Compositions and methods of enhancing immune cell therapies by RUNX2 modulation

Abstract

The present disclosure relates generally to compositions and methods of enhancing potency of immune cell therapies by Runx2 modulation.

Description

COMPOSITIONS AND METHODS OF ENHANCING IMMUNE CELL THERAPIES BY RUNX2 MODULATIONRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 595,612, filed on November 2, 2023, and U.S. Provisional Application No. 63 / 640,437, filed on April 30, 2024, the contents of which is incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. W81XWH-19-1- 0196 awarded by The Department of Defense. The government has certain rights in the invention.FIELD

[0003] The present disclosure relates generally to compositions and methods of immune cell therapies by Runx2 modulation.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0004] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is “UNCO- 060_001WO_SeqList_ST26.xml”. The XML file is about 32,768 bytes in size, was created on October 17, 2024, and is submitted electronically via the USPTO Patent Center.BACKGROUND

[0005] Adoptive T cell therapy is an emerging immunotherapy for treating hematologic and solid malignancies. The three main modalities of adoptive T cell therapies are based on the use of autologous T cells isolated from tumors or peripheral blood, extensively expanded for days or weeks in vitro with IL-2 as a growth factor and transfused back to cancer patients. Tumor-infiltrating lymphocyte (TIL) therapy uses T cells isolated from solid tumors. Genetically engineered T cell receptor (TCR) therapy uses autologous peripheral T cells expressing a specific TCR recognizing a tumor antigen presented by MHC / HLA. Chimeric antigen receptor (CAR) T cell therapy uses autologous peripheral T cells redirected by the CAR molecule to recognize tumor antigens independent of MHC molecules (see, e.g., June etal. (2015) Sci Transl Med (7):280ps7; Rohaan et al. (2019) Virchows Arch (474):449-61; Sterner et al. (2021) Blood Cancer J (11):69)). Among the three, currently only CAR T cell therapy has been approved by FDA for hematological cancers (see, e.g., Davila et al. (2014) Sci Transl Med (6):224ra25; Lee et al. (2015) Lancet (385):517-28; Neelapu et al. (2017) N Engl J Med (377):2531-44).

[0006] Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has been highly successful in treating relapsed and treatment-refractory B-lineage hematologic malignancies. However, many patients do not achieve complete remission or relapse. Poor response or lack of remission durability results from cancer cell resistance or suboptimal CAR T cell function (see, e.g., Labanieh, et al. (2023) Nature (614);635-648). Thus, further studies into the immunobiology of these engineered cells are warranted to enhance remissions and expand therapeutic potential to other hematologic and solid tumors. CAR T cells are commonly generated from a heterogeneous population of peripheral blood T cells that varies between patients, likely impacting the quality of a CAR T cell product (see, e.g., Sommermeyer et al. (2016) Leukemia (30); 492). Although it has been difficult to track cell fate through the manufacturing process and into patients, previous reports have shown differential function of CAR T cell products generated from memory versus naive T cells sorted by surface marker phenotypes, which are not always an accurate representation of cellular differentiation state (see, e.g., Sommermeyer et al. (2016) Leukemia (30); 492, Qin et al. (2019). Sci Transl Med (11); Aldoss et al. (2023). Clin Cancer Res (29); 742-753).

[0007] During acute infections, naive CD8+ T cells become activated through the T cell antigen receptor (TCR) by antigen-presenting cells displaying cognate antigen and costimulatory ligands and subsequently enter a highly regulated differentiation trajectory. A phase of rapid expansion and differentiation into effector cells is followed by contraction and formation of long-lived memory cells that rapidly respond to future exposures. However, if the pathogen is not cleared, antigen-specific T cell populations will receive recurring antigen stimulation. In this setting, rather than forming functional memory, T cells differentiate down a trajectory characterized by progressive dysfunction, preventing immune-mediated pathology, but simultaneously failing to clear the challenge. These differentiation trajectories (and resulting functional characteristics imbued on T cells) are controlled epigenetically in traditional T cell responses to viral infections and tumors. These programs are defined by progressive changes to the epigenome, associated with DNA methylation and histone modifications which are driven by a variety of transcription factors (TFs) and modulated by antigen receptor signaling (see, e.g., Frias et al. (2021) Immunol Rev (300); 9-21). Thesemolecular modifications alter chromatin accessibility and transcriptional profiles which characterize cellular differentiation state and functional capacity.SUMMARY

[0008] The present disclosure provides a modified immune cell comprising (i) a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and (ii) a polynucleotide encoding RUNX2, wherein the polynucleotide encoding RUNX2 comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0010] In some embodiments, the antigen binding domain binds a tumor-associated antigen.

[0011] In some embodiments, the tumor associated antigen is selected from a group consisting of CD19, CD22, CD20, CD138, BCMA, CD33, CD123, FLT, CLL, CD56, CD34, CD117, CD14, CD133, CD44v6, CD47, CD64, CD96, CD97, CD99, CD45, CD9, Mucl, Lewis- Y, IL1RAP, FR-beta, CD5, CD7, CD38, CD30, B7-H3, HER2, CD44v6, CEA, c-Met, EGFRvIII, Epcam, EphA2, FR-alpha, GD2, GPC3, IL13R-alpha2, ILl lR-alpha, Ll-CAM, mesothelin, MUC1, MUC16, NKGD2 and PSCA.

[0012] In some embodiments, the tumor-associated antigen is CD 19.

[0013] In some embodiments, the immune cell is a T cell, a Natural Killer (NK) cell, a Natural Killer (NK)-like cell, a Cytokine Induced Killer (CIK) cell, a hematopoietic progenitor cell, a peripheral blood (PB) derived T cell, an induced pluripotent stem cell (iPSC)-derived effector cell, or an umbilical cord blood (UCB) derived T cell.

[0014] In some embodiments, the immune cell is a T cell.

[0015] In some embodiments, the T cell is a CAR T cell.

[0016] In some embodiments, the T cell is a CD8+ CAR T cell.

[0017] In some embodiments, the CD8+ CAR T cell is derived from memory cells, naive cells, or effector cells.

[0018] In some embodiments, the CD8+ CAR T cell is derived from naive cells.In some embodiments, the modified immune cell expresses an amount of the polynucleotide encoding RUNX2 sufficient to induce in the modified immune cell at least one of: (a) increased RUNX2 expression by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2; (b) enhanced potency by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2; (c) increased target cell clearance by at least 5% relative to an immune cell not comprising the polynucleotide encodingRUNX2; or (d) decreased T cell exhaustion by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2.

[0019] The present disclosure also provides a composition comprising the modified immune cell disclosed herein and a pharmaceutically acceptable carrier.

[0020] The present disclosure also provides a composition comprising a population of cells, wherein a plurality of cells of the population comprises (i) a chimeric antigen receptor or an engineered T cell receptor; and (ii) a polynucleotide encoding RUNX2.

[0021] In some embodiments, at least 50% of the cells in the population comprise the CAR or TCR and the polynucleotide encoding RUNX2.

[0022] The present invention also provides a polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor or an engineered T cell receptor and a nucleic acid sequence encoding RUNX2.

[0023] The present disclosure also provides a vector encoding the polynucleotide disclosed herein.

[0024] In some embodiments, the vector is a viral vector, a non-viral vector, or a transposon.

[0025] In some embodiments, the vector comprises at least one of (a) a sequence encoding RUNX2; (b) a polycistronic element sequence; (c) a reporter gene sequence; or (d) a sequence encoding a CAR or TCR.

[0026] In some embodiments, (a) the sequence encoding RUNX2 comprises a sequence of SEQ ID NOs: 1-2; (b) the polycistronic element sequence comprises a sequence of any one of SEQ ID NOs: 3-9; and / or (c) the reporter gene sequence comprises a sequence of any one of SEQ ID NOs: 10-13.

[0027] The present invention also provides a method of improving an immune cell therapy, comprising delivering an exogenous polynucleotide encoding RUNX2 to a therapeutic cell.

[0028] In some embodiments, the method results in at least one of: (a) RUNX2 upregulation by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2; (b) enhanced potency by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2; (c) increased target cell clearance by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2; or (d) decreased T cell exhaustion by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2.

[0029] The present invention also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject the modified immune cell or the composition disclosed herein.

[0030] In some embodiments, the cancer is a liquid tumor or a solid tumor.

[0031] In some embodiments, the liquid tumor is leukemia.

[0032] In some embodiments, the leukemia is B cell leukemia.

[0033] The present invention also provides a recombinant viral vector comprising: (i) a viral capsid comprising a viral capsid protein; (ii) a first nucleic acid sequence encapsulated within the viral capsid, wherein the first nucleic acid sequence encodes a chimeric antigen receptor or an engineered T cell receptor; and (iii) a second nucleic acid sequence encapsulated within the viral capsid, wherein the second nucleic acid sequence encodes RUNX2.

[0034] In some embodiments, the recombinant viral vector comprises an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a vaccinia viral vector, or a vesicular stomatitis viral vector.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGs. 1A-1M show that antigen experience history directs multiple aspects of in vitro functional capacity of murine CD8+ CAR T cells. FIG. 1A shows E2A-PBX murine leukemia models engineered to knockout CD 19, followed by reintroduction of CD 19 at different levels to generate a range of antigen density clones. FIG. IB depicts a schematic of the vaccine model for generating memory CD8+ OI-I T cells. FIG. 1C shows intracellular cytokine staining of IFNY and TNFa after a 6-hour co-culture assay. FIG. ID shows degranulation as measured by CD 107a expression after a 4-hour co-culture assay. FIG. IE shows the quantification of % IFNY+ cells. FIG. IF shows the quantification of % TNFa+ cells. FIG. 1G shows the quantification of % IFNY+ / TNFa+ cells. FIG. 1H shows the quantification of CD 107a data (% positive cells). FIG. II shows % cytotoxicity, quantified after a 6 hour co-culture assay. FIG. 1 J shows cell-cycle entry as measured by Ki-67 staining after an 18-hour co-culture assay. FIG. IK shows proliferation as measured by dilution of CellTrace Violet dye after a 72-hour co-culture assay. FIG. IL shows quantification of % Ki67-negative cells as measured by Ki67 staining after a 18 hour co-culture assay. FIG. IM shows quantification of % CellTrace-Lo cells after a 72 hour co-culture assay. All in vitro assays were performed with n=3 technical replicates, and are representative of 3 independent experiments. Data represent mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] FIGs. 2A-2J show that memory derived CD8+ CAR T cells (CAR8MD) exhibit enhanced cytotoxicity and clearance of CD19Loleukemia in vivo (high CAR dose). FIG. 2A depicts a schematic of the timeline for in vivo experiments. FIGs. 2B-2C show early T cell expansion (day +4) or persistence (day +11) after the infusion of transduced T cells againstwildtype (WT) leukemia (FIG. 2B) and CD19Loleukemia (FIG. 2C). FIG. 2D shows clearance of WT and CD19Loleukemia at day +11 after CAR infusion. FIGs. 2E-2F show intracellular cytokine staining of interferon gamma (FIG. 2E) or granzyme B (FIG. 2F) in CAR T cells from whole bone marrow restimulated ex vivo with leukemia. FIGs. 2G-2I show intranuclear transcription factor staining of IRF4 (FIG. 2G), EOMES (FIG. 2H), or T- bet (FIG. 21) on CAR+ T cells from mice bearing the indicated leukemia at day +4 after CAR infusion. Violin plot data represent mean with quartiles. FIG. 2J shows the survival of mice after treatment with le6 EGFR+ CAR or control T cells. Data was collected from 2 pooled, independent experiments with n=10 mice per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0037] FIGs. 3A-3I show that naive derived CD8+ CAR T cells (CAR8ND) exhibit enhanced expansion capacity and clearance of WT leukemia in vivo (low CAR dose). FIGs. 3A-3B show early T cell expansion (day +4) or persistence (day +11) after infusion of transduced T cells against WT leukemia (FIG. 3A) and CD19Loleukemia (FIG. 3B). FIG. 3C shows clearance of WT and CD19Loleukemia at day +11 after CAR infusion. FIGs. 3D-3E show intracellular cytokine staining of interferon gamma (FIG. 3D) or granzyme B (FIG. 3E) in CAR T cells from whole bone marrow restimulated ex vivo with leukemia. FIGs. 3F-3H show intranuclear transcription factor staining of IRF4 (FIG. 3F), EOMES (FIG. 3G), or T- bet (FIG. 3H) on CAR+ T cells from mice bearing the indicated leukemia at day +4 after CAR infusion. Violin plot data represents the median with quartiles. FIG. 31 shows the survival of mice after treatment with le6 EGFR+ CAR or control T cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data represent + / - SD. Data was collected from 2 independent pooled experiments, total n=10 mice per group.

[0038] FIGs. 4A-4E show that prior antigen experience imprints chromatin accessibility states that follow unique patterns during CAR transduction and reinfusion. FIG. 4A depicts a schematic showing the layout for printed ATAC-seq / RNA-seq experiments. FIG. 4B shows chromatin accessibility at Gzmb. Gzmc. IFNy, Tcf7, and Pdcdl gene loci for naive and memory-derived T cells at each timepoint. FIG. 4C shows ChromVAR deviation z-scores between indicated populations at differentially accessible regions between Effector and Memory T cells after LCMV-Armstrong infection. FIG. 4D shows motif-associated ChromVAR deviation z-scores between indicated populations. FIG. 4E shows K-mean clustering of relative ATAC-seq signal at differentially accessible regions (top, data from two biological replicates) and motif enrichment in each cluster vs. all regions (bottom). Data represents the mean + / - range of two biological replicates.

[0039] FIGs. 5A-5E show that prior antigen experience drives differential CAR8 transcriptomic states which follow unique patterns during CAR transduction and reinfusion. FIG. 5A shows volcano plots of significant differentially expressed genes between naive and memory-derived cells at each of the three time points. FIG. 5B shows normalized enrichment scores from a gene set enrichment analysis (GSEA) of differentially enriched gene sets between the indicated CD8+ T cell subsets after LCMV- Arm strong acute viral infection. FIG. 5C shows GSEA plots at each time point. FIG. 5D shows the top differentially expressed transcription factors at the “PreCAR” timepoint generated using Ingenuity Pathway Analysis (IP A). FIG. 5E shows DESeq2-normalized counts of the Runx family transcription factors at each time point for naive and memory-derived cells. All statistics were performed using DESeq2 with a filtering threshold at 10, log2foldchange >2 and padj <0.05.

