any of claims 10-20, wherein the cell is obtained from a sample from a subject, such as a tumor sample, a lymph node or blood.

22. The cell of any of claims 10-21, wherein the engineered cell is expanded ex vivo.

23. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1-7, the polynucleotide of claim 8, or the engineered cell of any one of claims 10-22, and a pharmaceutically acceptable buffer, diluent or excipient.28.24 A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of a cell according to any one of claims 10-23.

25. The method of claim 24, wherein the cell is allogeneic or autologous to the patient.

26. The method of any one of claims 24 or claim 25, wherein the cell is administered systemically.

27. The method of any one of claims 24-26, further comprising administering a second anticancer therapy to the patient.

28. The method of claim 27, wherein the second anti-cancer therapy comprises a comprises surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

29. The method of any one of claims 24-28, wherein the cell is a T cell or a tumor infiltrating lymphocyte.

30. The method of claim 29, wherein the T cell is a CAR-T cell.

31. The method of any of claims 24-30, wherein the cancer is a solid tumor.

32. The method of any of claims 24-31, wherein the administering reduces the number of cancerous cells in the patient, reduces and / or eliminates the tumor burden in the patient, or result in an enhanced cancer treatment compared to administration of unmodified T cells, and / or results in lower expression of inhibitory receptors in the subject in comparison to administration of unmodified T cells.

33. A method of inducing a naive T cell expression signature, increasing CAR T persistence, increasing CAR T proliferation, and / or reinvigorating exhausted CAR T cells, the method comprising a recombinant fusion protein comprising (a) a P-catenin protein or fragment that comprises, consists, or consists essentially of a putative transactivation domain; (b)(i) a TCF1 protein or fragment that binds to the truncated - catenin protein and retains TCFl-driven T cell memory phenotype and / or sternness; (ii) a LEF1 protein or fragment that binds to the truncated P-catenin protein and retains LEFl-driven T cell memory phenotype and / or sternness; and optionally (c) a destabilizing domain (DD) attached to said TCF1 / LEF1 protein or fragment, wherein the order of elements starting from the amino-terminus is (b) to (a) or (c) to (b) to (a).

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