Compositions and methods comprising 3' UTR stem loop mediated transgene modulation

Abstract

The present disclosure provides compositions and methods for dynamic control of transgene expression by inclusion of optimized 3' UTR stem loops that are bound by Regnase-1 (Reg1) and / or Roquin-1 (Roq1).

Description

[0001] COMPOSITIONS AND METHODS COMPRISING 3’ UTR STEM LOOP MEDIATED TRANSGENE MODULATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 622,949, filed January 19, 2024, which application is hereby incorporated herein by reference in its entirety.

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The present application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML file, created on January 8, 2025, is named 046483-7444WO1 Sequence Listing.xml and is 24,576 bytes in size.

[0005] BACKGROUND OF THE INVENTION

[0006] Adoptive cell transfer (ACT) therapy using gene-modified immune effector cells, e.g., T cells, has become a desirable chassis for engineering regulatable transgene expression. This is largely due to the burgeoning of the cell therapy field following earlier therapeutic successes in hematological malignancies using autologous T cells genetically modified with chimeric antigen receptors (CARs) (Porter, et al., 2011, N Engl J Med, 365, 725-733; Grupp, etal., 2013 , N Engl J Med, 368, 1509-1518; Maude, etal., 2014, N Engl J Med, 371, 1507- 1517; Maude, et al., 2018, N Engl J Med, 378, 439-448; Melenhorst, et al., 2022, Nature, 602, 503-509). However, most techniques used to modulate gene expression typically leverage molecular actuators, such as small molecule drugs, to induce changes in gene expression (Siddiqui, et al., 2022, Curr Opin Biotechnol, 78, 102823). While this type of external, user-dependent control is beneficial in many contexts, there may be instances in which cell-autonomous control, or modulation that does not require user-given external inputs to actuate, is preferable. These instances include situations when it is unclear when to invoke control or the desired modulation is in response to a specific cell state, neither of which can be adequately monitored in vivo.

[0007] Early iterations of CAR-T cells were not engineered with systems to dynamically modulate transgene expression; however more recent efforts have led to the development of drug-regulatable CAR-T cells, which can be leveraged to mitigate toxicity either through regulation of the CAR itself or other effector payloads (Weber, et al., 2019, Blood Adv, 3, 711-717; Choe, etal., 2021, Sci Transl Med, 13; Labanieh, etal., 2022, Cell, 185, 1745-1763 el722; Li, et al., 2022, Cancer Cell, 40, 1294-1305 el294). These systems are useful, yet methods to modulate transgene expression in a cell-autonomous way remain limited in effector immune cells, a cell type in which drug-independent regulation may be particularly useful because of their current and potential therapeutic applications.

[0008] Currently, the most notable way to dynamically modulate transgene expression in T cells without small molecule drugs is by using the nuclear factor of activated T cells (NF AT) promoter, which can enable activation-specific transgene expression (Guo, et al., 2022, ACS Synth Biol, 11, 1440-1453). Promoter-level control of transgene expression is powerful; yet there exists a need for other layers of regulation which would be similarly potent and have the potential to be used orthogonally. The present invention addresses this need.

[0009] SUMMARY OF THE INVENTION

[0010] In some aspects, the invention provides a vector comprising:

[0011] (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and

[0012] (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg-1) and / or Roquin-1 (Roq-1) when Reg-1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0013] In some embodiments, the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

[0014] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

[0015] In some embodiments, the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop. In some embodiments, the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0016] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0017] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0018] In some embodiments, the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0019] In some embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

[0020] In some embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a Pl-Ll region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

[0021] In some embodiments, G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson-Crick G-C base pairing.

[0022] In some embodiments, the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

[0023] In some embodiments, the vector further comprises a WPRE.

[0024] In some embodiments, the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

[0025] In some embodiments, the exogenous receptor is a CAR, wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen. In some embodiments, the exogenous receptor is a CAAR, wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody -binding fragment thereof.

[0026] In some embodiments, the exogenous receptor is a TCR.

[0027] In some embodiments, the exogenous receptor is a switch receptor (e.g., PD1-CD28).

[0028] In some embodiments, the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

[0029] In some embodiments, the vector is a viral vector, (e.g., a lentiviral vector).

[0030] In some aspects, the invention provides a modified cell comprising the vector disclosed herein.

[0031] In some aspects, the invention provides a modified cell comprising a vector, wherein the modified cell is selected from the group consisting of a T cell, a

[0032] CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell; and wherein the vector comprises:

[0033] (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and

[0034] (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0035] In some embodiments, the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

[0036] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

[0037] In some embodiments, the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop. In some embodiments, the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0038] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0039] In some embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0040] In some embodiments, the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0041] In some embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

[0042] In some embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a Pl-Ll region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

[0043] In some embodiments, G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson-Crick G-C base pairing.

[0044] In some embodiments, the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

[0045] In some embodiments, the vector further comprises a WPRE.

[0046] In some embodiments, the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

[0047] In some embodiments, the exogenous receptor is a CAR, wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen. In some embodiments, the exogenous receptor is a CAAR, wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody -binding fragment thereof.

[0048] In some embodiments, the exogenous receptor is a TCR.

[0049] In some embodiments, the exogenous receptor is a switch receptor (e.g., PD1-CD28).

[0050] In some embodiments, the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

[0051] In some embodiments, the vector is a viral vector, (e.g., a lentiviral vector).

[0052] In some embodiments, the modified cell is a human cell.

[0053] In some aspects, the invention provides a pharmaceutical composition comprising a population of the modified cell disclosed herein and at least one pharmaceutically acceptable carrier.

[0054] In some aspects, the invention provides a method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the modified cell disclosed herein or the pharmaceutical composition disclosed herein to the subject.

[0055] In some embodiments, the disease, disorder, or condition is selected from a cancer, an autoimmune disease, and a neurodegenerative disorder or condition.

[0056] In some embodiments, the subject is a human.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0059] FIG. 1A: Conceptual schematic of certain embodiments of the invention illustrating stem loop mediated dynamic transgene expression control triggered by T cell stimulation in endogenous and engineered T cell contexts. Top, in resting T cells, endogenous regulatory RBPs, particularly Regnase-1 and Roquin-1, regulate the expression of transcripts encoding inflammatory factors by acting on stem loops located at the 3’UTR. Upon stimulation and activation of T cells, Regnase-1 and Roquin-1 are degraded, increasing expression of inflammatory factors. Bottom, this system can be harnessed to regulate transgenes of interest by inserting regulated stem loops in the 3’UTR of transgenes, resulting in engineered regulation.

[0060] FIG. IB: Transcript levels of candidate genes bearing stem loops targeted by Roql in resting and activated human T cells. Genes in red font (ICOS, IER3, NFKBID, PPP1R10, and TNF) highlight top candidates with a large transcriptional upregulation between resting and activated cells. Bold dashed lines represent median. Thin dashed lines represent interquartile ranges. Multiple unpaired t-tests were used for statistical analysis. Data shown is from the Database of Immune Cell Expression, Expression quantitative trait loci (eQTLs) and Epigenomics.

[0061] FIG. 2A: Single loop regulated stem loop designs (SEQ ID NOs: 1-4) based on the target endogenous TNFa stem loop.

[0062] FIG. 2B: Double loop regulated stem loop designs (SEQ ID NOs: 5-8) based on the TNFa stem loop joined to the target endogenous ICOS stem loop.

[0063] FIG. 2C: Energetic predictions of WT loop and tLoop structures from the RNAfold web server. Shown are charts for minimum free energy (MFE) predictions, thermodynamic ensemble (TE) free energy predictions, MFE structure frequency predictions, and ensemble diversity predictions.

[0064] FIG. 3: Regulatory activity of stem loop designs on transgene expression in primary human T cells. Representative flow plots showing CARO 19 expression 24 hours after mRNA electroporation in CD8 T cells from donor ND580.

[0065] FIG. 4: Top, characterization of CAR-positive cells 24 hours after electroporation of CAR019-encoding mRNAs bearing different stem loops in CD8 T cells. Each data point in each experimental group represents an independent donor (n=4). Bottom, CAR expression kinetics over time after mRNA electroporation. Data shown is pooled from two independent donors (n=2). Bars represent S.D.