[0040] FIGs. 6A-6L show that Runx2 overexpression enhances naive-derived CD8+ CAR T cell potency and resistance to dysfunction and enhances effector-like genomic profile while preserving memory-like characteristics. FIGs. 6A-6B show co-transduction of memory (FIG. 6A) or naive (FIG. 6B) CD8+ T cells with CAR and pMIG-Empty, or pMIG-RUNX2 and intracellular staining for RUNX2. FIGs. 6C-6D show CAR T cell and leukemia proportions for memory (FIG. 6C) and naive-derived (FIG. 6D) CAR T cells co-transduced with RUNX2 or pMIG control. FIGs. 6E-6F show the proportion of CAR T cells displaying PD1+ / TOX+ phenotype for memory (FIG. 6E) or naive-derived (FIG. 6F) CAR T cells. FIGs. 6G-6H show volcano plots of significant differentially expressed genes between RUNX2-CAR8 and pMIG-CAR8 at a PostCAR timepoint for memory (FIG. 6G) or naive- derived (FIG. 6H) CAR T cells. FIGs. 6I-6J show top enriched HALLMARK genesets between RUNX2-CAR8 and pMIG-CAR8 at a PostCAR timepoint for memory (FIG. 61) or naive-derived (FIG. 6J) CAR T cells. FIG. 6K shows chromatin accessibility at Gzma and Tcf7 and gene loci for RUNX2 or pMIG naive or memory-derived T cells at the PostCAR timepoint. FIG. 6L shows ChromVAR deviation z-scores at PostCAR timepoint between indicated RUNX2 / pMIG populations at differentially accessible regions between effector and memory T cells after LCMB-Armstrong infection. Data in FIGs 6C-6F are from 2 pooled, independent experiments with n=9 mice per condition. RNAseq data in FIGs. 6G-6J were performed using pooled replicates using the DESeq2 R package, with a filtering threshold at 10 with greater than 2-fold change and adjusted p value <0.05 with a filtering threshold at 10, log2foldchange <2, and padj <0.05. Data represents mean + / - SD. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001.

[0041] FIGs. 7A-7Q show RUNX2 overexpression enhances in vitro function of human CD4+ and CD8+ CAR T cells. FIG. 7A shows expression of the hl928z CAR in CD4+ or CD8+ cells co-transduced with hRUNX2, EGFR (control), or Mock (untransduced). FIG. 7B shows intracellular staining for hRUNX2 in CD4+ or CD8+ cells co-transduced with hRUNX2, EGFR (control) or Mock (untransduced). FIG. 7C shows degranulation as measured by CD 107a expression and intracellular cytokine staining of IFNy after a 5.5 hour co-culture assay. FIG. 7D shows intracellular cytokine staining of IL-2 and TNFa after a 5.5 hour co-culture assay. FIG. 7E shows proliferation as measured by the dilution of CellTrace Violet dye after a 72 hour co-culture assay. FIG. 7F shows the quantification of CAR and leukemia counts for RUNX2 overexpressing naive-derived CAR T cells compared to pMIG control. FIG. 7G shows the % cytotoxicity after a 5.5 hour co-culture assay. FIGs. 7H-7Q show GzmA (FIG. 7H), GzmB (FIG. 71), Perforin (FIG. 7J), Granulysin (FIG. 7K), IFNY (FIG. 7L), IL-2 (FIG. 7M), IL-4 (FIG. 7N), IL-17A (FIG. 70), TNFa (FIG. 7P), and IL-6 (FIG. 7Q) protein concentrations as measured by cell culture supernatant by multiplexed ELISA after a 16 hour co-culture assay. All in vitro assays show data from 3 independent biological donors. . Data represents mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0042] FIGs. 8A-8J show E2A-PBX / mCD19 antigen density model and murine anti-CD19 CAR T cells, and additional statistical comparisons of in vitro data (related to FIGS. 1 A-1M). FIG. 8A depicts a schematic showing the anti -mouse CD 19 CAR contained in pMSCV backbone. FIG. 8B shows co-expression of CAR and EGFR on murine CAR T cells. FIG. 8C shows the engineering of murine leukemia with lentiviral vectors containing hUbC or hEFla promoters driving the CD 19 transgene. FIG. 8D shows the survival of mice after treatment with le6 EGFR+ (EGFR8, non-CAR expressing) naive or memory-derived CD8+ T cells. FIG. 8E shows the fold expansion of T cells during transduction protocol: During Stim (while on anti-CD3 / CD28 beads from day 1 to day 4), Post Stim (continued expansion from day 4 to day 6 after removing anti-CD3 / CD28 beads), and Composite (total fold expansion from day 1 to day 6). FIG. 8F shows the mean fluorescence intensity of the IFNy+ cell population. FIG. 8G shows the mean fluorescence intensity of the TNGa+ cell population. FIG. 8H shows the mean fluorescence intensity of the CD107a+ population. FIG. 81 shows the statistical comparisons of Ki67Neg(% Ki67Negof EGFR+), Ki67Lo(% Ki67Loof EGFR+, MFI Ki67Loof EGFR+) and Ki67Hi(% Ki67Hiof EGFR+, MFI Ki67Hiof EGFR+) populations. FIG. 8J shows statistical comparisons of CellTraceLo(% CellTraceLoof EGFR+, MFI CellTraceLoof EGFR+) and total EGFR+ (GFMI CellTrace, GFMICellTrace with zoomed axis) populations. Data represent the mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0043] FIGs. 9A-9G show that polyclonal pathogen-elicited CAR8MD function similarly to vaccine-elicited CAR8MD (related to FIGS. 1 A-1M). FIG. 9A depicts a schematic showing the LCMV model for generating memory CD8+ T cells. FIG. 9B shows intracellular cytokine staining of ZFNy and TNFa after a 6-hour co-culture assay. FIG. 9C shows quantifications of proportions of IFNy+ and TNFa+ cells of a EGFR+ population. FIG. 9D shows quantifications and proportions of IFNy+ / TNFa+ double positive cells of a EGFR+ population. FIG. 9E shows the % cytotoxicity, quantified after a 6 hour co-culture assay. FIG. 9F shows proliferation as measured by the dilution of CellTrace Violet (CTV) dye after a 72-hour co-culture assay. FIG. 9G shows the quantification of CellTrace assays, proportions of CTVLocells, MFI of CTVLocells. All assays were performed with n=7 technical replicates and are representative of 3 independent experiments. Data represents the mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0044] FIGs. 10A-10D show CAR T cells and leukemia counts per tibia for in vivo data (related to FIGS 2A-2J and FIGS. 3A-3I). FIG. 10A shows CAR counts for le6 CAR dose experiments. FIG. 10B shows leukemia counts for le6 CAR dose experiments. FIG. 10C shows CAR counts for 3e5 CAR dose experiments. FIG. 10D shows leukemia counts for 3e5 CAR dose experiments. Data were pooled from 2 independent experiments with n=10 mice per condition, apart from the le6 CAR dose day 11 timepoint, which contains data from one experiment with n=5 mice per condition. Data represents mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0045] FIGs. 11A-11E show the basic characterization of in vivo model and additional in vivo effector / memory phenotyping at high CAR dose (related to FIGS. 2A-2J). FIG. 11A shows the basic flow cytometry gating strategy for in vivo experiments. FIG. 11B shows proportions of CAR8 with the short-lived effector cell (SLEC, IL7Ra- / KLRGl+) or memory precursor effector cell (MPEC, IL7Ra+ / KLRGl-) phenotypes at the indicated timepoint against WT leukemia. FIG. 11C shows proportions of CAR8 with the short-lived effector cell (SLEC, IL7Ra- / KLRGl+) or memory precursor effector cell (MPEC, IL7Ra+ / KLRGl-) phenotypes at the indicated timepoints against CD19Loleukemia. FIG. 11D shows proportions of CAR8 with the effector memory precursor (EMP, CD27+ / CD62L-) or central memory precursor (CMP, CD27+ / CD62L+) phenotypes at the indicated timepoint against WT leukemia. FIG. HE shows proportions of CAR8 with the effector memory precursor (EMP, CD27+ / CD62L-) or central memory precursor (CMP, CD27+ / CD62L+) phenotypes atthe indicated timepoint against CD19Loleukemia. Data were pooled from 2 independent experiments with n=10 mice per condition. Data represents mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0046] FIGs. 12A-12L show additional in vivo effector / memory and exhaustion phenotyping at low CAR dose (related to FIGS. 3A-3I). FIG. 12A shows proportions of CAR8 with the short-lived effector cell (SLEC, IL7Ra- / KLRGl+) or memory precursor effector cell (MPEC, IL7Ra+ / KLRGl-) phenotypes at the indicated timepoint against WT leukemia. FIG. 12B shows proportions of CAR8 with the short-lived effector cell (SLEC, IL7Ra- / KLRGl+) or memory precursor effector cell (MPEC, IL7Ra+ / KLRGl-) phenotypes at the indicated timepoints against CD19Loleukemia. FIG. 12C shows proportions of CAR8 with the effector memory precursor (EMP, CD27+ / CD62L-) or central memory precursor (CMP, CD27+ / CD62L+) phenotypes at the indicated timepoint against WT leukemia. FIG. 12D shows proportions of CAR8 with the effector memory precursor (EMP, CD27+ / CD62L-) or central memory precursor (CMP, CD27+ / CD62L+) phenotypes at the indicated timepoint against CD19Loleukemia. FIGs. 12E-12L show proportions of CAR8 with the PD1+ / TOX+ phenotype (FIGs. 12E, 12G), the PD1+ / CD39+ phenotype (FIGs. 12F, 12H), the TCF1+ / TIM3- phenotype (FIGs. 121, 12K), and the TCF1- / TIM3+ phenotype (FIGs. 12J, 12L) at 11 days post-CAR injection against either WT (FIGs. 12E, 12F, 121, 12 J) or CD19Loleukemia (FIGs. 12G, 12H, 12K, 12L). Data was collected from 2 pooled, independent experiments with n=10 mice per condition. Data represents mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0047] FIGs. 13A-13D show additional analyses of ATAC-seq data (related to FIGS. 4A- 4E). FIG. 13A shows inter-replicate Elicidian distance of voom-normalized ATAC-seq counts per peak between biological replicates. FIG. 13B shows pairwise comparisons of differentially accessible chromatin regions within conditions between different time points of the same condition, or between different conditions at each timepoint. Data points represent the mean of voom-normalized ATAC-seq counts per peak between biological replicates of each group. FIG. 13C shows a heatmap of motif-associated ChromVAR deviation z-scores patterns of motif-associated ATAC-seq signal for the indicated transcription factors. FIG. 13D shows the representative gating for sorting of the cells in sequencing experiments.

[0048] FIGs. 14A-14F show additional analyses for RNA-seq data (related to FIGS. 5A-5E). FIG. 14A shows the normalized enrichment scores from GSEA of differentially enriched gene sets between indicated CD8+ T cell subsets after LCMV-Armstrong acute viral infection. FIG. 14B shows the top transcriptional activators predicted to be activated anddriving differential transcriptional states between naive vs. memory-derived cells at the indicated time points, as predicted by Qiagen Ingenuity Pathway Analysis (IP A). FIG. 14C shows the IPA activation map for the Cebpb transcription factor, the top predicted driver of transcription states in memory-derived cells at the PostCAR and Tumor timepoints. FIGs. 14D-14F shows the top differentially expressed transcription factors at the PreCAR (FIG. 14D), PostCAR (FIG. 14E), and Tumor (FIG. 14F) time points. All statistics were performed using DESeq2 with a filtering threshold at 10, log2foldchange >2 and padj >0.05.

[0049] FIGs. 15A-15E show the characterization of le5 CAR T cell dose in vivo experiments and in vitro comparison of BATF, JUN overexpressing cells to pMIG (related to FIGS. 6A-6L). FIG. 15A shows leukemia burden. FIG. 15B shows proportions of CAR8 with PD1+ / TOX+ phenotype. Data represents 1 experiment with n=5 mice per condition. FIGs. 15C-15D show the quantification of intracellular cytokine staining of IFNy and TNFa after a 6-hour co-culture assay, % positive of EGFR+, for memory (FIG. 15C) or naive- derived (FIG. 15D) cells co-transduced with BATF, JUN, or pMIG). Data represents 3 independent experiments. FIG. 15E shows the proliferation as measured by the dilution of CellTrace Violet dye of EGFR+ cells after a 72-hour co-culture assay for memory or naive- derived cells co-transduced with BAFT, JUN, or pMIG. Data represents the mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0050] FIGs. 16A-16N show overexpression of BATF or c-JUN in functional enhancement of naive-derived cells in the presence of phenotypic exhaustion (related to FIGs. 6A-6L). FIGs. 16A-16B show co-transduction of memory (FIG. 16A) or naive (FIG. 16B) CD8+ T cells with CAR and pMIG-Empty, pMIG-BATF, or pMIG- JUN. FIGs. 16C-16N show Ragl~ / _mice that were given leukemia on day -3, followed by le4 pMIG-Runx2 or pMIG-Empty co-transduced with CAR8 on day 0. Bone marrow was analyzed by flow cytometry on day 11 Post-CAR. FIGs. 16C-16D show CAR T cell and leukemia proportions for memory (FIG. 16C) and naive-derived (FIG. 16D) CAR T cells co-transduced with BATF, JUN, or pMIG control. FIGs. 16E-16F show the proportion of memory (FIG. 16E) and naive-derived (FIG. 16F) CAR T cells displaying PD1+ / TOX+ phenotype. FIGs. 16G-16H show CAR and leukemia counts for BATF or JUN overexpressing memory (FIG. 16G) or naive-derived (FIG. 16H) CAR T cells compared to pMIG control. FIGs. 16I-16N show the proportions of EGFR+ cells from BATF, JUN, or pMIG CAR8 memory (FIGs. 161, 16J, 16M) and naive- derived (FIGs. 16K, 16L, 16N) cells with the PD1+ phenotype (FIGs. 161, 16K), the PD1+ / CD39+ phenotype (FIGs. 16J, 16L), and the TCF1 / TIM3 phenotype (FIGs. 16M,16N). Data are from 3 pooled, independent experiments with n=13 mice per condition. Data represents the mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0051] FIGs. 17A-17Q show in vitro characterization, additional in vivo data, and additional genomic comparisons for RUNX2-overexpressing CAR8. FIGs. 17A-17B show the quantification of intracellular cytokine staining of IFNy and TNFa after a 6 hour co-culture assay, % EGFR+ memory (FIG. 17A) or naive-derived (FIG. 17B) cells co-transduced with RUNX2 or pMIG. FIG. 17C shows proliferation as measured by dilution of CellTrace Violet dye dilution of EGFR+ cells after a 72 hour co-culture assay, for memory or naive-derived cells co-transduced with RUNX2 or pMIG. FIGs. 17D-17E show CAR and leukemia counts for RUNX2 overexpressing memory (FIG. 17D) and naive-derived (FIG. 17E) CAR T cells compared to pMIG control. FIGs. 17F-17I show proportions of EGFR+ cells from RUNX2 or pMIG CAR8 in memory derived (FIGs. 17D, 17F, 17G) or naive-derived (FIGs. 17E, 17H, 171) cells with the PD1+ phenotype (FIGs. 17F, 17H), the PD1+ / CD39+ phenotype (FIGs. 17G, 171), the TCF1 / TIM3 phenotype (FIGs. 17J-17K). FIG. 17L shows a PC A plot for RUNX2 and pMIG cells, both memory and naive-derived groups, generated via variabce stabilization transformation (VST) function from the R package DESeq2 and the removeBatchEffect function from the R package Limma to account for different collection dates. FIG. 17M shows inter-replicate Euclidian distance of voom-normalized ATAC-seq counts per peak between biological replicates, plotted using multidimensional scaling. FIG. 17N shows pairwise comparisons of differentially accessible chromatin regions within conditions between RUNX2 and pMIG CAR T cells in memory or naive-derived groups. FIG. 170 shows co-staining of hl928z CAR with EGFR in CD4+ or CD8+ cells cotransduced with hRUNX2-IRES-EGFR, EGFR (control), or Mock (untransduced). FIG. 17P shows degranulation as measured by CD 107a expression and intracellular cytokine staining of IFNy after a 5.5 hour co-culture assay. FIG. 17Q shows intracellular cytokine staining of IL-2 and TNFa after a 5.5 hour co-culture assay. Data represents the mean + / - SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.DETAILED DESCRIPTION

[0052] As used herein, the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." As used herein "another" may mean at least a second or more.

[0053] As used herein, the term "about" is used to indicate the value ± 10%.

[0054] The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism; or in relation to a cell, the term refers to a cell that was isolated and subsequently introduced into a cell population or to an organism. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid that occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is one that is introduced via viral transduction. The term “exogenous” is used interchangeably with the term “heterologous”.

[0055] By "expression construct" or "expression cassette" is used to mean a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements (such as promoters, enhancers or a structure functionally equivalent thereof) that direct gene expression in one or more desired cell types, tissues or organs. Additional elements, such as a transcription termination signal, may also be included.

[0056] A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide, or the protein expressed by said polynucleotide, to be delivered to a host cell, either in vitro or in vivo.

[0057] A "plasmid," a common type of vector, is an extra-chromosomal DNA molecule separate from the chromosomal DNA that is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double-stranded.

[0058] An "origin of replication" ("ori") or "replication origin" is a DNA sequence, that when present in a plasmid in a cell is capable of maintaining linked sequences in the plasmid and / or a site at or near where DNA synthesis initiates. As an example, an ori for EBV (Ebstein-Barr virus) includes FR sequences (20 imperfect copies of a 30 bp repeat), and preferably DS sequences; however, other sites in EBV bind EBNA-1, e.g., Rep* sequences can substitute for DS as an origin of replication (Kirshmaier and Sugden, 1998). Thus, a replication origin of EBV includes FR, DS, or Rep* sequences or any functionally equivalent sequences through nucleic acid modifications or synthetic combinations derived therefrom. For example, methods of the present disclosure may also use genetically engineered replication origin of EBV, such as by insertion or mutation of individual elements.

[0059] The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (IRES), enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell. Not all of these control elements need to be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0060] By "operably linked" with reference to nucleic acid molecules, is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and a functional effector element) are connected in such a way as to permit transcription of the nucleic acid molecule. "Operably linked" with reference to peptide and / or polypeptide molecules means that two or more peptide and / or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is preferably chimeric, i.e., composed of molecules that are not found in a single polypeptide in nature.

[0061] As used herein, the term "subject" or "subject in need thereof refers to a mammal, preferably a human being, male or female at any age that is in need of a therapeutic intervention, a cell therapy or a tissue transplantation. Typically, the subject is in need of therapeutic intervention, cell or tissue transplantation (also referred to herein as recipient) due to a disorder or a pathological or undesired condition, state, or syndrome, or a physical, morphological or physiological abnormality that is amenable to treatment via therapeutic intervention, cell or tissue transplantation.

[0062] An "immune response" is a response of a cell of the immune system, such as an NK cell, B cell, or a T cell, or innate immune cell to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0063] As used herein, the term "antigen" is a molecule capable of being bound by an antibody, T cell receptor, Chimeric Antigen Receptor and or engineered immune receptor. An antigen may generally be used to induce a humoral immune response and / or a cellular immune response leading to the production of B and / or T lymphocytes.

[0064] The terms "tumor-associated antigen," "tumor antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0065] "Treating" or “treatment of’ a disease or condition refers to executing a protocol or treatment plan, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease or the recurrence of the disease. Desirableeffects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission, increased survival, improved quality of life or improved prognosis. "Treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and includes protocols or treatment plans that have only a marginal effect on the patient.

[0066] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, a reduction in the growth rate of the cancer, or prevention of metastasis or recurrence. Treatment of cancer may also refer to prolonging the survival of a subject with cancer.

[0067] "Antigen recognition moiety” or “antigen recognition domain" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody-like molecule or fragment thereof and the antigen is a tumor antigen.

[0068] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required, e.g., by the FDA Office of Biological Standards.

[0069] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0070] The term "T cell" refers to T lymphocytes, and includes, but is not limited to, y / 5 T cells, a / p T cells, NK T cells, CD4+ T cells and CD8+ T cells. CD4+ T cells include THO, Thl and TH2 cells, as well as regulatory T cells (Treg). There are at least three types of regulatory T cells: CD4+ CD25+Treg, CD25 TH3 Treg, and CD25 TRI Treg. "Cytotoxic T cell" refers to a T cell that can kill another cell. The majority of cytotoxic T cells are CD8+ MHC class I-restricted T cells, however, some cytotoxic T cells are CD4+. In some embodiments, the T cell of the present disclosure is CD4+ or CD8+.

[0071] The term "antigen presenting cells (APCs)" refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. APCs can be intact whole cells such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to 2-microglobulin.

[0072] The term "culturing" refers to the in vitro maintenance, differentiation, and / or propagation of cells in suitable media. By "enriched" it meant a composition comprising cells present in a greater percentage of total cells than is found in the tissues where they are present in an organism.Immune Cells

[0073] Certain embodiments of the present disclosure concern immune cells that express an immune receptor (e.g., a chimeric antigen receptor or an engineered T cell receptor). In some embodiments of the present disclosure, the immune cells express an engineered T cell receptor (TCR). In some embodiments of the present disclosure, the immune cells express a chimeric antigen receptor (CAR). Examples of such immune cells include, but are not limited to, T cells (e.g., regulatory T cells, CAR T cells, CD8+ CAR T cells, CD4+ CAR T cells, CD4+ T cells, CD8+ T cells, peripheral blood (PB) derived T cells, umbilical cord blood (UCB) derived T cells, or gamma-delta T cells), NK cells, NK-like cells, invariant NK cells, NKT cells, cytokine-induced killer (CIK) cells, stem cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells, hematopoietic progenitor cells, or induced pluripotent stem (iPSC) cells). In some embodiments, the T cell Is a tumor-infiltrating lymphocyte (TIL). In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. Also provided herein are methods of producing and engineering the immune cells and methods ofusing and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

[0074] The immune cells may be enriched or purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues and organs from which the immune 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. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing. In some embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some embodiments, immune cells isolated from cord blood have enhanced immunomodulation capacity, such as measured by CD4+-positive or CD8+-positive T cell suppression. In specific aspects, the immune cells are 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., from 2 or more donor subjects).

[0075] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells may be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor not suffering from a disease associated with reduce immune cell activity. The immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells reside in said subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as from pooled cord blood. The population of immune cells can be derived from induced pluripotent stem cells (iPSCs) and / or any other stem cell known in the art. In some aspects, the iPSCs and / or stem cells used to derive the population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity, thus these IPSCs and / or stem cells will be autologous to the subject in need of therapy. Alternatively, the iPSCs and / or stem cells can be obtained from a donor and therefore be allogeneic to the subject in need of therapy.

[0076] When the population of immune cells is obtained from a donor distinct from the subject to be administered the population of immune cells, the cells obtained should be subject-compatible in that they can be introduced into the subject. Allogeneic donor cells aremay or may not be human leukocyte antigen (HLA)-compatible. To be rendered subjectcompatible, allogeneic cells can be treated to reduce immunogenicity.T cells

[0077] T cells are a type of white blood cell that plays a central role in the adaptive immune system. T cells are critical for the immune response and are involved in recognizing and eliminating infected or abnormal cells. T cells originate from hematopoietic stem cells in the bone marrow and undergo maturation in the thymus. T cells express cell surface markers which are critical in recognizing and interacting with antigens. Non-limiting examples of cell surface markers include T cell receptor (TCR), CD3, CD4+, and CD8+.

[0078] There are several types of T cells, namely: 1) Helper T cells (e.g. CD4+ cells), 2) Cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T cells or killer T cell), 3) Memory T cells ((i) stem memory TSCM cells, like naive cells, are CD4+5RO-, CCR7+, CD4+5RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not ZFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNy and IL-4), 4) Regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), 5) Natural Killer T cells (NKT), 6) Mucosal-Associated Invariant T cells (MAIT cells) and 7) Gamma Delta T cells.

[0079] The T cells used in the compositions and methods described herein can come from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is herein incorporated by references in its entirety.

[0080] In some aspects, the present disclosure provides a population of immune cells (e.g. T cells). In some aspects, the present disclosure provides a population of modified immunecells that express a genetically engineered T cell receptor or chimeric antigen receptor. In some embodiments, the population of immune cells comprises CD4+ CAR T cells. In some embodiments, the population of immune cells comprises CD8+ CAR T cells. In some embodiments, the ratio of CD4+ CAR T cells vs CD8+ CAR T cells is 1 : 1, 2:1, 3:1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, or any ratio in between CD4+:CD8+ CAR T cells. In some embodiments, the population of immune cells comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% CD8+ CAR T cells. In some embodiments, the population of immune cells comprises 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or any ratio in between CD4+ CAR T cells. In some embodiments, the modified immune cell comprises a chimeric antigen receptor and an exogenous polynucleotide encoding RUNX2. In some embodiments, the exogenous polynucleotide encoding RUNX2 comprises SEQ ID NO: 1 or SEQ ID NO: 2.Engineered T Cell Receptors

[0081] The immune cells of the disclosure (e.g., autologous, or allogenic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, stem cells (e.g., MSCs, HPCs, HSCs, or iPS cells) can be genetically engineered to express antigen receptors such as engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Multiple engineered TCRs may be added to a single cell type, such as T cells.

[0082] In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous. In some embodiments, the nucleic acids are not naturally occurring.

[0083] In some embodiments, the TCR contains an extracellular antigen-recognition domain that specifically binds to an antigen expressed on a cell (e.g., a cancer cell). In some embodiments, the antigen is a protein expressed on the surface of cells (e.g., on the surface of a cancer cell).

[0084] Illustrative engineered antigen receptors, including TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in PCT Publication Nos. WO 2000 / 14257, WO 2013126726, WO 2012 / 129514,WO 2014 / 031687, WO 2013 / 166321, and WO 2013 / 071154, U.S. Patent Application Publication Nos. US 2002 / 131960, US 2013 / 287748, and US 2013 / 0149337; and U.S. Patent Nos. 6,451,995, 7,446,190, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995,7,265,209, 7,354,762, 7,446,190, 7,446,191, 8,324,353, and 8,479, 118; International Patent Application Publication No.: WO 2014 / 055668 Al, and European Patent Application Publication No. EP2537416; and / or those described by Sadelain et al., 2013; Turtle et al., 2012; Wu et al., 2012, each of which is incorporated herein by reference in its entirety for examples of TCRs that may be expressed in the immune cells disclosed herein.

[0085] In some aspects, the present disclosure provides a population of genetically modified immune cells (e.g., T cells) engineered to express a T cell receptor (TCR) and / or a polynucleotide encoding a TCR, wherein the TCR comprises two chains (alpha and beta) each comprising variable and constant regions, a peptide binding groove, a transmembrane domain, and an intracellular signaling domain comprising at least one CD3 complex.

[0086] In some aspects, the present disclosure provides a population of engineered immune cells, wherein a plurality of the engineered immune cells of the population comprises a TCR disclosed herein. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express the TCR. In some embodiments, each TCR polypeptide is expressed at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 copies per cell. In some embodiments, the nucleic acid encoding the TCR is integrated into the genome at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 copies per cell. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express the TCR and an exogenous nucleic acid encoding RUNX2. In some embodiments, the exogenous polynucleotide encoding RUNX2 comprises SEQ ID NO: 1 or SEQ ID NO: 2.Genetically Engineered Chimeric Antigen Receptors

[0087] A "chimeric antigen receptor" is also known as an artificial cell receptor, a chimeric cell receptor, or a chimeric immunoreceptor. Chimeric antigen receptors (CARs) are engineered receptors, which graft a selected specificity onto an immune effector cell. CARs typically have an extracellular domain (ectodomain), a transmembrane domain and an intracellular (endodomain) domain. In some embodiments, the ectodomain comprises anantigen-binding domain and a stalk region. In some embodiments, the ectodomain comprises an antibody binding domain that recognizes CD 19.

[0088] A “stalk region”, which encompasses the terms "spacer region" or "hinge domain" or “hinge”, is used to link the antigen-binding domain to the transmembrane domain. As used herein, the term "stalk region" generally means any oligonucleotide or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain of a CAR. In embodiments, it is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition.

[0089] The term "functional portion," when used in reference to a CAR, refers to any part or fragment of a CAR described herein, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). In reference to a nucleic acid sequence encoding the parent CAR, a nucleic acid sequence encoding a functional portion of the CAR can encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.