[0066] FIG. 5: CAR expression in CD4 T cells after electroporation of CAR019-encoding IVT mRNAs bearing different stem loops. Representative flow plots showing CAR expression 24 hours after mRNA electroporation in CD4 T cells from donor ND580.

[0067] FIG. 6: CAR expression in CD4 T cells after electroporation of CAR019-encoding IVT mRNAs bearing different stem loops. Top, characterization of CAR-positive cells 24 hours after electroporation of CAR mRNAs bearing different stem loops in CD4 T cells. Each data point in each experimental group represents an independent donor (n=4). Right, CAR expression kinetics over time after mRNA electroporation. Data shown is pooled from two independent donors (n=2). Bars represent S.D.

[0068] FIG. 7: The impact of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on stem loop regulation and dependence on Regnase-1 and Roquin-1 activity. Conceptual schematic depicting a generalized 2-promoter bicistronic vector system and rationale of use. Top, a reporter under a standard promoter will largely yield consistent expression while a regulated transgene will demonstrate modulated expression due to transcript degradation (dotted). Bottom, to control for potential transgene expression differences due to variable transduction, transgene expression can be normalized to reporter expression. P = promoter.

[0069] FIG. 8: Schematic illustration of specific constructs tested with variable stem loops to determine the effect of the WPRE.

[0070] FIG. 9: Impact of the WPRE on stem loop regulation in CD4 T cells. Top, comparison of normalized CARO 19 expression with and without the WPRE using different stem loops in CD4 T cells. Each data point in each experimental group represents an independent donor (n=2 or 3). Bottom, representative flow plots showing CAR and GFP expression from donor ND451.

[0071] FIG. 10: Impact of the WPRE on stem loop regulation in T cells in independent human donors. Representative flow plots showing CARO 19 and GFP expression in CD4 T cells from donor ND615.

[0072] FIG. 11: Representative flow plots showing CARO 19 and GFP expression in CD4 T cells from donor ND578.

[0073] FIG. 12: Impact of the WPRE on stem loop regulation in CD4 T cells. Characterization of ON / OFF ratios for different stem loops with and without the WPRE in CD4 T cells. Bars represent S.D. Multiple unpaired t-tests were used for statistical analysis.

[0074] FIG. 13: The impact of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on stem loop regulation and dependence on Regnase-1 and Roquin-1 activity. Top, comparison of normalized CAR019 expression with and without the WPRE using different stem loops in CD8 T cells. Each data point in each experimental group represents an independent donor (n=2 or 3). Bottom, representative flow plots showing CARO 19 and GFP expression from donor ND451. FIG. 14: Impact of the WPRE on stem loop regulation in T cells in independent human donors. Representative flow plots showing CAR and GFP expression in CD8 T cells from donor ND615.

[0075] FIG. 15: Representative flow plots showing CAR and GFP expression in CD8 T cells from donor ND 578.

[0076] FIG. 16: The impact of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on stem loop regulation and dependence on Regnase-1 and Roquin-1 activity. Characterization of ON / OFF ratios for different stem loops with and without the WPRE in CD8 T cells. Bars represent S.D. Multiple unpaired t-tests were used for statistical analysis.

[0077] FIG. 17: Top, comparison of normalized CAR / GFP between mock unedited and Regnase-1 and Roquin-1 double knockout (DKO) CD4 T cells. Each data point in each experimental group represents an independent donor (n=4). Bottom, representative flow plots showing CAR and GFP expression from donor ND578.

[0078] FIG. 18: The impact of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on stem loop regulation and dependence on Regnase-1 and Roquin-1 activity. Top, comparison of normalized CAR / GFP between mock unedited and Regnase-1 and Roquin-1 double knockout (DKO) CD8 T cells. Each data point in each experimental group represents an independent donor (n=4). Bottom, representative flow plots showing CARO 19 and GFP expression from donor ND578.

[0079] FIG. 19: Quantification of indel percentages at the editing loci of Regnase-1 and Roquin-1 for independent donors (n=4). The red percentage displayed above one of the bars denotes the lower indel frequency of the two genes and is the gating percentage (top X% of ICOS expression) used to select the population of cells most likely to have successful Regnase-1 and Roquin-1 double knockout.

[0080] FIG. 20: Representative gating strategy used (left to right) to isolate desired cell populations for expression analysis. As a well-established regulatory target of Regnase-1 and Roquin-1, ICOS was used as a marker to select the most probable Regnase-1 and Roquin-1 knockout cells.

[0081] FIG. 21: Representative flow plots showing CARO 19 and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CAR and GFP expression in mock and DKO CD4 T cells from donor ND410. FIG. 22: Representative flow plots showing CARO 19 and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CAR and GFP expression in mock and DKO CD8 T cells from donor ND410.

[0082] FIG. 23: Representative flow plots showing CARO 19 and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CARO 19 and GFP expression in mock and DKO CD4 T cells from donor ND607.

[0083] FIG. 24: Representative flow plots showing CAR and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CAR and GFP expression in mock and DKO CD8 T cells from donor ND607.

[0084] FIG. 25: Representative flow plots showing CARO 19 and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CARO 19 and GFP expression in mock and DKO CD4 T cells from donor ND608.

[0085] FIG. 26: Representative flow plots showing CARO 19 and GFP expression in mock and DKO T cells in independent human donors. Flow plots showing CAR and GFP expression in mock and DKO CD8 T cells from donor ND608.

[0086] FIG. 27: Top, normalized ICOS expression in mock CD4 and CD8 T cells. Bottom, representative flow plots of ICOS expression in mock and DKO CD4 (left) and CD8 (right) T cells from donor ND578. 2-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis. Individual data points in each experimental group represent independent donors (n=4).

[0087] FIG. 28: Flow plots showing ICOS expression in mock and DKO CD4 (left) and CD8 (right) T cells from donor ND410.

[0088] FIG. 29: Flow plots showing ICOS expression in mock and DKO CD4 (left) and CD8 (right) T cells from donor ND607.

[0089] FIG. 30: Flow plots showing ICOS expression in mock and DKO CD4 (left) and CD8 (right) T cells from donor ND608.

[0090] FIG. 31A: Regulation by stem loops enables stimulation-induced upregulation of transgene expression. Conceptual schematic depicting regulation of Regnase-1 and Roquin-1 activity and downstream stem loop regulated transgene expression through TCR stimulation and potential regulation from stimulation through engineered receptors.

[0091] FIG. 31B: Regulation by stem loops enables stimulation-induced upregulation of transgene expression. Conceptual schematic of the restimulation assay and time points used to measure CAR expression. FIG. 32: CAR019 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND578.

[0092] FIG. 33: Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND578.

[0093] FIG. 34: Regulation by stem loops enables stimulation-induced upregulation of transgene expression. CARO 19 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND578.

[0094] FIG. 35: Regulation by stem loops enables stimulation-induced upregulation of transgene expression. Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND578.

[0095] FIG. 36: CAR019 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND451.

[0096] FIG. 37: Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND451.

[0097] FIG. 38: CAR019 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND451.

[0098] FIG. 39: Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND451.

[0099] FIG. 40: CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND500.

[0100] FIG. 41: Regulation by stem loops enables stimulation-induced upregulation of transgene expression. CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND500.

[0101] FIG. 42: CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND508. FIG. 43: CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND508.

[0102] FIG. 44: CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND572.

[0103] FIG. 45: CARO 19 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND572.

[0104] FIG. 46: Tumor cell killing at 48 hours and 96 hours for CAR-regulated T cells in donor ND451 (left) and donor ND615 (right).

[0105] FIG. 47: Functional tuning and application of stem loop modulation to other transgenes in human CAR-T cells. Schematic illustration of specific constructs tested with variable promoter strengths to determine the tunability of stem loop regulation.

[0106] FIG. 48: Top, comparison of CARO 19 MFI using different promoter strengths in CD4 T cells. Bottom, representative flow plot of CARO 19 and GFP expression under different promoters in CD4 T cells for donor ND580. Individual data points in each experimental group represent independent donors (n=3).

[0107] FIG. 49: Functional tuning and application of stem loop modulation to other transgenes in human CAR-T cells. Top, comparison of CARO 19 MFI using different promoter strengths in CD8 T cells. Bottom, representative flow plot of CARO 19 and GFP expression under different promoters in CD8 T cells for donor ND580. Individual data points in each experimental group represent independent donors (n=3).