[0090] The immune cells of the disclosure (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, stem cells (e.g., MSCs, HPCs, HSCs, or iPS cells) can be genetically engineered to express antigen receptors such as engineered CARs. In some embodiments, the immune cells of the present disclosure are T cells. In some embodiments, T cells are engineered to express a CAR. Multiple CARs may be added to a single cell type, such as T cells. In some embodiments, the T cells of the present disclosure are CAR T cells. In some embodiments, the CAR T cells of the present disclosure are CD8+ CAR T cells. In some embodiments, the CD8+ CAR T cells of the present disclosure are derived from memory cells. In some embodiments, the CD8+ CAR T cells of the present disclosure are derived from naive cells. In some embodiments, the CD8+ CAR T cells of the present disclosure are derived from effector cells.

[0091] In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0092] In some embodiments, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen (e.g., a cancer cell, or an infected cell). In someembodiments, the antigen is a protein expressed on the surface of cells (e.g., on the surface of a cancer cell, or an infected cell).

[0093] Illustrative engineered antigen receptors, including CARs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in PCT Publication Nos. WO 2000 / 14257, WO 2013126726, WO 2012 / 129514, WO 2014 / 031687, WO 2013 / 166321, WO 2013 / 071154, and WO 2013 / 123061, U.S. Patent Application Publication Nos. US 2002 / 131960, US 2013 / 287748, and US 2013 / 0149337; and U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,190, 7,446,191, 8,324,353, and 8,479, 118; International Patent Application Publication No.: WO 2014 / 055668 Al, and European Patent Application Publication No. EP2537416; and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012, each of which is incorporated herein by reference in its entirety for examples of CARs that may be used in the compositions and methods of this disclosure.

[0094] In some aspects, the present disclosure provides a population of genetically modified immune cells (e.g. T cells) engineered to express a chimeric antigen receptor (CAR) and / or a polynucleotide encoding a CAR, wherein the CAR comprises (a) an ectodomain comprising an antigen binding domain; (b) a transmembrane domain; (c) at least one costimulatory domain; and (d) an intracellular signaling domain.

[0095] In some embodiments, the genetically engineered cells include additional CARs, including activating or stimulatory CARs, co-stimulatory CARs (see, e.g., PCT Publ. No. WO 2014 / 055668), and / or inhibitory CARs (iCARs, see, e.g., Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) recognition domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. For example, once an antigen is recognized by the ectodomain, the intracellular signaling components transmit an activation signal to the T cell that induces the T cell to destroy a targeted tumor cell.

[0096] The present disclosure provides a population of engineered T cells, wherein a plurality of the engineered T cells of the population comprise any chimeric stimulatory receptor (CAR) disclosed herein. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cell in the population comprise the CAR. In some embodiments, each CAR polypeptide is expressed at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 copies per cell. In some embodiments, the nucleic acid encoding the CAR is integrated into the genome at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 copies per cell. In some embodiments, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cells in the population comprise the CAR and an exogenous nucleic acid encoding RUNX2. In some embodiments, the exogenous polynucleotide encoding RUNX2 comprises SEQ ID NO: 1 or SEQ ID NO: 2.Target Antigens

[0097] Provided herein are immune cells (e.g., T cells) expressing an immune receptor (e.g., a CAR or engineered TCR) that targets a cancer cell. In some embodiments, the cancer cell is located in a tumor. In some embodiments, the cancer cell is not located in a tumor.

[0098] In some embodiments, the immune cells (e.g., T cells) expressing an immune receptor (e.g., a CAR or engineered TCR) target an infected cell. In some embodiments, the infected cell is an infected host cell. In some embodiments, the infected cell is an infected host immune cell.

[0099] Among the antigens that may be targeted by the genetically engineered antigen receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are aberrant or misregulated immune responses such as cancers, autoimmune disorders, diseases of immunity, and conditions characterized by chronic inflammation. Aberrant or pathological immune activation underlies diseases, such as autoimmune diseases, solid transplant rejection, transplantation graft rejection, allergy, asthma, diabetes mellitus and rheumatoid arthritis and T cell leukemia.

[0100] In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or nontargeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0101] Any suitable antigen may find use in the present method. Exemplary target antigens include, but are not limited to, antigens expressed on the surface of cancer cells or infected cells described herein.

[0102] In some embodiments, the antigen binding domain of the present disclosure binds a tumor-associated antigen. In some embodiments, the tumor associated antigen comprises CD19, CD22, CD20, CD138, BCMA, CD33, CD123, FLT, CLL, CD56, CD34, CD117, CD14, CD133, CD4+4v6, CD4+7, CD64, CD96, CD97, CD99, CD4+5, CD9, Mucl, Lewis- Y, IL1RAP, FR-beta, CD5, CD7, CD38, CD30, B7-H3, HER2, CD4+4v6, CEA, c-Met, EGFRvIII, Epcam, EphA2, FR-alpha, GD2, GPC3, IL13R-alpha2, ILl lR-alpha, Ll-CAM, mesothelin, MUC1, MUC16, NKGD2 or PSCA. In some embodiments, the tumor associated antigen is CD 19.T cell activity

[0103] In some embodiments, a population of genetically engineered T cells as disclosed herein exhibits T cell functions (e.g., effector functions). In some embodiments, the population is cytotoxic to cancer cells. In some embodiments, the population is cytotoxic to infected cells. Effector function of a genetically engineered T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. In some embodiments, the population exhibits one or more T cell effector functions at a level that is at least 2-3-fold, at least 3-4-fold, at least 4-5-fold, at least 5-10-fold, at least 10-15-fold, at least 15-20-fold, or more than 20-fold higher than the functions exhibited by a population of T cells not expressing the immune receptor.Methods of Improving Cell Therapies

[0104] In another aspect, provided herein are methods of improving a cell therapy, e.g., an adoptive cell therapy. Such methods may result in enhanced potency of a therapeutic cell. In some embodiments, a method of improving a cell therapy may result from the upregulation of RUNX2 in therapeutic cell. In some embodiments, a method of improving a cell therapy comprises a step of introducing into a therapeutic cell, an exogenous polynucleotide encoding RUNX2, wherein the exogenous polynucleotide increases the expression of RUNX2, e.g., upregulating RUNX2 in comparison to the cell prior to the introduction of the exogenous nucleic acid.

[0105] In some embodiments, a therapeutic cell comprising an exogenous polynucleotide encoding RUNX2, wherein the exogenous polynucleotide encoding RUNX2 increases the expression of RUNX2, further comprises a chimeric antigen receptor (CAR) or an engineeredT cell receptor (TCR). CARs or TCRs may be readily inserted into and expressed by immune cells, (e.g., T cells). In certain embodiments, cells (e.g., immune cells such as T cells) are obtained from a donor subject. In some embodiments, the donor subject is a human patient afflicted with cancer. In other embodiments, the donor subject is a human patient not afflicted with cancer. In some embodiments, an engineered cell is autologous to a subject. In some embodiments, an engineered cell is allogeneic to a subject. Preferably, the methods of improving therapeutic cells and therapeutic populations of cells do not substantially affect the antigen specificity of the receptor comprised by the cells. Thus, in preferred embodiments, a method of improving a therapeutic cell described herein results in an improved cell with substantially the same antigen specificity. “Substantially the same antigen specificity” means that the type of antigen recognized by the receptor is unchanged and that the affinity of a receptor for the antigen is decreased by at most 10% compared to the receptor of the cell prior to improvement.

[0106] In some embodiments, a method of improving a cell therapy described herein results from the upregulation of RUNX2 expression in a therapeutic cell relative to a therapeutic cell prior to improvement. In some embodiments, a modified immune cell exhibiting upregulated RUNX2 expression also exhibits enhanced potency relative to an immune cell comprising normal RUNX2 expression. The potency of the immune cell may be measured using any suitable method known in the art or described herein. Non-limiting examples of measuring the potency of an immune cell therapy include cytotoxicity assays, cytokine release assays, proliferation assays, in vivo efficacy studies, expression of activation markers, and tumor antigen clearance assays.

[0107] In some embodiments, a modified immune cell exhibiting upregulated RUNX2 expression also exhibits increased target cell clearance relative to an immune cell comprising normal RUNX2 expression. Target cell clearance may be measured using any suitable method known in the art or described herein. Non-limiting examples of measuring target cell clearance include cytotoxicity assays, cytokine release assays, proliferation assays, in vivo efficacy studies, expression of activation markers, and tumor antigen clearance assays.

[0108] In some embodiments, a modified immune cell exhibiting upregulated RUNX2 expression also exhibits decreased T cell exhaustion relative to an immune cell comprising normal RUNX2 expression. T cell exhaustion may be measured using any suitable method known in the art or described herein. Non-limiting examples of measuring T cell exhaustion include functional assays, analysis of phenotypic markers, and analysis of exhaustion-specific transcriptional signatures.

[0109] Some embodiments of the disclosure also provide a method of improving a cell therapy comprising introducing into a modified immune cell comprising a chimeric antigen receptor or an engineered T cell receptor, and an exogenous polynucleotide encoding RUNX2.

[0110] In some embodiments, a method of improving a cell therapy results in RUNX2 expression upregulation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3- fold, at least 4-fold, or at least 5-fold relative to the expression of RUNX2 in an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in RUNX2 expression upregulation by about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3 -fold, about 3 -fold to about 4- fold, or about 4-fold to about 5-fold relative to the expression of RUNX2 in an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in RUNX2 expression upregulation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the expression of RUNX2 in the immune cell prior to improvement. In some embodiments, a method of improving a cell therapy results in RUNX2 expression upregulation by about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3-fold, about 3-fold to about 4-fold, or about 4-fold to about 5-fold relative to the expression of RUNX2 in the immune cell prior to improvement. Expression of RUNX2 can be determined using any suitable method known in the art or described herein, including, for example, Western Blotting, ELISA, qPCR, or RNA sequencing.[OHl] In some embodiments, a method of improving a cell therapy results in an increase in potency of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4- fold, or at least 5-fold relative to the potency of an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in an increase in potency of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3-fold, about 3-fold to about 4-fold, or about 4-fold to about 5- fold relative to the potency of an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in an increase in potency of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3- fold, at least 4-fold, or at least 5-fold relative to the potency of the immune cell prior to improvement. In some embodiments, a method of improving a cell therapy results in an increase in potency of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3-fold, about 3-fold to about 4-fold, or about 4-fold to about 5-fold relative to the potency of the immune cell prior to improvement. The potency of a therapeutic cell can be determined using any suitable method known in the art or described herein, including, for example, comparing target cell clearance of the modified immune cells described herein with that of an unmodified immune cell (e.g., an immune cell not comprising the exogenous polynucleotide encoding RUNX2), or comparing cell enrichment or production of the modified immune cells described herein with that of an unmodified immune cell (e.g., an immune cell not comprising the exogenous polynucleotide encoding RUNX2). A non-limiting method of measuring target cell clearance or cell enrichment / production includes flow cytometry.

[0112] In some embodiments, a method of improving a cell therapy results in an increase in target cell clearance of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3- fold, at least 4-fold, or at least 5-fold relative to the target clearance by an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in an increase in target cell clearance potency of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3-fold, about 3-fold to about 4-fold, or about 4-fold to about 5-fold relative to the target clearance by an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy results in an increase in target cell clearance of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to the target clearance of the immune cell prior to improvement. In some embodiments, a method of improving a cell therapy results in an increase in target cell clearance potency of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 2-fold to about 3-fold, about 3-fold to about 4-fold, or about 4-fold to about 5-fold relative to the target clearance of the immune cell prior to improvement. Target cell clearance may be assessed using any suitable method known in the art or described herein, including, for example, flow cytometry, qPCR, immunohistochemistry, bioluminescence imaging (BLI), positron emission tomography (PET), cytokine release assay, or ELISA.

[0113] In some embodiments, a method of improving a cell therapy described herein results in a decrease in T cell exhaustion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least about 95% relative to the exhaustion of an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy described herein results in a decrease in T cell exhaustion of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 95% relative to the exhaustion of an immune cell not comprising the exogenous polynucleotide encoding RUNX2. In some embodiments, a method of improving a cell therapy described herein results in a decrease in T cell exhaustion of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least about 95% relative to the exhaustion of the immune cell prior to improvement. In some embodiments, a method of improving a cell therapy described herein results in a decrease in T cell exhaustion of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 95% relative to the exhaustion of the immune cell prior to improvement. T cell exhaustion may be assessed using any suitable method known in the art or described herein, including, for example, detection of exhaustion marker expression, functional assays, exhaustion gene expression profiling, metabolic profiling, longitudinal clinical monitoring, or immunohistochemistry.

[0114] In some embodiments, the cell therapy being improved by the method described herein is an FDA-approved CAR T cell therapy. In some embodiments, the cell therapy beingimproved by the method described herein is Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), Breyanzi (lisocabtagene maraleucel), Tecartus (brexucabtagene autoleucel), or Abecma (idecatagene vicleucel).Gene Delivery and Cell Modification

[0115] Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting the expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

[0116] Expression cassettes included in vectors useful in the present disclosure contain (in a 5'-to-3 ' direction) a transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, and a transcriptional termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein-encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation.Promoters and Enhancers

[0117] The expression constructs provided herein may comprise a promoter to drive the expression of the CAR or TCR and the exogenous polynucleotide encoding RUNX2. A promoter used in the context of the present disclosure includes constitutive, inducible, and tissue-specific promoters. A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. Additional promoter elements regulate the frequency of transcriptional initiation. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.

[0118] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as beta-actin promoter, GADPH promoter, metallothionein promoter; and concatenated response element promoters, such as cyclic AMP response element promoters (ere), serum response element promoter (sre), phorbol ester promoter (TP A) and response element promoters (tre) near a minimal TATA box. It is also possible to use human growth hormone promoter sequences(e.g., the human growth hormone minimal promoter described at Genbank, accession no. X05244, nucleotide 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007). In certain embodiments, the promoter is EFl, EFl alpha, MND, CMV IE, dectin- 1, dectin-2, human CD1 1c, F4 / 80, SM22, RSV, SV40, Ad MLP, betaactin, MHC class I, MHC class II promoter, U6 promoter or Hl promoter, however any other promoter that is useful to drive expression of therapeutic gene is applicable to the practice of the present disclosure.Initiation Signals and Linked Expression

[0119] A specific initiation signal also may be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0120] In certain embodiments, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap-dependent translation and begin translation at internal sites. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.