[0108] FIG. 50A: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. Tumor cell killing at 18 hours for CAR-regulated T cells in donor ND602 under varying promoter strengths.

[0109] FIG. 50B: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. Tumor killing after 18 hours after co-culture with CAR-T cells with different promoters in donor ND615. Bars represent S.D.

[0110] FIG. 51: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. Schematic illustration of specific constructs tested to evaluate regulation of the therapeutic payload IL 18. FIG. 52: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. IL18 production under different stimulation conditions after 48 hours in donor ND616. Multiple unpaired t-tests were used for statistical analysis.

[0111] FIG. 53: IL18 production under different stimulation conditions after 48 hours in donor ND567. Multiple unpaired t-tests were used for statistical analysis.

[0112] FIG. 54: IL18 production under different stimulation conditions after 48 hours in donor ND607. Multiple unpaired t-tests were used for statistical analysis.

[0113] FIG. 55: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. Schematic illustration of specific constructs tested to evaluate the regulation of Bcl-xL

[0114] FIG. 56: Top, Bcl-xL expression under different stimulation conditions after 48 hours in CD4 T cells. Bottom, representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD4 T cells in donor ND578. 2-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis. Individual data points in each experimental group represent independent donors (n=3).

[0115] FIG. 57: Representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD4 T cells in donor ND579.

[0116] FIG. 58: Representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD4 T cells in donor ND585.

[0117] FIG. 59: Functional tuning and application of stem loop modulation to other transgenes in human CAR019-T cells. Top, Bcl-xL expression under different stimulation conditions after 48 hours in CD8 T cells. Bottom, representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD8 T cells in donor ND578. 2-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis. Individual data points in each experimental group represent independent donors (n=3).

[0118] FIG. 60: Representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD8 T cells in donor ND579.

[0119] FIG. 61: Representative flow plots showing Bcl-xL expression under different stimulation conditions after 48 hours in CD8 T cells in donor ND585.

[0120] FIG. 62: Schematic illustration of specific construct tested to evaluate the regulation of CAR M5. FIG. 63: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor TMP484. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor TMP484.

[0121] FIG. 64: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor TMP484. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor TMP484.

[0122] FIG. 65: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND518. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND518.

[0123] FIG. 66: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND518. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND518.

[0124] FIG. 67: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND608. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND608.

[0125] FIG. 68: Top, CAR M5 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND608. Bottom, representative flow plots of CAR M5 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND608.

[0126] FIG. 69: CAR M5 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND518.

[0127] FIG. 70: CAR M5 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND518.

[0128] FIG. 71: CAR M5 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND608. FIG. 72: CARM5 MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND608.

[0129] FIG. 73: Schematic illustration of specific construct tested to evaluate the regulation of a BCMA-targeting VHH CAR (a camelid single domain antibody-based CAR).

[0130] FIG. 74: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor TMP484. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor TMP484.

[0131] FIG. 75: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor TMP484. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor TMP484.

[0132] FIG. 76: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND518. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND518.

[0133] FIG. 77: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND518. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND518.

[0134] FIG. 78: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND608. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND608.

[0135] FIG. 79: Top, BCMA VHH CAR MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND608. Bottom, representative flow plots of BCMA VHH CAR expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND608.

[0136] FIG. 80: BCMA VHH CAR MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND518. FIG. 81: BCMA VHH CAR MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND518.

[0137] FIG. 82: BCMA VHH CAR MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD4 T cells for donor ND608.

[0138] FIG. 83: BCMA VHH CAR MFI fold change dynamics after primary stimulation and restimulation with CD3 / CD28 activator (left) and antigen (right) with representative flow plots below in CD8 T cells for donor ND608.

[0139] FIG. 84: CAR019 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD4 T cells for donor ND615.

[0140] FIG. 85: Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD4 T cells for donor ND615.

[0141] FIG. 86: CAR019 MFI fold change dynamics over time after CD3 / CD28 (left) and antigen (right) stimulation in CD8 T cells for donor ND615.

[0142] FIG. 87: Representative flow plots of CARO 19 expression following no (left), CD3 / CD28 (middle), and antigen (right) stimulation over time in CD8 T cells for donor ND615.

[0143] FIG. 88: Representative flow plot of CARO 19 and GFP expression under different promoters in CD4 T cells (top) and CD8 T cells (bottom) for donor ND602.

[0144] FIG. 89: Representative flow plot of CARO 19 and GFP expression under different promoters in CD4 T cells (top) and CD8 T cells (bottom) for donor ND612.

[0145] FIG. 90: Representative flow plot of CARO 19 and GFP expression under different promoters in CD4 T cells (top) and CD8 T cells (bottom) for donor ND615.

[0146] DETAILED DESCRIPTION

[0147] Modulation of gene expression is desirable for various applications in both basic and translational sciences, including the regulation of armaments in the context of CAR- and TCR-T, NKT, NK or macrophage cell therapies. However, most techniques used to modulate gene expression typically leverage molecular actuators, such as small molecule drugs as switches, to induce changes in gene expression (Siddiqui, et al., 2022, Curr Opin Biotechnol, 78, 102823). While this type of external, user-dependent control is beneficial in many contexts, there are instances in which cell-autonomous control, or modulation that does not require user-given external inputs to actuate, is preferable. These instances include situations when it is unclear when to invoke control, such as in an in vivo setting when cells are more difficult to observe, or when desired modulation is in response to a specific cell state such as activation through a CAR or TCR. The RNA binding proteins Regnase-1 and Roquin-1 impose post transcriptional regulation of gene expression in lymphocytes by targeting stem loop structures in the 3’ untranslated regions (UTRs) of the mRNAs of proinflammatory genes, targeting these transcripts for sequestration or degradation. This system enables lymphocytes to have pools of proinflammatory mRNAs that are inactive until T cell activation which induces the MALT-1 paracaspase to target and degrade Regnase-1 and Roquin-1 proteins, alleviating the repression of proinflammatory genes. MALT-1 levels eventually decline after T cell activation restoring the cellular levels of Regnase-1 and Roquin-1 which silence pro-inflammatory gene expression and return T cells to their basal state, curtailing the duration of inflammatory gene expression.

[0148] In the present disclosure, the regulatory roles of RNA binding proteins Regnase-1 (Regl) and Roquin-1 (Roql) on stem loops were harnessed to program stimulation-induced transgene upregulation in primary human T cells. By encoding novel optimized stem loops targeted by Regnase-1 and Roquin-1 in the transgene 3’ UTR, it is demonstrated herein that loops can facilitate transgene repression through Regnase-1 and Roquin-1 activity, enable dynamic upregulation through stimulation, and be orthogonally tuned. This system was applied herein to test modulation of payloads in CAR-T cells.

[0149] The present invention is an alternative to the use of the nuclear factor of activated T cells (NF AT) promoter, which can enable activation-specific transgene expression (Guo, et ACS Synth Biol, 11, 1440-1453), but which requires more coding capacity, and has often proven ‘leaky’, in that expression of the regulated transgene is often detected in the absence of antigen-specific T cell activation. In the present invention, stem loops based on select putative stem loop targets of Regl and Roql were designed and encoded into the 3’ UTR. For initial proof-of-concept, the effect of stem loops on in vitro transcribed mRNA was tested, using the clinical stage CD19-targeting CAR (CARO 19) as a model transgene. The stem loops were then encoded into lentiviral constructs to determine whether their function was affected by the presence of other expression modulating elements in the 3’ UTR, namely the woodchuck post-transcriptional regulatory element (WPRE) widely used in cell therapy settings. To evaluate the dynamics of transgene modulation mediated by stem loops, the expression of regulated CARO 19 was measured after stimulation with CD3 / CD28 or antigenbearing tumor cells. Finally, promoter-level expression tuning of regulated CARO 19 was explored and stem loop modulation was applied to other molecular payloads including other CARs, and alternative transgene cargo, namely IL- 18 and BclxL. As demonstrated herein, 3’ UTR stem loops targeted by Regl and Roql are useful to dampen transgene expression and to program stimulation-induced upregulation in human T cells. Thus, it is contemplated herein to harness the strong regulatory capabilities of Regl and Roql in T cells, including NK and NKT cells and / or stem cells which have been differentiated to T cells, to modulate expression of transgenes of interest.

[0150] Accordingly, in one aspect, the present invention provides a vector comprising:

[0151] (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and

[0152] (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0153] In another aspect, the present disclosure provides a modified cell comprising a vector, wherein the modified cell is selected from the group consisting of a T cell, a CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell; and wherein the vector comprises:

[0154] (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and

[0155] (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene. In another aspect, the present disclosure provides a pharmaceutical composition comprising a population of the modified cell disclosed herein and at least one pharmaceutically acceptable carrier.

[0156] In another aspect, the present disclosure provides a method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the modified cell disclosed herein or the pharmaceutical composition disclosed herein to the subject.

[0157] It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0158] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by Green and Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0159] Methods and techniques using T Cells with exogenous receptors such as chimeric antigen receptors (e.g., CAR T cells) are described in, e.g., Ruella, et al., J. Clin. Invest., 126(10):3814-3826 (2016) and Kalos, et al., 3 (95), 95ra73: l-l l (2011), the contents of which are hereby incorporated by reference in their entireties.

[0160] Definitions

[0161] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0162] That the disclosure may be more readily understood, select terms are defined below.

[0163] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0164] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0165] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0166] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0167] “Allogeneic” refers to any material derived from a different animal of the same species.

[0168] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0169] “Autoantibody” refers to an antibody that is specific for an autoantigen.

[0170] By “autoantigen” is meant an endogenous antigen that stimulates production of an autoimmune response, such as production of autoantibodies. Autoantigen also includes a selfantigen or antigen from a normal tissue that is the target of a cell-mediated or an antibody- mediated immune response that may result in the development of an autoimmune disease.

[0171] The term “autoimmune disease,” as used herein, is defined as a disorder or condition that results from an antibody mediated autoimmune response against autoantigens. An autoimmune disease results in the production of autoantibodies that are inappropriately produced and / or excessively produced to a self-antigen or autoantigen.

[0172] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0173] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0174] A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0175] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0176] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0177] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0178] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0179] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0180] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8- 10 amino acids. One skilled in the art understands that generally the overall three- dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0181] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0182] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term “ex vivo " as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0183] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0184] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0185] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0186] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0187] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0188] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0189] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0190] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0191] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0192] In the context of the present invention, the following abbreviations for the commonly occurring nucleotides and nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0193] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0194] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0195] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0196] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0197] The term “quiescent” refers to non-proliferating, non-dividing, or resting cells, e.g., cells in the GO phase of the cell cycle. Cells may naturally be in a quiescent state. Cell quiescence may be artificially induced using methods or agents generally known in the art, e.g., by serum starvation. In some embodiments, a population of quiescent immune effector cells (e.g., T cells) comprises cells which have been synchronized in the GO phase of the cell cycle, e.g., by serum starvation. In some embodiments, quiescent immune effector cells (e.g., T cells) are cells that are not activated, e.g., not activated via T cell receptor (TCR) or coreceptors (e.g., CD3 and / or CD28). In some embodiments, quiescent immune effector cells are engineered to express a CAR / CAAR in the absence of T cell activation, e.g., without exposure to any stimulus of T cell activation, e.g., without ex vivo exposure to any stimulus of T cell activation.

[0198] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such crossspecies reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0199] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0200] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti- CD2 antibody.

[0201] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used herein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is a human.

[0202] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0203] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y / 8) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0204] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0205] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0206] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0207] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0208] The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0209] Any compositions or method provided herein can be combined with one or more of any of the other compositions and / or methods provided herein. Nucleic Acids and Expression Vectors

[0210] In some aspects, the present disclosure provides a nucleic acid comprising (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter, and (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg-1) and / or Roquin-1 (Roq-1) when Reg-1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene. In certain embodiments, the nucleic acid is mRNA, e.g. in vitro transcribed (IVT) mRNA.

[0211] In some aspects, the present disclosure provides a vector comprising (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter, and (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg-1) and / or Roquin-1 (Roq-1) when Reg-1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0212] In certain embodiments, the vector further comprises a WPRE.

[0213] In certain embodiments, the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

[0214] In certain embodiments, the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop. In certain embodiments, the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop.

[0215] In certain embodiments, the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10. In certain embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10. In certain embodiments, the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10. In certain embodiments, the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0216] In certain embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa. In certain embodiments, the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a Pl-Ll region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

[0217] In certain embodiments, G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson-Crick G-C base pairing. In certain embodiments, the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

[0218] Table 1: Exemplary stem loop sequences The nucleic acids and vectors disclosed herein allow for T cells expressing an exogenous receptor (e.g., CAR / TCR T cells) to control expression of transgenes in relation to antigen-specific activation, keeping transgene expression very low or absent prior to T cell activation, followed by a burst of expression and then a curtailment back to baseline. Such a profile of gene expression may be especially valuable where regulated transgenes demonstrate a limited therapeutic window if expressed continuously. It is contemplated herein that the transgene can comprise any gene of interest. In certain embodiments, the transgene is selected from a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

[0219] In some embodiments, the exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the exogenous receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the exogenous receptor is a T cell receptor (TCR). In some embodiments, the exogenous receptor is a switch receptor. In some embodiments, the exogenous receptor is a dominant negative receptor.

[0220] As is known in the art, a CAR comprises an extracellular antigen binding domain whereas a CAAR comprises an extracellular autoantigen domain comprising an autoantigen or autoantibody-binding fragment thereof. Additionally, as is known in the art, a TCR also binds an antigen.

[0221] CARs and CAARs

[0222] The extracellular domain of the CAR or CAAR (z.e., “CAR / CAAR”) is operably linked to another domain of the CAR / CAAR, such as a hinge, a transmembrane domain and / or an intracellular domain, each described elsewhere herein, for expression in the cell, e.g., a T cell.

[0223] The extracellular domain of the CAR / CAAR can be combined with any of the transmembrane domains described herein or known in the art, any of the intracellular domains or cytoplasmic domains described herein or known in the art, or any of the other domains described herein or known in the art that may be included in a CAR / CAAR of the present invention, such as a hinge domain or a spacer sequence. The CAR / CAAR of the present invention may also include a leader sequence. The CAR / CAAR of the present invention may also include one or more spacer domains or linkers as described herein or as known in the art which may serve to link one domain of the CAR / CAAR to the next domain.

[0224] Antigen Binding Domain

[0225] The extracellular domain of a CAR comprises an antigen binding domain which serves to recognize (i.e., bind to) a specific target antigen, which antigens may include proteins, carbohydrates, and glycolipids. In certain embodiments, the CAR of the invention comprises an extracellular antigen binding domain having affinity for a tumor antigen. The CAR further comprises a transmembrane domain and an intracellular domain comprising at least one costimulatory domain and a signaling domain as described elsewhere herein or known in the art. In some embodiments, the CAR comprises a CD28 costimulatory domain and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a 4- IBB costimulatory domain and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a CD28 costimulatory domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.

[0226] Suitable tumor antigens which the CAR targets (i.e., binds to) are known in the art and include, but are not limited to, alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 1 (IL13Ral), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Mullerian inhibiting substance type 2 receptor (MISIIR), Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), Pl 6, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is selected from prostate specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUCl), Glycosyl-phosphatidylinositol (GPI)- linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), folate receptor alpha (FRa), mesothelin, New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin 13 receptor subunit alpha 1 (IL13Ral), and interleukin 13 receptor subunit alpha 2 (IL13Ra2). In some embodiments, the tumor antigen is selected from PSMA, mesothelin, and EGFR.

[0227] The antigen binding domain of a CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody (mAb), a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-domain antibody, a full length antibody or any antigen-binding fragment thereof, a Fab, and a single-chain variable fragment (scFv). In some embodiments, the antigen binding domain comprises an aglycosylated antibody or a fragment thereof or scFv thereof.