[0121] Additionally, certain 2A sequence elements could be used to create linked- or coexpression of genes in the constructs provided in the present disclosure. For example, cleavage sequences could be used to co-express genes by linking open reading frames to form a single cistron. Exemplary cleavage sequences include but are not limited to T2A, P2A, E2A and F2A. In a preferred embodiment, the cleavage sequence comprises a P2A sequence.Origins of Replication

[0122] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, a replication origin of another extra-chromosomally replicating virus as described above or an autonomously replicating sequence (ARS) can be employed.Selectable and Screenable Markers

[0123] In some embodiments, cells containing a construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0124] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers.

[0125] In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. In some embodiments, the reporter genes such as tEGFR are used. Further examples of selection and screenable markers are well known to one of skill in the art. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.

[0126] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence, transiently integrates a nucleic acid sequence, produces site-specific integration a nucleic acid sequence, or produces a biased integration of a nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0127] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence. In some embodiments, the stable chromosomal integration can be a random integration, a sitespecific integration, or a biased integration. In some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site- directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enables genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. In some embodiments, the site-specific integrationoccurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

[0128] In some embodiments, the site-specific transgene integration occurs at a site that disrupts the expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to any immunosuppressive gene, and genes involved in allo-rej ection.

[0129] In some embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0130] In some embodiments of the methods of the disclosure, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In some embodiments, enzymes create single-strand breaks. In some embodiments, enzymes create double-strand breaks. In some embodiments, examples of break-inducing enzymes include but are not limited to, transposases, integrases, endonucleases, meganucleases, megaTALs, CRISPR-Cas9, CRISPR-CasX, transcription activator-like effector nucleases (TALEN) or zinc finger nucleases (ZFN). In some embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, or as a nucleoprotein complex with a guide RNA (gRNA).Vectors

[0131] The present disclosure also envisions a polynucleotide comprising a nucleic acid sequence encoding an immune receptor and a nucleic acid sequence encoding RUNX2. In some embodiments, the nucleic acids encoding the immune receptor (e.g., a TCR or a CAR) and RUNX2 of the present disclosure are delivered to a given cell (e.g., a T cell) via a vector. In some embodiments, the vector as described herein can be a viral vector, a non-viral vector,or a transposon. In some embodiments, the vector is a viral vector. In some embodiments of the present disclosure, the nucleic acid sequence encoding an immune receptor (e.g., a TCR or a CAR) and a nucleic acid sequence encoding RUNX2 is delivered to a given cell (e.g., a T cell) via separate vectors. In some embodiments, a nucleic acid sequence encoding the immune receptor (e.g., a TCR or a CAR) as disclosed herein is encoded on a first vector, and the nucleic acid sequence encoding RUNX2 as disclosed herein is encoded on a second vector. The present disclosure also provides a viral vector comprising (i) a viral capsid comprising a viral capsid protein; (ii) a first nucleic acid sequence encapsulated within the viral capsid, wherein the first nucleic acid sequence encodes an immune receptor (e.g., a TCR or a CAR); and (iii) a second nucleic acid sequence encapsulated within the viral capsid, wherein the second nucleic acid sequence encodes RUNX2. In some embodiments, the viral vector can be an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a vaccinia viral vector, or a vesicular stomatitis viral vector. In some embodiments, the exogenous polynucleotide encoding RUNX2 comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0132] Introduction of a nucleic acid, such as DNA or RNA, into the immune cells of the current disclosure may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection); by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome-mediated transfection and receptor-mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium- mediated transformation; by desiccation / inhibition-mediated DNA uptake, and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s), or organism(s) may be stably or transiently transformed.

[0133] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the disclosure.

[0134] In some embodiments, the vectors as disclosed herein comprise at least one of (a) a sequence encoding TUNX2; (b) a polycistronic element; (c) a reporter gene; (d) a sequence encoding an immune receptor; or (e) any combination thereof. In some embodiments, the sequence encoding RUNX2 comprises the sequence of SEQ ID NOs: 1 or 2. In some embodiments, the polycistronic element comprises the sequence of any one of SEQ ID NOs: 3-9. In some embodiments, the reporter gene comprises the sequence of any one of SEQ ID NOs: 10-13. In some embodiments, the sequence encoding the immune receptor sequence comprises a sequence encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) targeting at least one tumor-associated antigen.

[0135] In some embodiments, the vectors as disclosed herein comprise at least one of the sequences as outlined in Table 1 or any combination thereof.Table 1: Expression Vector ElementsPharmaceutical Compositions

[0136] Also provided herein are pharmaceutical compositions and formulations comprising the modified immune cells as described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a population of cells, wherein a plurality of the cells of the population comprises the modified immune cell as described herein. In some embodiments, the composition comprises a population of cells, wherein a plurality of cells of the population comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between of the modified immune cell as disclosed herein.

[0137] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required, e.g., by the FDA Office of Biological Standards.

[0138] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22ndedition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non- ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos.2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.Methods of Treatment

[0139] In some embodiments, the present disclosure provides methods for immunotherapy comprising administering an effective amount of the immune cells (e.g. modified T cells) of the present disclosure. A therapeutically effective amount of immune cells for use in adoptive cell therapy is an amount that achieves a desired effect in a subject being treated. For instance, this can be the amount of immune cells necessary to inhibit advancement, or to cause regression of a cancer, or which is capable of relieving symptoms caused by cancer, such as pain and inflammation. It can be the amount necessary to relieve symptoms associated with inflammation, such as pain, edema, and elevated temperature.

[0140] In some embodiments, the method of treatment results in a beneficial change in the tumor microenvironment. Examples of beneficial changes in the tumor environment are known in the art and include and is not limited to, for example, decreases in hypoxia and decreases in vascularization. See, e.g., Benavente et al., Front. Oncol., 23 October 2020, which is incorporated herein by reference in its entirety.

[0141] The cells and pharmaceutical compositions described herein may be used to treat a disorder or disease in a subject in need thereof. Thus, in another aspect, provided herein is a method of treating cancer in a subject, comprising administering to the subject a modified immune cell comprising a chimeric antigen receptor or an engineered T cell receptor and an exogenous polynucleotide encoding RUNX2. In some embodiments, the exogenous polynucleotide encoding RUNX2 comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0142] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. A “hematological tumor” also refers to a “liquid tumor”. In some embodiments, the cancer is a CD22-positive cancer. In some embodiments, the cancer has a low expression of CD22 (e.g., a CD22 low cancer). In some embodiments, the cancer is a CD 19-positive cancer. In some embodiments, the cancer has a low expression of CD 19 (e.g., a CD 19 low cancer).

[0143] Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include but are not limited to tumors of the bone marrow, T or B cell malignancies, myeloid malignancies, leukemias, lymphomas, blastomas, and myelomas. Further examples of cancers that may be treated using the methods provided herein include but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0144] 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; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; 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; T lymphoblastic leukemia; T lymphoblastic lymphoma; B cell leukaemia; Hodgkin's disease; Hodgkin’s lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; B cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; 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; hairy cell leukemia; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia, acute lymphoblastic leukemia (ALL); acute lymphoblastic lymphoma; acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasms; chronic myeloblasts leukemia; diffuse large B cell lymphoma (DLBCL); peripheral T cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL). In some embodiments, the cancer comprises a liquid tumor. In some embodiments, the liquid tumor is a leukemia or a lymphoma. In some embodiments, the leukemia or lymphoma is B cell leukemia or B cell lymphoma.

[0145] Particular embodiments concern methods of treatment of leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually immature white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms and / or by the cell type of origin (myeloid or lymphoid). In some embodiments, the leukemia is an antigen-low leukemia. In some embodiments, the leukemia is a CD22-low leukemia.

[0146] In some embodiments, the cancer is breast cancer, sarcoma, melanoma, or lung cancer.

[0147] The terms “subject” and “patient” are used interchangeably herein. In some embodiments, the subject treated in accordance with the methods described herein is a human patient, e.g., a human adult.

[0148] A person of skill in the art will be able to determine the appropriate duration and dose of treatment for a cell comprising a CAR or TCR and an exogenous polynucleotide encoding RUNX2 provided herein and / or for a pharmaceutical composition provided herein.

[0149] The cell comprising a CAR or TCR and an exogenous polynucleotide encoding RUNX2 provided herein or the pharmaceutical composition provided herein may be administered by any suitable route of administration, including, for example, intravenous, intrathecal, intraocular, subcutaneous, intraperitoneal, intramuscular, intracerebral, intraventricular, or intratracheal administration. In preferred embodiments, the cell comprising a CAR or TCR and an exogenous polynucleotide encoding RUNX2 is provided herein, or the pharmaceutical composition provided herein is administered intravenously.

[0150] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, such as an individual who has cancer or an infection. The cells then enhance the individual's immune system to attack or directly attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the immune cells. In cases where the individual is provided with two or more doses of the immune cells, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more weeks.

[0151] The cell comprising a CAR or TCR and an exogenous polynucleotide encoding RUNX2 provided herein, or the pharmaceutical composition provided herein may be administered for any suitable duration, for example, until symptoms improve, or for apredetermined duration such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks.

[0152] The efficacy of a treatment for cancer may be assessed by any suitable method known in the art or described herein, including, for example, by monitoring tumor growth or measuring the time to tumor recurrence. In some embodiments, a method of treatment described herein induces tumor regression. In some embodiments, the tumor regresses by at least 10%, by at least 20%, by at least 30% within about one week, about 2 weeks, about one month, about 2 months, about 3 months, about 6 months, about 9 months, or about 12 months after the first administration of the bispecific binding agent or functional fragment thereof or of the pharmaceutical composition.

[0153] In some embodiments, a method of treatment described herein results in regression of the tumor to undetectability and delays tumor recurrence. In some embodiments, the tumor recurrence is delayed by at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least 18 months, at least 24 months, at least 3 years, at least 4 years or at least 5 years after the tumor becomes undetectable.

[0154] The efficacy of a method of treatment described herein may be assessed in comparison to an untreated subject having a comparable diagnosis or to a subject having a comparable diagnosis who is receiving standard-of-care therapy. In some embodiments, the efficacy of a method described herein is compared to the subject treated in accordance with a method described herein prior to the first administration.Combination Therapies

[0155] In some embodiments, the compositions and methods of the present embodiments involve an immune cell population in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0156] The immune cells may be administered in combination with one or more other therapeutic agents for the treatment of the immune-mediated disorder. Combination therapies can include, but are not limited to, one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine),immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokine antagonists (for example, anti-TNF agents such as infliximab, adalimumab, golimumab, natalizumab, anti-IL- 6 such as tocilizumab and sarilizumab, anti- 12 / 23 such as ustekinumab), cytokines (for example, interleukin- 10 or transforming growth factor-beta), anti -trafficking agents (for example, anti-integrins such as vedolizumab and SIP inhibitors such as ozanimod, etrasimod, and fmgolomid), hormones (for example, estrogen), or a vaccine. In addition, immunosuppressive or tolerogenic agents including but not limited to anti-thymocyte globulin, calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., rapamycin, sirolimus); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4+, CD4+0, CD 154, CD4+5, IVIG, or B cells); chemotherapeutic agents (e.g., fhidarabine, cyclophosphamide, bendamustine, cytarabine, doxorubicin, methotrexate, prednisone, dexamethasone, etoposide, mercaptopurine, nelarabine, or clofarabine); irradiation; tyrosine kinase inhibitors (e.g., imatinib, dasatinib, ibrutinib, or idelalisib); monoclonal antibodies (e.g., rituximab, Obinutuzumab, alemtuzumab, ofatumumab, blinatumomab); BCL2 inhibitors (e.g., venetoclax); Pi3K inhibitors (e.g., duvelisib); allogenic or autologous stem cell transplant; checkpoint inhibitors (e.g., pembrolizumab, nivolumab, oratezolizumab); or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceutical agents can be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the cells and the agent can be by the same route or by different routes, and either at the same site or at a different site.

[0157] The immune cells may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as radiation therapy, chemotherapy, or immune therapy (e.g., immune checkpoint therapy). The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the immune cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. Insome situations, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0158] Various combinations may be employed. For the example below an immune cell therapy is "A" and an anti-cancer therapy is "B":

[0159] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B.

[0160] B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A.

[0161] B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A.Dosage Regimens

[0162] In one embodiment, the immune cells (e.g., T cells) are modified by engineering / introducing immune receptors and exogenous polynucleotide encoding RUNX2 into said immune effector cells and then infused into a subject. In some embodiments, immune cells are modified by engineering / introducing a chimeric receptor and an exogenous polynucleotide encoding RUNX2 into the immune naive cells and then infused within about 0 days, within about 1 day, within about 2 days, within about 3 days, within about 4 days, within about 5 days, within about 6 days or within about 7 days into a subject.

[0163] In one embodiment, the modified immune cells are targeted to the cancer cells via regional delivery directly to the tumor tissue. For example, in ovarian or renal cancer, the modified immune cells can be delivered intraperitoneally (IP) to the abdomen or peritoneal cavity. Such IP delivery can be performed via a port or pre-existing port placed for delivery of chemotherapy drugs. Other methods of regional delivery of modified immune cells can include catheter infusion into a resection cavity, ultrasound-guided intra-tumoral injection, hepatic artery infusion or intrapleural delivery. As another example, in leukemia, the modified immune cells can be delivered intravenously directly into the bloodstream.