[0228] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The variable heavy (VH) and light (VL) chains are either joined directly or joined by a peptide linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. In some embodiments, the antigen binding domain (e.g., tumor antigen binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C- terminus, VL - linker - VH or VH - linker -VL. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0229] The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen etal., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In one embodiment, an antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are separated by a linker sequence. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Set. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao, et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter, et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh, et al., J Imunol 2009 183(4):2277-85; Giomarelli, et al., Thromb Haemost 2007 97(6):955-63; Fife, et al., J Clin Invst 2006 116(8):2252-61; Brocks, et al., Immunotechnology 1997 3(3):173-84; Moosmayer, et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter, et al., J Biol Chem 2003 25278(38):36740-7; Xie, et al., Nat Biotech 1997 15(8):768-71 ; Ledbetter, et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho, et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0230] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0231] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0232] In other embodiments, the antigen binding domain comprises an antibody mimetic protein such as, for example, designed ankyrin repeat protein (DARPin), affibody, monobody, (i.e., adnectin), affilin, affimer, affitin, alphabody, avimer, Kunitz domain peptide, or anticalin. Constructs with specific binding affinities can be generated using DARPin libraries e.g., as described in Seeger, et al., Protein Sci., 22: 1239-1257 (2013).

[0233] In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof, or a humanized murine antibody or a fragment thereof.

[0234] In certain embodiments, the antigen binding domain comprises a heavy chain variable region that comprises three heavy chain complementarity determining regions (HCDRs) and a light chain variable region that comprises three light chain complementarity determining regions (LCDRs). In certain embodiments, the antigen binding domain comprises a linker.

[0235] In certain embodiments, the CAR comprises an extracellular antigen binding domain that binds a tumor antigen selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD 117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 1 (IL13Ral), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE- A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Mullerian inhibiting substance type 2 receptor (MISIIR), Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), New York esophageal squamous cell carcinoma- 1 (NY-ESO- 1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is selected from PSMA, mesothelin, and EGFR.

[0236] Autoantigens

[0237] Chimeric autoantibody receptors (CAARs) can be used to target B cells that express autoantibody -based B cell receptors, which after activation and autoantibody secretion, may cause an autoantibody-mediated condition or disease. The extracellular domain of the CAAR comprises an autoantibody binding domain otherwise referred to as an autoantigen or an autoantibody -binding fragment thereof. Exemplary CAARs suitable for use in the invention are known in the art and described, for example, in WO2015 / 168613, WO2017 / 181101, WO2018 / 127585, WO2019 / 213434, WO2019 / 236593, WO2020 / 231999, and WO2023 / 015239, each of which is incorporated by reference in its entirety herein.

[0238] Transmembrane Domain

[0239] CARs / CAARs of the present invention comprise a transmembrane domain that connects the extracellular domain to the intracellular domain of the CAR / CAAR. The transmembrane domain of the CAR / CAAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof). In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR / CAAR.

[0240] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR / CAAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

[0241] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR / CAAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0242] In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8a.

[0243] In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0244] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in the CAR / CAAR, such as a hinge domain or region.

[0245] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR / CAAR of the present invention may also include a hinge region. The hinge region of the CAR / CAAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR / CAAR. The hinge region is an optional component for the CAR / CAAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

[0246] In some embodiments, the CAR / CAAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek, et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek, et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations.

[0247] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0248] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

[0249] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0250] For example, hinge regions include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region (see, e.g., Yan, et al., J. Biol. Chem. (2012) 287: 5891-5897). In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

[0251] Intracellular Signaling Domain

[0252] The CAR / CAAR of the present invention also includes an intracellular signaling domain. The terms “intracellular signaling domain,” “signaling domain,” “intracellular domain,” and “ICD” are used interchangeably herein. The intracellular signaling domain of the CAR / CAAR is responsible for activation of at least one of the effector functions of the cell in which the CAR / CAAR is expressed (e.g., immune cell). The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell.

[0253] Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the immune cell (z.e., the T cell), as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability. Examples of the intracellular signaling domain include, without limitation, the C, chain of the T cell receptor complex or any of its homologs, e.g., r] chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 5 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may comprise an intracellular signaling domain of a protein selected from human CD3 zeta chain, FcyRIII, FcsRI, DAP10, DAP12, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

[0254] In one embodiment, the intracellular signaling domain of the CAR / CAAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, such as any synthetic sequence thereof, that has the same functional capability, and any combination thereof.

[0255] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), 0X9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDlib, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co- stimulatory molecule that has the same functional capability, and any combination thereof. Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains ofNKp30 (B7-H6) (see, e.g., Zhang, et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer, et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP 10, and CD3z.

[0256] Intracellular signaling domains suitable for use in the CAR / CAAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR / CAAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR / CAAR, but is instead diffused in the cytoplasm.

[0257] Intracellular signaling domains suitable for use in the CAR / CAAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of the CAR / CAAR comprises 3 ITAM motifs.

[0258] In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (ITAMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis, et al, Front. Immunol. (2014) 5:254).

[0259] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an IT AM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0260] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase- binding protein; killer activating receptor associated protein; killer-activating receptor- associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); 0KT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T-cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in a CAR / CAAR of the present disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a CAR / CAAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR / CAAR includes a cytoplasmic signaling domain of human CD3 zeta.

[0261] While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire molecule. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0262] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR / CAAR.

[0263] In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB. In certain embodiments, the intracellular domain comprises an intracellular domain of CD3(^ or a variant thereof. In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB and an intracellular domain of CD3(^.

[0264] Amino acid and nucleotide sequences of CARs / CAARs, and domains thereof, are well known in the art. Exemplary sequences are provided, e.g., in W02020 / 210678, WO2023 / 158978, WO2015 / 168613, WO2017 / 181101, WO2019 / 213434, WO2019 / 236593, WO2020 / 231999, WO2023 / 015239 each of which is incorporated by reference in its entirety herein. Tolerable variations of the individual CAR / CAAR domain sequences (leader, antigen binding domain, hinge, transmembrane, and / or intracellular domains) will be known to those of skill in the art. For example, in certain embodiments the CAR / CAAR domain comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any naturally-occurring or known sequence.

[0265] T cell Receptors

[0266] In some embodiments, the exogenous receptor is a T cell receptor (TCR). In some embodiments, the TCR targets (i.e., has antigenic specificity for) an antigen, for example, a tumor antigen. As used herein, the phrase “having antigenic specificity,” or like phrase, means that the TCR can specifically bind to and recognize the antigen, or an epitope thereof.

[0267] Natural TCRs are generally hetero dimers. In humans, in 95% of T cells, the TCR comprises an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR comprises gamma and delta (y / 8) chains (encoded by TRG and TRD, respectively). Natural TCR complexes are an octameric assembly of type-I singlespanning membrane proteins arranged into four dimeric modules: the variable ligand-binding TCRaP module (in most T cells) (or the TCR y / 8 module) and the three invariant signaling modules CD35s, CD3ys, and CD3(^ dimer. The TCRaP module binds to pMHC ligands on APC or target cell surfaces, but these proteins lack intrinsic signaling capability and, as such, rely on the signaling modules to transmit information through their cytoplasmic immunoreceptor tyrosine-based activation motifs (IT AMs). See, e.g., Chandler et al. Int J Mol Sci (2020) 21 :7424, which is incorporated by reference herein. Natural TCRs can be cloned and modified using standard molecular biology and genetic engineering techniques known in the art.

[0268] Various engineered TCR forms are also known in the art, including TCR mimic antibodies (Chang et al., Expert Opin Biol Ther. (2016) 16(8):979-87), TCR-like CARs and TCR-CARs (Walseng et al., Sci Rep. (2017) 7: 1-10; Akatsuka et al., Front Immunol. (2020) 11 :257; Poorebrahim et al., Cancer Gene Ther. (2021) 28(6):581-589. Unlike CARs, TCRs are not restricted to the cell surface antigens, but can detect and bind to the peptides presented by MHC molecules (pMHCs). This feature provides a wide range of potential targets for TCRs such as tumor-specific neoepitopes. Of note, redirection of TCR-based CARs on the highly tumor-specific neoepitopes can prevent “off-tumor” toxicities that are commonly associated with CAR therapies.

[0269] In some embodiments, the TCR is a natural TCR. In some embodiments, the TCR is a modified TCR. In some embodiments, the TCR is an engineered TCR, such as a TCR mimic or antibody-like structure, a CAR-like TCR, or a CAR-TCR. In some embodiments, the TCR is a murine TCR. In some embodiments, the TCR is a human TCR. In some embodiments, the TCR is a hybrid TCR having one or more portions of a human TCR (e.g., a constant portion or a variable portion) and one or more portions of a murine TCR (e.g., a constant portion or a variable portion). Alternatively, the portion can be a few amino acids of a human TCR, such that the TCR, which is mostly murine, is “humanized.” Methods of making such hybrid TCRs are known in the art (see, for example, Cohen et al., Cancer Res., (2006) 66:8878-8886).

[0270] Switch Receptors

[0271] In some embodiments, the exogenous receptor is a switch receptor, i.e., a fusion protein that when displayed on a cell can convert a negative signal into a positive signal in the cell. The switch receptor is a chimeric protein in that the protein comprises at least two domains, wherein the first domain is a polypeptide that is associated with a negative signal and the second domain is a polypeptide that is associated with a positive signal. Switch receptors are known in the art. See, e.g., W02013 / 019615 and WO2016 / 122738, each of which is incorporated herein by reference in its entirety. In some embodiments, the switch receptor is a PD1-CD28 switch receptor.

[0272] Dominant Negative Receptors

[0273] In some embodiments, the exogenous receptor is a dominant negative receptor. In some embodiments, the dominant negative receptor is a dominant negative TGFbRII. Dominant negative variants of TGFbRII are known in the art. See, e.g., Wieser, et al., 1993, Mol. Cell. Biol., 13:7239-7247; Kloss, et al., 2018, Afo / . Ther., 26(7): 1855-1866; Li, et al., 2022, Biomedicine & Pharmacotherapy, 148: 112754).

[0274] In certain embodiments, the vector is a viral vector. Viral vectors suitable for use are, e.g., without limitation, a lentiviral vector, a retroviral vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In certain embodiments, the viral vector is a lentiviral vector.

[0275] In certain embodiments, the first nucleotide sequence is situated 5’ to the second nucleotide sequence. In certain embodiments, the first nucleotide sequence is situated 3’ to the second nucleotide sequence. In some embodiments, a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0276] In certain embodiments, the nucleic acid encoding an exogenous receptor or a transgene of the invention is operably linked to (i.e., under control of) a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase- 1 (PGK) promoter. In certain embodiments, the promoter is an EFla promoter. In certain embodiments, the promoter is a Ubc promoter.

[0277] In some embodiments, the first promoter and / or the second promoter is the EF-1 alpha (aka EF-1 a or EFla) promoter or an attenuated variant thereof.

[0278] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cellspecific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon, et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon, et al. (2003) Blood 101 :3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Neri (p46) promoter; see, e.g., Eckelhart, et al., Blood (2011) 117: 1565.

[0279] For expression in a bacterial cell, suitable promoters include, but are not limited to, lad, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadi azole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0280] A nucleic acid and / or nucleotide sequence of the present disclosure may be present within an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0281] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li, et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto, et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali, et al., Hum. Gene Ther. (1998) 9: 81- 86, Flannery, et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary, et al., Gene Ther. (1997) 4:683 690, Rolling, et al., Hum. Gene Ther. (1999) 10: 641-648; Ali, et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski, et al., J. Vir. (1989) 63: 3822-3828; Mendelson, et al., Virol. (1988) 166: 154-165; and Flotte, et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi, et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi, et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0282] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook, et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0283] In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0284] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce the exogenous receptor and transgene into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a exogenous receptor and transgene. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the exogenous receptor and transgene encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for a exogenous receptor and transgene further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor- 1 -alpha promoter (EFla promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes (e.g., a exogenous receptor and transgene encoding nucleic acid sequence). Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5 ’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding for an exogenous receptor and transgene.

[0285] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication.

[0286] Consequently, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication- competent virus. In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding an exogenous receptor (e.g., a CAR / CAAR / TCR) and a transgene of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao, et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding an exogenous receptor and transgene of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding an exogenous receptor and / or transgene of the present disclosure.

[0287] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0288] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei, et al., 2000 FEBS Letters 479: 79-82).

[0289] In some embodiments, a nucleic acid of the present disclosure is provided for the production of an exogenous receptor and / or a transgene as described herein, e.g., in a mammalian cell. In some embodiments, a nucleic acid of the present disclosure provides for amplification of the exogenous receptor-encoding nucleic acid and / or vector. In some embodiments, a cell comprising the isolated nucleic acid and / or vector is provided, wherein the cell is selected from a bacterial cell, a fungal cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, and a human cell. In some embodiments, the cell is a mammalian or human cell and is an immune cell or precursor cell thereof, such as a T cell.

[0290] Modified Cells

[0291] The present invention further provides a modified cell comprising a vector disclosed herein, the modified cell being a T cell or any other immune effector cell which expresses Regl and / or Roql. In certain embodiments, the cell is selected from the group consisting of a T cell, a CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell. In certain embodiments, the modified cell is an autologous cell. In certain embodiments, the modified cell is an autologous cell obtained from a human subject. In certain embodiments, the modified cell is a T cell. In certain embodiments, the modified cell is a human cell.

[0292] In certain aspects, the modified cell is selected from the group consisting of a T cell, a CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell, and the modified cell comprises a vector of the present invention, i.e., a vector comprising (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg-1) and / or Roquin-1 (Roq-1) when Reg-1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0293] Thus, modified cells of the present invention harness the strong regulatory capabilities of Regl and Roql to modulate expression of transgenes of interest as described herein.

[0294] Methods of Treatment

[0295] In one aspect, the present invention includes a method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the modified cell described herein or a pharmaceutical composition comprising the modified cell or a population of the modified cell described herein to the subject. In certain embodiments, the disease, disorder, or condition is selected from a cancer, an autoimmune disease, and a neurodegenerative disorder or condition. It is further contemplated herein that the method of treatment disclosed herein can include administering a population of modified cells that express the exogenous receptor and the transgene, which is under control of the 3’ UTR stem loop disclosed herein, and that can be expressed by a viral vector for gene therapy, ie., a gene that expresses the exogenous receptor and a gene that expresses the transgene.

[0296] In certain embodiments, the subject is a human.

[0297] Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive immune cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10): 577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338; Lee et al., Int J. Mol Sci. (2021) 22(9):4590; Banerjee et al., JCO Clin Cancer Inform. (2021) 5:668-678; Robbins et al., Stem Cell Res Ther. (2021) 12(1):350; Wrona et al., Int J Mol Sci. (2021) 22(11) : 5899; Atrash and Moyo, Onco Targets Ther. (2021) 14:2185-2201; Martinez Bedoya et al., Front Immunol. (2021) 12:640082; Morgan et al., Front Immunol. (2020) 11 : 1965; Chicaybam et al., Cancers (Basel) (2020) 12(9):2360; and Rafiq et al., Nat Rev Clin Oncol. (2020) 17(3): 147-167. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0298] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0299] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0300] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0301] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0302] The modified immune cell of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Exemplary cancers include but are not limited to B-cell malignancies such as B-cell lymphomas and leukemias and the like, as well as colorectal cancer, breast cancer, ovarian cancer, renal cancer, non-small cell lung cancer, melanoma, lymphoma, and hepatocellular cancers. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is a leukemia and / or a lymphoma. In certain embodiments, the cancer cells express CD 19. The cells to be administered may be autologous, with respect to the subject undergoing therapy.

[0303] The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0304] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0305] In some embodiments, the populations or sub-types of cells, such as CD8+and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+to CD8+ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0306] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+to CD8+cells, and / or is based on a desired fixed or minimum dose of CD4+and / or CD8+cells.

[0307] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0308] In some embodiments, the dose of total cells and / or dose of individual subpopulations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about 1. IxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0309] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 105and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD8+cells / kg, or 1 x 106CD4+and / or CD8+cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about I x lO6, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about IO10and 1011T cells, between about 108and 1012or between about IO10and 1011CD4+cells, and / or between about 108and 1012or between about IO10and 1011CD8+cells.

[0310] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1 :5 and less than about 5 : 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1 : 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1:2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0311] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0312] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0313] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0314] In certain embodiments, the modified cells of the invention (e.g., a modified cell comprising a CAR) may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the modified cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO- 4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cell may be administered in combination with an anti-PD-Ll antibody or antigenbinding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.