[0164] In one embodiment, a subject in need thereof can begin therapy with a first dose of modified immune cells delivered via IV followed by a second dose of modified immune naive cells delivered via IV. In one embodiment, a subject in need thereof can begin therapy with a first dose of modified immune cells delivered via IP followed by a second dose of modified immune cells delivered via IV. In a further embodiment, the second dose of modified immune cells can be followed by subsequent doses which can be delivered via IV or IP.Articles of Manufacture or Kits

[0165] An article of manufacture or a kit comprising immune cells is also provided herein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual having cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or poly olefin), or a metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation, and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent. Suitable containers for the one or more agents include, for example, bottles, vials, bags and syringes.EXAMPLESExample 1: T cell antigen experience prior to transduction with a CAR directs in vitro proliferative and effector capacities of CD8+ CAR T cells

[0166] Anti-murine-CD19 CAR T cells containing CD28 and CD3zeta (Wang, X. et al. (2011) Blood (118); 1255-1263) were generated (FIG. 8A). CAR expression correlated strongly with EGFR expression, allowing for use of EGFR as a marker for long term tracking of CAR+ cells (FIG. 8B). CAR T cells were used to target murine leukemia driven by the E2A-PBX1 fusion protein (E2A-PBX) (Qin, H. et al. (2018) Blood (132) 1899-1910, Jacoby, E. et al. (2016) Nat Commun (7) 12320, Yang, Y. et al. (2017) Sci Transl Med (9)) modified to differing CD 19 densities (FIGs. 1A, 8C). Memory OT-I T cells generated using a well characterized ovalbumin vaccination model (Ivanova, D.L. et al. (2023) Cell Rep (42), 112911, Klarquist, J. et al. (2021) Cell Rep (36); 109591, Klarquist, J. et al. (2018) Sci Immunol (3)) (FIG. IB) were used to produce memory-derived CD8+ CAR T cells (CAR8MD) for comparison to naive-derived CD8+ OT-I CAR T cells (CAR8ND). AS no difference was seen between memory or naive-derived CAR-ZEGFR+ T cells (FIG. 8D), naive-derived were used as a control (EGFR8) were used in subsequent experiments.CAR8ND expanded more during manufacturing (FIG. 8E). Following CAR stimulation, a greater proportion of CAR8MD cells produced both TNFa and IFNy , and despite no differences in degranulation (as measured by CD 107a), CAR8MD were also more cytotoxic, most pronounced in response to low target antigen (FIGs. 1C-1H; 8E-8H). The proportion of IFNy+ cells was greater in CAR8MD and the proportion of TNFa+ cells was slightly increased in CAR8ND (FIGS. IE & IF). CAR8ND outperformed CAR8MD in cell cycle entry (lower % Ki67 expression) and extended proliferative capacity across antigen densities (FIGs. 1 J-1M, 8I-8J)

[0167] To compare polyclonal antigen-experienced and naive T cells more analogous to human CAR T cells, pathogen-elicited polyclonal T cells were generated by infecting WT C57BL / 6 mice with the common acute viral infection model LCMV- Arm strong. Memory (CD8+ / CD44+ / CD49dHi) and naive (CD8+ / CD44- / CD49dLo / CD62L+) T cell populations were FACS-sorted from the same mice 28 days after LCMV infection and used for CAR T cell manufacturing (FIG. 9A). LCMV-CAR8MD demonstrated superior effector function and CAR8ND demonstrated superior proliferative capacity (FIGs. 9B-9G).Example 2: Treatment of leukemia-bearing mice with a high CAR+ cell dose reveals enhanced cytotoxic profile and clearance of antigen-low leukemia by memory-derived CAR8

[0168] Mice were engrafted with WT (35,000 antigens per cell) or CD19LO (10,000 antigens per cell) leukemia followed 3 days later by a dose of le6 CAR T cells (FIG. 2A). The CD19LO clone was chosen based on the differential in vitro responses (FIGs. 1A-1M). Ragl- deficient hosts enabled CAR T cell expansion without irradiation and limited CAR T cell exposure to CD 19 density on leukemia rather than that on endogenous B cells. There was no difference in CAR T cells in the marrow at peak expansion (day 4) but CAR8ND had increased CAR T cells in mice bearing WT or CD19LO leukemia post-contraction (day 11; FIGs. 2B-2C, 10A-10B). Despite no significant difference in CD19LO leukemia at day 11, 4 / 10 mice treated by CAR8ND had detectable leukemia at >15% of live bone marrow cells while all 10 mice treated with CAR8MD had minimal leukemic burdens (<5%) (FIG. 2D).

[0169] Upon ex vivo restimulation of CAR8 in the bone marrow, it was found that while IFNy production was variable, GZMB production was markedly greater in CAR8MD (FIGS. 2E-2F). CAR8MD had significantly higher proportions of cells bearing phenotypes of shortlived effector cell (SLEC, IL7Ra- / KLRGl+) and effector memory precursor (EMP, CD27+ / CD62L-), fewer cells in the central memory precursor phenotype (CMP,CD27+ / CD62L+), and no change in memory precursor effector cell (MPEC, IL7Ra+ / KLRGl-) populations (FIGs. 11A-11E). Additionally, early expression of effector- associated transcription factors (IRF4, T-bet and EOMES) was greater in CAR8MD (FIGS. 2G-2I). Mice bearing WT high-antigen leukemia showed no survival difference after treatment with CAR8MD versus CAR8ND. Mice bearing CD19LO leukemia treated with CAR8MD showed a significant survival benefit, with 20% of mice surviving (FIG. 2J).Example 3: Treatment of leukemia-bearing mice with a low CAR+ cell dose reveals enhanced proliferative capacity and clearance of WT leukemia by naive-derived CAR8

[0170] CAR8ND expanded to significantly higher numbers in the bone marrow by day 4 regardless of leukemia antigen density, mirroring in vitro proliferative assays (FIGs. 3A-3B, 10C, 11-1 J). CAR8ND mediated enhanced clearance and survival in mice bearing WT leukemia (FIGs. 3C, 31, 10D) Ex vivo IFNy production was greater in CAR8MD. There was no difference in GZMB production or expression of IRF4, T-bet or EOMES (FIGs. 3D-3H). CAR8MD consistently demonstrated significantly higher proportions of SLECs at day 4, but no difference at day 11, and no differences were seen in the MPEC population (FIGs. 12A- 12B) While EMP and CMP patterns mimicked high-dose experiments, the differences were much less pronounced, indicating that CAR8ND cells became more “effector-like” upon greater proliferative drive (FIGs. 12C-12D). However, these effects did not result in survival benefit against CD19LO leukemia (FIG. 31). CAR8MD expressed higher levels of exhaustion- associated markers against WT leukemia with failure of CAR8MD to control leukemia (FIGs. 12E-12F, 12I-12J) It was found that CD19LO leukemia drove similar exhaustion phenotypes in both CAR8 populations, demonstrating that even chronic antigen exposure to low density antigens, can drive dysfunction (FIGs. 12G-12H, 12K-12L).Example 4: Epigenetic profiling of naive and memory-derived CAR8 shows differential chromatin accessibility at binding sites for bZIP, Tcf, Runx, and other TF families

[0171] Bulk ATAC-seq was performed on naive and memory-derived cells at three time points: ex vivo prior to CAR transduction (Day - 5, “PreCAR”), in vitro after CAR transduction (Day 0, “PostCAR”), and after reinfusion into mice bearing CD19LO leukemia (Day 4, “Tumor”) (FIG. 4A). Experimental replicates showed tight concordance of chromatin accessibility at each condition and timepoint (FIG. 13A). Broadly, the data showed several thousand differentially accessible regions between either cell type compared to itself across timepoints, and between naive and memory-derived cells at each timepoint(FIG. 13B). Predictably, there was higher chromatin accessibility in CAR8MD at effector / activation loci (Gzmb, Gzmc, Pdcdl) and in CAR8ND was found at the Tcf7 loci (FIG. 4B). ChromVAR22was used to associate these changes in chromatin accessibility to previously defined datasets and potential transcription factor (TF) activities.

[0172] Comparison to LCMV-Armstrong-elicited T cells (Scott-Browne, J. P. et al. (2016) Immunity (45) 1327-1340) showed that memory-derived CAR8 acquired effector-associated changes in chromatin accessibility during CAR generation were maintained after transfer into tumor-bearing mice and had reduced memory-associated chromatin accessibility. Conversely, naive-derived cells maintained chromatin accessibility at regions associated with memory T cells and showed minimal skewing toward an effector-like profile (FIG. 4C; Scott-Browne, J. P. et al. (2016) Immunity (45) 1327-1340). ChromVAR was used to compare chromatin accessibility at regions containing DNA sequence motifs bound by different TFs (FIG. 4D). Distinct patterns of motif-associated chromatin accessibility between conditions and across timepoints were found (FIG. 4E). bZIP and Irf-family TF motifs became progressively enriched in memory cells, Tcf family motifs became enriched in naive cells, while E2A family motifs progressively converged. Motifs for Runx family members were always more accessible in memory-derived cells (FIGs. 4D-4E, 13C).Example 5: Prior antigen experience directs distinct transcriptomic patterns of naive and memory derived CAR8

[0173] Bulk RNA-seq was performed at the same time points as for ATAC-seq (FIG. 4A). Genes associated with self-renewal (Lefl, Sell, Id3, Tc , U7r) were upregulated in the naive- derived cells and effector / activation genes (Prfl, IFN , Gzmb, Id2, Pdcdl, Tbx21) were upregulated in the memory-derived cells (FIG. 5A). Gene set enrichment analysis (GSEA) showed progressive bias toward more effector-like (less memory -like) in memory-derived CAR8 cells, and toward more memory-like (less naive-like) in naive-derived CAR8 cells (FIGs. 5B-5C, 14A)

[0174] PreCAR top differentially expressed genes and Ingenuity Pathway Analysis (IP A) demonstrated many expected differences including upregulation of Bhlhe40, Klf4, Tbx21, Id2 and bZIP family members (Jun, JunB, Fos, Cebpb) in the memory cells, while Zebl, Myb and Lefl, encoding TFs associated with self-renewal, were upregulated in the naive-derived cells (FIGs. 14B-F, 5D). Notably, among the Runx family, which showed uniquely stable differential motif accessibility (FIG. 4D), Runx2 was among the most differentially expressed TF genes with marked overexpression in memory derived cells (FIG. 14D).ChromVAR-implicated TFs showed high initial expression in memory cells at the PreCAR timepoint, followed by a convergence in expression at the PostCAR and Tumor timepoints, as seen with bZIP family members Jun, Fos and Atf3, along with the gene Tbx21, encoding canonical effector TF T-bet (FIG. 5D). Among the Runx family, Runxl and Runx3 gene expression tracked relatively closely between memory and naive-derived cells at each timepoint (FIG. 5E).Example 6: Overexpression of bZIP family members BATF or c-JUN enhances leukemia clearance by naive-derived CAR T cells while allowing progression into phenotypic exhaustion

[0175] The bZIP -family showed the greatest differences between memory-derived and naive- derived T cells in motif-associated chromatin accessibility, indicating importance of this family in T cell function. Overexpression of BATF and c-JUN enhanced proportions of memory and naive-derived CAR T cells in the marrow at a sub-curative CAR dose (le5) against which both, CAR8ND and CAR8MD exhibit markers of exhaustion and fail to control leukemia (FIGs. 15A-15B, 16C-16D, 16G-16H), although neither TF impacted in vitro function (FIGs. 15C-15E). There was no difference between BATF-CAR8 or cJUN-CAR8 and control CAR8 in leukemia clearance by CAR8MD, while both TFs enhanced clearance by CAR8ND (FIGS. 16C-16D, 16G-16H). While BATF slightly reduced PD1+ and PD1+ / CD39+ CAR8ND, transcription factor signatures of exhaustion (PD1+ / TOX+, TCF1+ / TIM3-, TCF1- / TIM3+) were unchanged, and both overexpressed TFs decreased progenitor-exhausted CAR8MD (TCF1+ / TIM3-) (FIGs. 16E-16F, 16I-16N).Example 7: RUNX2 overexpression boosts leukemia clearance, CAR T cell potency and CAR proportions in bone marrow and redirects cells from phenotypic exhaustion

[0176] Co-transduction of naive CD8+ T cells with CAR-EGFR and murine RUNX2-GFP (in tandem with pMSCV-IRES-eGFP (pMIG)) resulted in a large proportion of cells expressing both EGFR and GFP and increased RUNX2 expression by intracellular staining (FIGs. 6A-6B). While RUNX2-CAR8 functioned similarly in vitro relative to pMIG-CAR8 for both CAR8MD and CAR8ND (FIGS. 17A-17C), RUNX2 overexpression in CAR8ND strongly enhanced leukemia clearance and increased CAR proportions and counts in the marrow at 11 days post-CAR infusion (FIGs. 6C-6D, 17D-17E).

[0177] While there was no difference in the PD1+ proportion. Mice treated with RUNX2- CAR8ND (but not RUNX2-CAR8MD) exhibited reduced proportion of PD1+ / TOX+ cells, alower proportion of PD1+ / CD39+ cells and TCF1- / TIM3+ cells, suggesting that RUNX2 overexpression counteracts the differentiation trajectory toward terminal exhaustion (FIGs. 6E-F, 17F-K). RUNX2-overexpression resulted in a significant reduction in the PD1+ / CD39+ exhaustion phenotype of RUNX2-CAR8MD and reduction in leukemia counts in marrow (FIG. 161) but no difference in other metrics (FIGs. 6E, 17D).Example 8: Overexpression enhances in vitro function of human CD4+ and CD8+ CAR T cells

[0178] The impact of RUNX2 overexpression in bulk human CD4+ / CD8+ CAR T cells was tested. pLenti-RUNX2-IRES-EGFR construct was generated and co-transduced with a human anti-CD19-28z CAR (hCAR) (FIGs. 7A-7B, 170). Despite lower RUNX2 overexpression compared to murine data, across 3 independent human donors RUNX2-overexpression increased proportions of CD8+ cells producing IL-2 and INFg, while CD4+ cells showed increases in CD107a+, ZFNy+, and IL-2+ cells (FIGs. 7C-7D, 17P-17Q). RUNX2- overexpression slightly enhanced proliferation of CD4+ CAR T cells (FIGs. 7E-7F) and bulk RUNX2-hCAR showed >30% increase in cytotoxicity in 2 of 3 donors against both WT and CD19LO leukemia (FIG. 7G). A multiplex ELISA was performed to more broadly characterize RUNX2-hCAR and it was found that RUNX2 overexpression significantly enhanced secretion of effector molecules (GzmA, GzmB, Perforin, and Granulysin), while trending toward a Th 1 -like profile (increased IFNy , decreased IL-4, IL- 17 A) and decreased IL-6 (FIGs. 7H-7Q)Methods for Examples 1-8Mouse Strains

[0179] B6.129S6-Rag2tmlFwa Tg(TcraTcrb)l lOOMjb (“OT-I,” Model #: 2334-F) mice were obtained from Taconic Biosciences. B6.SJL-Ptprca Pepcb / BoyJ (“PepBoy,” Strain #:002014), B6.129S7-RagltmlMom / J (“ U / ",” Strain #:002216), C57BL / 6J mice (“B6,” Strain #:000664) were obtained. Female mice were used for all experiments with B6 background mice. All mice were bred and / or maintained in the animal facility at the University of Colorado Anschutz Medical Campus. All experiments were performed in compliance with the study protocol approved by University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC).Mouse DNA Constructs

[0180] The murine anti-CD19 scFv was Flag-tagged to enable CAR detection, and all IT AMs in the CD3zeta domain were kept intact. A truncated human EGFR reporter protein was incorporated following a 2A skip sequence to provide an additional method for detection of CAR-transduced cells. The DNA was codon optimized, ordered from ThermoFisher GeneArt, and cloned into the MSCV-IRES-GFP backbone (Addgene plasmid # 20672), using Xhol and Clal enzyme sites. A control plasmid with just the truncated EGFR reporter in the MSCV backbone was generated using similar methods.Human DNA Constructs