[0315] Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009); Herman et al. J. Immunological Methods, 285(1): 25-40 (2004); Kiesgen et al., Nat Protoc. (2021) 16(3): 1331-1342; and Maldini et al., J Immunol Methods (2020) 484-485: 112830. In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0316] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0317] In some embodiments, the subject can be administered a conditioning therapy prior to adoptive cell therapy (e.g., CAR T cell therapy). In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to adoptive cell therapy (e.g., CAR T cell therapy) may increase the efficacy of the adoptive cell therapy (e.g., CAR T cell therapy). Methods of conditioning patients for adoptive cell therapy (e.g., CAR T cell therapy) are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0318] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0319] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0320] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0321] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0322] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

[0323] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0324] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0325] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0326] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL-10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0327] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

[0328] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

[0329] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0330] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high-dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee, et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu, et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey, et al. (2016) Cancer Discov, 6(6):664-679).

[0331] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0332] In one aspect, the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject any one of the modified immune or precursor cells disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject a modified immune or precursor cell generated by any one of the methods disclosed herein.

[0333] Sources of Immune Cells

[0334] In certain embodiments, a source of immune cells (e.g. T cells) is obtained from a subject for ex vivo manipulation and / or in vivo transduction. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. Methods for in vivo transduction of immune cells for expression of the exogenous receptor described herein are described, e.g., in Pfeiffer, et al., EMBO Mol Med. (2018) 10(1 l):e9158; Weidner, et al., Nat Protoc. (2021) 16(7):3210-3240; Frank, et al., Blood Advances (2020) 4(22):5702-5715; Nawaz, et al., Blood Cancer J. (2021) 11(6): 119.

[0335] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0336] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a macrophage, or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0337] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

[0338] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0339] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0340] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0341] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0342] In one embodiment, immune are obtained cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg -free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0343] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0344] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0345] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0346] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111®11) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (markerlow) of one or more markers. For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and / or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0347] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy. In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and a CD8+ T cell subpopulation, e.g., a sub-population enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0348] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0349] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti- CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0350] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0351] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0352] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CDl lb, CD16, HLA-DR, and CD8.

[0353] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (z.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used, n yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0354] T cells can also be frozen after the washing step, which does not require the monocyte-removal step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0355] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0356] In certain embodiments, T regulatory cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, the Tregs are isolated by flow-cytometry sorting. The sample can be enriched for Tregs prior to isolation by any means known in the art. The isolated Tregs can be cryopreserved, and / or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13 / 639,927, contents of which are incorporated herein in their entirety.

[0357] Expansion of Immune Cells

[0358] After modification of cells, i.e., transduction of the cells with the vector disclosed herein, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge, et al., Transplant Proc . (1998) 30(8): 3975-3977; Haanen, etal., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland, et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0359] Expanding T cells by the methods disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold.

[0360] Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.

[0361] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0362] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3 -L, IL-1, IL-3 and c-kit ligand.

[0363] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0364] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time. Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a PIO culture. The primary culture, z.e., the first culture following the isolation of cells from tissue, is designated PO. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (z.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0365] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N- acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37 °C) and atmosphere (e.g., air plus 5% CO2).

[0366] In certain embodiments, the medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. A cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold, or more. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555, 105, contents of which are incorporated herein in their entirety.

[0367] In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.

[0368] Pharmaceutical compositions and Formulations

[0369] Also provided are populations of immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing the exogenous receptor and / or transgene make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0370] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0371] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). 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).

[0372] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0373] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0374] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0375] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0376] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0377] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0378] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0379] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0380] EXPERIMENTAL EXAMPLES

[0381] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0382] Example 1 : Stem loop mediated transgene modulation in human T cells

[0383] Materials and Methods

[0384] Plasmid design and construction'. All plasmids used in this study were designed using Geneious Prime (Dotmatics) and generated via restriction cloning of synthesized DNA oligos (IDT) or gene fragments (Genscript) into pTRPE, a third-generation self-inactivating lentiviral vector, followed by sequence verification using Nanopore / Sanger sequencing (Plasmidsaurus / Genewiz) (Posey, et al., 2016, Immunity, 44, 1444-1454). Plasmids and sequencing primers are listed in the following tables, respectively.

[0385] Plasmids used in this study:

[0386] Sequencing primers used in this study:

[0387] In vitro transcription (IVT) of mRNA'. mRNA was transcribed in vitro using previously described methods (Singh, et al., (2014) Cancer Immunol Res, 2, 1059-1070). Briefly, plasmids encoding a 4-lBB-based, second generation CD19-targeting CAR (CARO 19) with variable stem loops in the 3’UTR region were linearized overnight, followed by in vitro transcription of mRNA using the T7 mMessage ULTRA kit (Thermo Fisher) per manufacturer instructions. mRNAs were purified using the RNeasy mini kit (Qiagen). mRNA electroporation into T cells'. T cells were washed three times with Opti- MEMTM (Gibco) reduced serum media and resuspended at IxlO8cells / mL in the same media. T cells were dosed at a concentration of 1 pg mRNA per 106cells. Cells were electroporated in a 2 mm cuvette using the ECM830 Square Wave Electroporator (Harvard Apparatus BTX) with the following settings: 500 V, 700 ps.

[0388] Primary T cell and cell line culture '. Healthy donor PBMCs were obtained after written informed consent under a University Institutional Review Board-approved protocol. The Human Immunology Core (HIC) at the Perelman School of Medicine processed donor PBMCs and isolated T cells. T cells were cultured in R10 media consisting of RPML1640 (Gibco, Life Technologies) with 10% FBS, 10 mM HEPES (Gibco), 1% GlutaMAXTM (Gibco), 1 mM Sodium Pyruvate (Gibco), 1% MEM NEAA (Gibco), and 1% penicillin / streptomycin. The cell lines used in this study (Nalm6 and HEK293T cells) were obtained from the ATCC, confirmed free from mycoplasma infection (Cambrex MycoAlert), authenticated by short tandem repeat profiling (ATCC), and used at passage numbers <25. Nalm6 cells were engineered to express GFP and luciferase by lentiviral transduction and were grown and maintained in R10 media.

[0389] Lentiviral vector production '. Lentiviral vector production was performed using previously established techniques (Kutner, et al., (2009) Nat Proloc, 4, 495-505). Briefly, HEK293T cells were transfected with lentiviral vectors encoding the dual-promoter reporter system with various transgenes and lentiviral packaging plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Supernatants were collected at 24 and 48 hours after transfection and concentrated using high-speed ultracentrifugation. To generate lentiviral stocks, concentrated batches of lentivirus were resuspended in cold R10 media and stored at -80 °C. CRISPR-Cas9 knockout. Single guide RNA (sgRNA) sequences targeting Regnase-1 and Roquin-1 from a previous study validating their efficiency were used to perform knockouts (Mai, et al., 2023, Proc Natl Acad Sci USA, 120, e2218632120). The sgRNA sequences were synthesized by Integrated DNA Technologies (IDT). Gene disruption was performed using a previously described protocol (Agarwal, et al., 2021, “Production of Human CRISP R-Engineered CAR-T Cells” J Vis Exp f Briefly, T cells were washed three times in Opti-MEMTM and resuspended in P3 nucleofection solution (Lonza) at IxlO8cells / mL. Each sgRNA (5 pg per 10xl06cells) was incubated with Cas9 nuclease (Aldevron, 10 pg per 10xl06cells) for 10 minutes at room temperature to generate ribonucleoprotein (RNP) complexes. For no knockout (Mock) groups, no sgRNA was used and for multiple knockout groups, RNPs targeting each gene were incubated separately before combining. Cells were electroporated in batches of 10xl06cells (100 uL) with a mix of RNP complex and 16.8 pmol of electroporation enhancer (IDT) in nucleofection cuvettes (pulse code EH111) using a 4D-Nucleofector X-Unit (Lonza).

[0390] Flow cytometry antibodies'. The following antibodies were used (from BioLegend): CD3-BV711 (clone OKT3); (from BD): CD4-PE-Cy5 (clone RPA-T4), CD8-APC-Cy7 (clone SKI), ICOS-BV650 (clone DX29); (from CytoArt): FMC63-PE (clone R19M). Live / Dead Fixable Aqua (Invitrogen) was used to delineate live and dead cells.