[0181] Human RUNX2-IRES-hEGFRt DNA was codon optimized and cloned into a lentiviral backbone using Xhol and BamHI restriction sites. A control plasmid with just the truncated human EGFR reporter was PCR amplified out of the MSCV construct and cloned into the lentiviral backbone.Cell Lines and Media

[0182] Murine T cells and leukemia were cultured in Complete Mouse Media (CMM), consisting of RPMI 1640 medium (Gibco) with 10% heat-inactivated fetal calf serum (Omega Bio), 1% nonessential amino acids (Gibco), 1% sodium pyruvate (Gibco), 1% penicillin / streptomycin (Gibco), 1% L-glutamine (Gibco), 1% HEPES buffer (Gibco) and 50uM 2-mercaptoethanol (Sigma-Aldrich). NALM GFP / Luciferace human B-ALL was cultured in Complete RPMI Media (cRPMI), consisting of RPMI 1640 medium (Gibco) with 10% heat-inactivated fetal calf serum (Omega Bio), 1% penicillin / streptomycin (Gibco) and 1% L-glutamine (Gibco). All cell lines were routinely tested for mycoplasma (at least on an annual basis). Human T cells were cultured in Human T cell Expansion Media (hTCEM), consisting of AIM-V medium (Gibco) with 5% heat inactivated fetal calf serum (Omega Bio), 1% L-glutamine (Gibco), and 1.5% HEPES buffer. Platinum E cells were purchased from Cell Biolabs and grown to create frozen stocks in Plat E Media containing DMEM, 10% heat-inactivated fetal calf serum (Omega Bio), 1% penicillin / streptomycin (Gibco), 1% L- glutamine (Gibco) with Ipg / mL puromycin and lOpg / mL blasticidin for selection. Transfections were performed without pen / strep or selection antibiotics in media. Lenti X cells were purchased from Takara and grown to create frozen stocks in Lenti X media containing DMEM, 10% heat-inactivated fetal calf serum (Omega Bio), 1% penicillin / streptomycin (Gibco), 1% L-glutamine (Gibco), and 1.5% HEPES buffer.Generation of retrovirus and lentivirus

[0183] For retrovirus, 1 le6 Platinum E cells (Cell Biolabs) were plated in a T-75 flask and allowed to adhere overnight. The following day, media was changed to fresh Plat E media and transfected with transfer plasmid DNA and lipofectamine 3000 and p3000 reagents in basal OptiMEM media (Gibco) according to the manufacturer’s protocol. Supernatant containing virus was collected 48 hours later and spun at 2000g to remove cellular particulate.

[0184] For lentivirus, 10e6 Lenti X cells (Takara) were plated in a T-75 flask and allowed to adhere overnight. The following day, media was changed to fresh OptiMEM+ media containing 5% heat-inactivated fetal calf serum (Omega Bio), 1% L-glutamine (Gibco), and 1% sodium pyruvate (Gibco). Cells were transfected with transfer plasmid DNA, plasmids encoding packing and envelope vectors (pMDLg / pRRE, pMD.2G, pRSV-Rev), and lipofectamine 3000 and p3000 reagents in basal OptiMEM media (Gibco) according to the manufacturer’s protocol. After 5 hours, media was replaced with 16mL fresh OptiMEM+. Supernatant containing virus was collected 48 hours later and spun at 2000g to remove cellular particulate, then spun for 90 minutes at 20,000g to concentrate.Mouse CAR Transduction

[0185] Briefly, spleens from 6-10 week-old donor mice were harvested and CD8+ T cells were isolated using EasySep Mouse CD8+ T cell Isolation Kit from STEMCell Technologies or bulk T cells were isolated using the Mouse CD3+ T Cell Enrichment Column Kit (R&D Biosciences, Cat No. MTCC-25). On day 1, T cells were activated on anti-CD3 / anti-CD28 Mouse T cell Activator DynaBeads (Invitrogen) at a 1 : 1 celkbead ratio and cultured at le6 / mL in CMM in the presence of rhIL-2 (40IU / mL) and rhIL-7(10ng / mL) from R&D Systems. On days 2 and 3, retroviral supernatant was added to Retronectin-coated (Takara Biosciences) 6 well plates and spun at 2000xg and 32°C for 2-3 hours. The supernatant was then removed and activated T cells were added to the wells at 1.67mL / well. On day 4, beads were removed and T cells were resuspended at le6 / mL in fresh media with cytokines. CAR transduction was determined post-de-beading by analyzing T cells by flow cytometry for a FLAGZEGFR double-positive population (or EGFR single positive for control T cells), and T cells were used in assays or infused into mice on day 5 or 6.Human CAR Transduction

[0186] Human PBMCs were isolated from Leukocyte Reduction System (LRS) Chambers from healthy blood donors at Children’s Hospital Colorado. Bulk T cells were isolated using EasySep™ Human T Cell Enrichment Kit from STEMCell Technologies and cryopreserved. On day 1, T cells were thawed and activated on Dynabeads Human T-Expander CD3 / CD28 (Gibco) at a 1 :3 celkbead ratio and cultured at le6 / mL in hTCEM in the presence of rhIL-2 (50IU / mL) from R&D Systems. On days 3 and 4, T cells were co-transduced with lentivirus encoding CAR and RUNX2, or CAR and EGFR, and Mock T cells were left un-transduced and given fresh media with cytokines. For transductions, le6 T cells were added to Retronectin-coated (Takara Biosciences) 24 well plates, along with concentrated lentiviral supernatant and LentiBOOST (SIRION Biotech) and plates were spun at 2000g and 32°C for 2-3 hours. On day 5, beads and supernatant were removed and cells were replated at le6 cells / mL in hTCEM with IL-2 (300IU / mL). On day 7, cells were replated at le6 cells / mL in hTCEM with IL-2 (300IU / mL), rhIL-7(10ng / mL, R&D Systems), and rhIL-15 (lOng / mL, STEMCell), and CAR and RUNX2 transduction were determined by flow cytometry. Cells were used for assays on day 8 post-transduction.Vaccine Model

[0187] The ovalbumin vaccine consists of lOOug whole ovalbumin protein (InvivoGen, Cat. code: vac-pova-100), 40ug anti -mouse CD40 (BioXCell, Catalog #BE0016-2) and 40ug Polyinosinic:polycytidylic acid [Poly (I: C)] (InvivoGen, Cat. code: tlrl-pic-5) per mouse, resuspended to 200uL total volume in PBS. CD8+ T cells were isolated from naive 6- to 8- week-old OT-I mouse splenocytes using the Mouse CD3+ T Cell Enrichment Column Kit (R&D Biosciences, Cat No. MTCC-25). PepBoy mice were given 5e3 OT-I T cells retro- orbitally and concurrently vaccinated intravenously. 3-4 weeks later, spleens from 5-20 vaccinated mice were pooled and CD45.2+ OT-I memory T cells were isolated using the EasySep Mouse CD8+ T cell Isolation Kit, followed by column isolation using biotinylated anti-mouse CD45.2 (BioLegend, Cat # 109804), LS Columns (Miltenyi Biotec, Order No. 130-042-401), and anti-Biotin MicroBeads (Miltenyi Biotec, Order No. 130-090-485). Naive T cells from 1-5 naive OT-I donors were isolated in parallel. T cells were then activated and transduced as described for downstream experiments.Generation of CD10T,OE2A-PBX and NALM6 Leukemia Cell Lines

[0188] CD 19 knockout leukemia was produced using CRISPR / Cas9. A previously validated murine CD19-targeting sgRNA from Integrated DNA Technologies was incubated with recombinant Cas9 from TakaraBio (Cat# 632641) to create an RNP complex. RNP was then electroporated into E2A-PBX using the Lonza 4D-Nucleofector X with nucleofector solution SG and pulse program CM-147. Electroporated cells were allowed to recover for 48 hours and then FACS-sorted twice to obtain a pure CD19 knockout cell line. This cell population was additionally single-cell cloned to create a CD 19 knockout single-cell clone prior to transduction with murine CD 19. A truncated / non-signaling murine CD 19 was cloned into the pLV.SP146.gp91.GP91.cHS4 plasmid (Addgene plasmid # 30480). Backbones were generated with the hEFla promoter (pLV.hEFla.cHS4) or the hUbC promoter (pLV.hUbC.cHS4) from the pLenti6 / UbC / mSlc7al plasmid (Addgene plasmid # 17224). VSV-G pseudotyped lentivirus was generated as described and E2A-PBX CD19KO underwent a single round of transduction using standard protocols, followed by single cell cloning to obtain clonally derived lines expressing defined levels of CD 19 target antigen.

[0189] NALM6 CD 19 knockout cells were generated using CRISPR / Cas9. CD19Lo-2,200 variant was generated using similar methods as described for the murine leukemia, with a truncated / non-signaling human CD 19 ordered from Twist Bioscience cloned into the pLV.SP146.gp91.GP91.cHS4 plasmid with hUbC promoter. Approximate mouse or human CD 19 antigen density on all cell lines were quantified using BD Quantibrite PE Fluorescnece Quantitation Kit, following the manufacturer’s protocol (BD Biosciences).Flow Cytometry

[0190] Flow cytometry analysis was performed using an LSR-Fortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences). Intracellular flow cytometry staining was performed using the TrueNuclear Transcription Factor Buffer Set (BioLegend) for ex vivo staining of transcription factors, Cytofix / Cytoperm Fixation / Permeablization Kit (BD Biosciences) for intracellular cytokine staining, and Mouse Foxp3 Buffer Set (BD Biosciences) for intracellular staining of Ki67 and Runx2.Murine CD107a Degranulation., Intracellular Cytokine Staining (ICCS), Ki67, and CellTrace Dilution in vitro Assays

[0191] In vitro assays were performed using a 1 : 1 effector to target cell ratio with le5 of each cell type in a 96-well round-bottom plate followed by analysis by flow cytometry at theindicated timepoints. Degranulation assays were performed by incubation for 4 hours in the presence of 2uM monensin and luL of CD 107a antibody. ICCS was performed by incubation for 6 hours, with luM monensin and 2.5uM Brefeldin A added at 1 hour in. Ki67 was performed by incubation for 18 hours, followed by intracellular staining for Ki67. CellTrace dilution assays were performed by staining T cells with CellTrace Violet (Thermo Fisher Scientific) per manufacturer protocols followed by incubation with target cells for 72 hours.Human CD107a Degranulation., Intracellular Cytokine Staining (ICCS), LEGENDPlex, and CellTrace Dilution in vitro Assays

[0192] In vitro assays were performed using a 1 : 1 effector to target cell ratio with le5 of each cell type in a 96-well round-bottom plate followed by analysis by flow cytometry at the indicated timepoints. Combined degranulation / ICCS assays were performed by incubation for 5.5 hours in the presence of 2uM monensin and luL of CD 107a antibody. CellTrace dilution assays were performed by staining T cells with CellTrace Violet (Thermo Fisher Scientific) per manufacturer protocols followed by incubation with target cells for 72 hours. Multiplexed ELISA using the LEGENDplex Hu CD8 / NK Panel V02 (BioLegend) was performed on supernatant collected after 16 hours co-culture. All human in vitro assays were performed in cRPMI Media.Murine and Human Killing Assays

[0193] In vitro killing assays were quantified from ICCS datasets as described above, with 1 : 1 effector to target cell ration with le5 of each cell type in a 96-well round bottom plate followed by analysis by flow cytometry at a 6 hour timepoint. Live leukemia proportions at 0 hours (Lo) were calculated by Lo= 100-9991 / TE) / (l+(l / TE)))*100) where TE represents transduction efficiency of CAR, with live leukemia and live T cell proportions quantified by Trypan Blue staining. Percent cytotoxicity (PC) for each replicate were calculated by PC = lOO*(l-(Le / Lo)) where Le represents live leukemia proportions at 6 hours, gated using viability dye.LCMV Infection and T Cell Isolation

[0194] 6 -week-old female C57BL / 6 mice were injected retro-orbitally with 2e5 PFU of LCMV-Armstrong. 4 weeks later, CD8+ T cells were isolated from 5 pooled spleens using the EasySep Mouse CD8+ T cell Isolation Kit from STEMCell Technologies and then FACS- sorted to obtain Memory (CD8+ / CD44+ / CD49dhi) and Naive (CD8+ / CD44- / CD49dlo / CD62L+) populations from the same mice. T cells were then transduced using the standard transduction protocol as described.In vivo Experiments in Rag! hosts

[0195] Ragl~ ~ hosts were inoculated with le6 E2A-PBX by tail vein I V. injection on day -3 followed by CAR T cells via retroorbital injection at either le5, 3e5 or le6 CAR+ cell dose on day 0. Bone marrow was harvested and analyzed by flow cytometry on day 4 or 11 postCAR infusion, or mice were euthanized at humane endpoints for survival experiments. Ex vivo stimulation for cytokine production was performed using le6 E2A-PBX WT to stimulate approximately 1.5e6 whole bone marrow cells from each individual mouse, with pooled bone marrow from each n=5 experimental group stimulated by E2A-PBX CD19Negas a negative control. Cells were co-cultured for 6 hours, with luM monensin and 2.5uM Brefeldin A added at 1 hour in and then analyzed by flow for cytokine production.Bulk ATAC and RNA Sequencing Experimental Setup and Workflows

[0196] OT-I CD8+ T cells were isolated from vaccinated or naive donors and CARs were transduced into T cells as described above. CAR8 Ragl ^ hosts were inoculated with le6 E2A-PBX CD 19 10,000 followed by le6 CAR8MD or CAR8ND on the timeline described above. At day 4 post-CAR infusion, bone marrow from 10 mice per CAR group was harvested and pooled. At each of 3 timepoints, CD8+ cells were isolated using the EasySep Mouse CD8+ T cell Isolation Kit from STEMCell Technologies and then FACS-sorted to obtain 50,000 cells per condition. ATAC-seq and RNA-seq were performed in triplicate on separate sorted aliquots of 50,000 cells at “Pre-CAR / Day -5” (ex vivo, directly after isolation of memory or naive CD8+ T cells from donor mice), “Post-CAR / Day 0” (in vitro, after CAR manufacturing) and “Tumor / Day 4” (ex vivo, after reinfusion into leukemia bearing mice). Experimental analyses were performed on the first technical replicate from 2 separate experimental replicates. For RNA-seq, cells were homogenized in QIAzol Lysis Reagent (Qiagen, Cat. No. 79306) and then frozen at - 80C for processing within 2 weeks. Samples were thawed and processed using the miRNeasy Micro Kit (Qiagen, Mat. No. 1071023), with on-column DNase treatment (RNase-Free DNase Set, Qiagen, Cat. No. 79254), both according to manufacturer protocols. RNA purity, quantity and integrity were determined with NanoDrop (ThermoFisher Scientific) and TapeStation 4200 (Agilent) analysis prior to RNA-seq library preparation. The Universal Plus mRNA-Seq library preparation kit with NuQuant was used (Tecan) with an input of 200ng of total RNA to generate RNA-seqlibraries. Paired-end sequencing reads of 150bp were generated on NovaSeq 6000 (Illumina) sequencer at a target depth of 40 million clusters / 80 million paired-end reads per sample. Raw sequencing reads were de-multiplexed using bcl2fastq. For ATAC-seq, cells were immediately processed using the Omni-ATAC protocol as previously described40. Briefly, sorted cells were washed once in IX PBS, lysed, and washed once in Wash Buffer, and then the transposition reaction was carried out at 32°C for 30 minutes on a thermomixer set to 1000 rpm. Transposed chromatin was then purified using the Zymo Clean and Concentrator 5 Kit (Zymo Research, Cat # D4013) using manufacturer protocols. DNA was then run on PCR for 12 total cycles with matched barcoding primers. PCR reactions were then size-selected using AMPure XP beads (Beckman Coulter Life Sciences, Product No: A63880) and checked for quality and size distribution using TapeStation 4200 with D5000 reagents (Agilent). Libraries were pooled at equimolar ratios for sequencing and paired-end sequencing reads of 150bp for the first replicate and 50bp for the second replicate were generated on NovaSeq 6000 (Illumina) sequencer at a target depth of 40 million clusters / 80 million paired-end reads per sample. Raw sequencing reads for replicate 1 were shortened to match the read lengths for replicate 2 using trimmomatic function CROP. Raw sequencing reads were demultiplexed using bcl2fastq.RNA-Seq Data Analysis

[0197] Quality of fastq files was accessed using FastQC (v.0.11.8), FastQ Screen (v.0.13.0)42 and MultiQC (v.1.8)43. Illumina adapters and low-quality reads were filtered out using BBDuk (v. 38.87). Trimmed fastqc files were aligned to the mmlO murine reference genome and aligned counts per gene were quantified using STAR (v.2.7.9a). Differential gene expression analysis was performed using the DESeq2 package. Pathway enrichment analysis was performed using GSEA (UC San Diego / Broad Institute), Metascape for gene mapping and IPA (Qiagen). Differential gene expression was plotted using GraphPad Prism or ggplot2 (R package). RNA-seq differential gene expression statistics were run using the DESeq2 R package, with filtering threshold at 10 with greater than 2-fold change and adjusted p value < 0.05.ATAC-Seq Data Analysis

[0198] Fastq files were used to map to the mmlO genome using the ENCODE ATAC-seq pipeline, with default parameters, except bam files used for peak calling were randomly downsampled to a maximum of 50 million mapped reads. Peaks with a MACS2computed qvalue of less than le-6 and a signal Value of more than 4 in at least one replicate were merged with bedtools function intersect and processed to uniform peaks with the functions getPeaks and resize from R package ChromVAR22. Reads overlapping peaks were enumerated with getCounts function from ChromVAR and normalized and log2 -transformed with voom from R package limma. Peaks with 3 or more normalized counts per million mapped reads at least one replicate were included to define a global peak set of 82,410 peaks. Pairwise Euclidean distances were computed between all samples using log2 -transformed counts per million mapped reads among the global peak set. Differentially accessible peaks were identified in pairwise comparisons based on fdr adjusted p values of less than 0.01, fold change of at least 4 and with an average of 3 normalized counts per million mapped reads using R package limma. Motif associated variability in ATAC-seq signal was computed with R package ChromVAR. Genome-wide visualization of ATAC-seq coverage was computed with deeptools function coveragebam, using manually computed scale factors based on the number of reads within the total peak set.Statistics

[0199] Statistical tests for all experiments except sequencing analyses were performed using GraphPad Prism v9.0 for Macintosh (GraphPad Software). Comparisons between three groups were made with ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test, Brown-Forsythe and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons test, or Kruskal -Wallis non-parametric test with Dunn’s multiple comparisons test were used depending on variance in standard deviations. Two-way ANOVA or mixed effects analysis with Tukey’s multiple comparisons test was used for in vitro experimental comparisons with multiple antigen densities and in vivo CAR expansion data. Two-tailed ordinary t-test, Welch’s t test, or Mann-Whitney test were performed for comparisons with two groups depending on normality of distributions. For multiple comparisons of two groups, multiple unpaired t tests or multiple Welch’s t tests, both with Holm-Sidak’s multiple comparisons test, were performed when appropriate depending on variance in standard deviations. Logrank (Mantel-Cox) test was used for survival curve comparisons. All data represented as mean + / - standard deviation. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Technical and experimental replicates in each dataset are indicated in figure legends.Discussion for Examples 1-8

[0200] The results presented in Examples 1-8 show a syngeneic murine model with antimouse CD 19 CAR T cells targeting murine pre-B cell leukemia enabling more natural T cell differentiation trajectories without xenogeneic TCR stimulation. The results also show a well- defined vaccine model for precise control of the antigen experience by clonotropic T cells prior to CAR transduction and confirmed findings in polyclonal pathogen-elicited cells. Memory T cells generated from this vaccine model share many characteristics with human memory T cells, including rapid induction of effector functions, high expression and dependence on TNF-superfamily members (CD27) and cytokine receptors such as IL-7Ra and IL-27Ra (and downstream JAK / STAT activation), as well as essential “self-renewal” associated transcriptional programs driven by FOXO1 and TCF1. With limiting T cell dose or low target antigen density as “stressors,” antigen experience was shown to dictate multiple functional outputs of CAR T cells. Memory-derived CD8+ CAR T cells exhibited stronger cytotoxic function across target antigen densities, while naive derived CAR T cells show greater proliferative capacity and more rapid cell cycle entry. This was associated with enhanced activity against low-antigen density leukemia by memory derived CAR T cells and enhanced antigen sensitivity of memory derived cells, but enhanced activity of naive-derived cells at limiting cell doses.

[0201] The examples presented herein also show that features of epigenetic and transcriptomic states are maintained through CAR manufacturing and associate with differences in functional profiles. Ex vivo genomic assays were performed on cells infused at a high CAR+ cell dose due to cell number limitations. It was found that significant differences in bZIP family transcription factors. Overexpression of BATF or c-JUN only conferred significantly enhanced leukemia clearance by naive-derived cells. This indicates that both bZIP TFs may exert their functional effect via binding to NFAT-bZIP or IRF-bZIP composite motifs, which show high accessibility in both cell types (Zhang, X. et al. (2022) Cancer Cell (40); 1407-1422. el407) and contrast with bZIP binding motifs, which have higher accessibility in memory-derived cells. Despite functional benefit, overexpression of either bZIP TF did not prevent phenotypic exhaustion, with no differences in exhaustion- associated TF profiles (TCF1- / TIM3+ and PD1+ / TOX+) relative to the control. Together, these data suggests that overexpression of bZIP TFs may preserve function during phenotypic exhaustion.

[0202] Epigenomic and transcriptomic assays were used to identify modulation of Runx- family TFs, particularly RUNX2, as having a higher impact in naive-derived cells comparedto memory, with distinctive motif-associated chromatin accessibility and transcriptomic patterns relative to all other TF families. RUNX2 overexpressing naive-derived CAR8 cells (but not memory-derived RUNX2 overexpressing CAR8 cells) mediated superior clearance of leukemia, higher proportions of cells in the marrow, and reduced proportions of cells displaying terminally exhausted phenotypes. This data suggests that RUNX2 overexpression, in contrast to bZIP family members, enhances functional potency of naive-derived CD8+ CAR T cells while preventing entry into the exhaustion differentiation.

[0203] Profiling of RUNX2-CAR8 by RNA-seq and ATAC-seq revealed an effector-like profile with preservation of self-renewal characteristics, consistent with functional read-outs. Dimensionality-reduction of genomic data showed greater magnitude changes from RUNX2- overexpression in naive-derived cells, though the primary driver of variation was still prior antigen experience. Together, these results suggest RUNX2 allows for enhanced effector function, while maintaining proliferative capacity, likely leading to the enhanced anti-tumor activity observed in vivo.

[0204] The top TF family (bZIP) dataset showing CAR8MD-biased motif accessibility showed similar patterns of chromatin accessibility in previous human and mouse datasets, and high peak-specific species conservation between mouse and human T cells in thorough analyses performed by Gennert, et al. (Gennert, D. G., et al. (2021) Front Immunol (12) 701924). The results show that Runx was unique among TF families, showing stably higher motif-associated chromatin accessibility. The mouse data reveals RUNX2-overexpression mediated chromatin remodeling at effector loci, predominantly in CAR8ND. Finally, in vitro functional differences from RUNX2-overexpression were species-conserved (but more evident in human).

[0205] The examples presented herein show a framework for the role of antigen experience on function of a CAR T cell in stress situations of limiting T cell dose or target antigen density and highlight the importance of considering this framework when assessing the impact of approaches to apply synthetic immunology to manipulate therapeutic immune effector cell functions.

Claims

CLAIMSWhat is claimed:

1. A modified immune cell comprising:(i) a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and(ii) a polynucleotide encoding RUNX2, wherein the polynucleotide encoding RUNX2 comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

2. The modified immune cell of claim 1, wherein the immune cell comprises a CAR and the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

3. The modified immune cell of claim 2, wherein the antigen binding domain binds a tumor associated antigen.

4. The modified immune cell of claim 3, wherein the tumor associated antigen is selected from a group consisting of CD19, CD22, CD20, CD138, BCMA, CD33, CD123, FLT, CLL, CD56, CD34, CD117, CD14, CD133, CD44v6, CD47, CD64, CD96, CD97, CD99, CD45, CD9, Mucl, Lewis- Y, IL1RAP, FR-beta, CD5, CD7, CD38, CD30, B7-H3, HER2, CD44v6, CEA, c-Met, EGFRvIII, Epcam, EphA2, FR-alpha, GD2, GPC3, IL13R- alpha2, ILl lR-alpha, Ll-CAM, mesothelin, MUC1, MUC16, NKGD2 and PSCA.

5. The modified immune cell of claim 4, wherein the tumor associated antigen is CD 19.

6. The modified immune cell of any one of claims 1-5, wherein the immune cell is a T cell, a Natural Killer (NK) cell, a Natural Killer (NK)-like cell, a Cytokine Induced Killer (CIK) cell, a hematopoietic progenitor cell, a peripheral blood (PB) derived T cell, an induced pluripotent stem cell (iPSC)-derived effector cell, or an umbilical cord blood (UCB) derived T cell.

7. The modified immune cell of claim 6, wherein the immune cell is a T cell.

8. The modified immune cell of claim 7, wherein the T cell is a CAR T cell.

9. The modified immune cell of claim 7 or 8, wherein the T cell is a CD8+ CAR T cell.

10. The modified immune cell of claim 9, wherein the CD8+ CAR T cell is derived from memory cells, naive cells, or effector cells.

11. The modified immune cell of claim 10, wherein the CD8+ CAR T cell is derived from naive cells.

12. The modified immune cell of any one of claims 1-11, wherein the modified immune cell expresses an amount of the polynucleotide encoding RUNX2 sufficient to induce in the modified immune cell at least one of(a) increased RUNX2 expression by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2;(b) enhanced potency by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2;(c) increased target cell clearance by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2; or(d) decreased T cell exhaustion by at least 5% relative to an immune cell not comprising the polynucleotide encoding RUNX2.

13. A composition comprising the modified immune cell of any one of claims 1-12 and a pharmaceutically acceptable carrier.

14. A composition comprising a population of cells, wherein a plurality of cells of the population comprises:(i) a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and(ii) a polynucleotide encoding RUNX2.

15. The composition of claim 14, wherein at least 50% of the cells in the population comprise the CAR or TCR and comprise the polynucleotide encoding RUNX2.

16. A polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor or an engineered T cell receptor and a nucleic acid sequence encoding RUNX2.

17. A vector comprising the polynucleotide of claim 16.

18. The vector of claim 17, wherein the vector is a viral vector, a non-viral vector, or a transposon.

19. The vector of claims 17 or 18, wherein the vector comprises:(a) a sequence encoding RUNX2;(b) a polycistronic element;(c) a reporter gene; and(d) a sequence encoding a CAR or a TCR.

20. The vector of claim 19, wherein(a) the sequence encoding RUNX2 comprises the sequence of SEQ ID NO: 1 or 2;(b) the polycistronic element comprises the sequence of any one of SEQ ID NOs: 3-9; and / or(c) the reporter gene sequence comprises the sequence of any one of SEQ ID NOs: 10-13.

21. A method of improving an immune cell therapy, comprising delivering an exogenous polynucleotide encoding RUNX2 to a therapeutic cell.

22. The method of claim 21, wherein the method results in at least one:(a) RUNX2 upregulation by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2;(b) enhanced potency by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2;(c) increased target cell clearance by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2; or(d) decreased T cell exhaustion by at least 5% relative to an immune cell not comprising the exogenous polynucleotide encoding RUNX2.

23. A method of treating cancer in a subject in need thereof, comprising administering to the subject the modified immune cell of any one of claims 1-12 or the composition of any one of claims 13-15.

24. The method of claim 23, wherein the cancer is a liquid tumor or a solid tumor.

25. The method of claim 24, wherein the liquid tumor is leukemia.

26. The method of claim 25, wherein the leukemia is B cell leukemia.

27. A recombinant viral vector comprising:(i) a viral capsid comprising a viral capsid protein;(ii) a first nucleic acid sequence encapsulated within the viral capsid, wherein the first nucleic acid sequence encodes a chimeric antigen receptor or an engineered T cell receptor; and(iii) a second nucleic acid sequence encapsulated within the viral capsid, wherein the second nucleic acid sequence encodes RUNX2.

28. The recombinant viral particle of claim 27, wherein the recombinant viral vector comprises an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a vaccinia viral vector, or a vesicular stomatitis viral vector.

Images