[0391] Intracellular staining for Bcl-xE. Cells were incubated with Bcl-xL rabbit-derived monoclonal antibody (Invitrogen; clone C.85.1) at a 1 : 1000 dilution in FACS buffer (2% FBS in IX PBS) for 30 minutes at 4 °C in the dark. Cells were washed in FACS buffer and then incubated with Alexa Fluor 488-conjugated AffiniPure Donkey anti-rabbit IgG (Jackson ImmunoResearch) at a 1 : 1000 dilution in IX intracellular staining permeabilization wash buffer (BioLegend) for 30 minutes at room temperature in the dark. Cells were washed and resuspended in FACS buffer before flow cytometry analysis.

[0392] Stimulation assays'. IxlO5T cells were plated in triplicate in 96-well plates in 100 pL R10 either alone, with 2.5 pL of CD3 / CD28 activator (STEMCELL Technologies), or with 0.5xl05Nalm6 tumor cells in a total volume of 200 pL. For each time point, a separate set of triplicate wells were plated for each experimental group. Cells were then assayed by flow cytometry at 24 hr, 48 hr, 72 hr, and 120 hr time points.

[0393] Restimulation assays'. T cells were plated as they were in stimulation assays, with at least four extra sets of cells plated to be rested and then restimulated as they would be stimulated in a stimulation assay. After the first 120 hr time point, replicate sets of cells from the same experimental group were pooled and rested for seven days in a 24-well plate. After seven days, cells were plated in the same fashion as a stimulation assay, with flow cytometry readouts at 24 hr, 48 hr, 72 hr, and 120 hr. Cells were only subject to the same stimulation condition that they were first exposed to - cells stimulated with CD3 / CD28 activator were restimulated with 2.5 pL of CD3 / CD28 activator and cells stimulated with tumor cells were restimulated with 0.5xl05tumor cells.

[0394] Cell Killing assays'. 0.5xl05CAR-T cells (CAR% normalized across different groups) were plated in triplicate and co-cultured with 0.5xl05Nalm6 tumor cells. After 18 hours, remaining tumor cells were counted by flow cytometry using CountB right absolute counting beads (Invitrogen).

[0395] IL18 ELISA: 0.5xl05CAR-T cells (CAR% normalized across different groups) were plated in triplicate and either cultured alone, with 2.5 pL of CD3 / CD28 activator (STEMCELL Technologies), or co-cultured with IxlO5Nalm6 tumor cells in 200 pL RIO. Supernatants were sampled at 24 hr and 48 hr and assayed neat with the total IL18 DuoSet ELISA kit (R&D Systems) according to manufacturer instructions.

[0396] The results of the experiments disclosed herein are now described.

[0397] Regnase-1 (Regl) and Roquin-1 (Roql)'. In T cells, post-transcriptional regulation of gene expression facilitated by RNA binding proteins (RBPs) constitutes a significant layer of control that shapes functional responses. In particular, the RBPs Regnase-1 (Regl) and Roquin-1 (Roql) are potent negative regulators of T cell inflammatory gene expression, and disruption of their endogenous function leads to hyperinflammatory pathology (Yu, et al., 2007, Nature, 450, 299-303; Matsushita, et al., 2009, Nature, 458, 1185-1190; Tavernier, et al., 2019, Nat Commun, 10, 4779; Behrens, et al., 2021, Nat Immunol, 22, 1563-1576). Recent studies have also shown that their disruption in therapeutic T cells enhances their antitumor activity, but also their toxicity (Wei, et al., 2019, Nature, 576, 471-476; Zheng, et al., 2021, Blood, 138, 122-135; Zhoa, et al., 2021, Cell Rep, 37, 110083; Mai, et al., 2023, Proc Natl Acad Sci USA, 120, e2218632120). Without wishing to be bound by theory, Both Regl and Roql bind to stem loops located at the 3’ UTR of mRNAs, many of which include factors associated with T cell activation, targeting them for destabilization and subsequent degradation in resting T cells (Vogel, et al., 2013, Immunity, 38, 655-668; Uehata, et al., 2013, Cell, 153, 1036-1049; Jeltsch, et al., 2014, Nat Immunol, 15, 1079-1089). Upon stimulation through the T cell receptor (TCR), Regl and Roql are degraded by mucosa- associated lymphoid tissue lymphoma translocation protein 1 (MALT1), transiently halting the inhibitory effects of Reg 1 and Roql activity on inflammatory gene expression (Uehata, et al., 2013, Cell, 153, 1036-1049; Jeltsch, el al, 2014, Nat Immunol, 15, 1079-1089) (FIG. 1A). Thus, it is contemplated herein to harness the strong regulatory capabilities of Regl and Roql in T cells, including NK and NKT cells and / or stem cells which have been differentiated to T cells, to modulate expression of transgenes of interest.

[0398] As disclosed herein, in order to leverage this endogenous regulatory system in primary human T cells to modulate the expression of transgenes, stem loops based on select putative stem loop targets of Regl and Roql were designed and encoded into the transgene 3’ UTR. For initial proof-of-concept, the effect of stem loops on in vitro transcribed mRNA was tested, using the clinical stage CD19-targeting CAR (CARO 19) as a model transgene. The stem loops were then encoded into lentiviral constructs to determine whether their function was affected by the presence of other expression modulating elements in the 3’ UTR, namely the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a large enhancer element that is widely used to increase transgene expression in cell therapy settings. To evaluate the dynamics of transgene modulation mediated by stem loops, the expression of regulated CARO 19 after stimulation with CD3 / CD28 or antigen-bearing tumor cells was measured. Finally, promoter-level expression tuning of regulated CARO 19 was explored and stem loop modulation was applied to other molecular payloads. Overall, it was determined that stem loops targeted by Regl and Roql are useful to dampen transgene expression and to program stimulation-induced upregulation in human T cells.

[0399] Design and testing of stem loops that mediate regulatory activity. To establish proof- of-concept of transgene modulation using stem loops targeted by Regl and Roql in primary human T cells, stem loop designs based on the well-characterized TNFa stem loop were used. Sequences of the designed stem loops used in this study are shown in the following table.

[0400] Designed stem loop sequences used in this study:

[0401] TNFa was the most dynamically upregulated gene in activated versus resting T cells among a set of gene candidates bearing stem loops targeted by Roql in their 3’ UTRs (FIG. IB). Previous work has also extensively detailed the biophysical interaction between the murine versions of Roql and the TNFa stem loop, which has high sequence and structural conservation with the human analog (Leppek, et al., 2013, Cell, 153, 869-881). While a minimal functional region of the constitutive decay element (CDE) was identified consisting of only the P2-L2 stem loop, the full P1-L1-P2-L2 structure (WT loop) was used herein as the basic design (FIGs. 2A - 2B). Next, the G-U wobble base pairing was converted into canonical Watson-Crick G-C base pairing in the Pl stem (tight loop or tLoop) to increase the folding stability of the loop, since their function is dependent on secondary structure (FIGs. 2A - 2C). The L2 bases known to interact with Roql were also altered from C-G-U to A-C-A (dead loop or dLoop) to abrogate Regl and Roql interaction as negative controls (FIGs. 2A - 2B) Because having additional loops could tighten regulation, double loop versions were designed by adding the Pl-Ll stem loop derived from ICOS, an established target of both Regl and Roql (Mai, et al., 2023, Proc Natl Acad Sci USA, 120, e2218632120; Leppek, et al., 2013, Cell, 153, 869-881; Mino, et al., 2015, Cell, 161, 1058-1073), and joining the loops with a poly-A linker (FIGs. 2A - 2B). Using CARO 19 as a model transgene, mRNAs bearing the described 3’UTR loop variants were electroporated into primary human T cells. In both CD4+ and CD8+ T cells, mRNAs bearing single or double functional loops (WT or tight) demonstrated reduced CARO 19 expression at 24 hours and onward, while those bearing dead versions showed comparable expression to the no loop control (FIGs. 3 - 6). Compared to the no loop control, the presence of the dead loops did not change CARO 19 expression dynamics, suggesting that the loops themselves do not inherently affect expression (FIG. 4, FIG. 6). These results established proof-of-concept that stem loops targeted by Regl and Roql can regulate transgene expression in primary human T cells. Since greater expression from functional single loops was observed compared to double loops, the more dynamically regulated and less “leaky” double loop variants were chosen to test in a constitutive setting using lentiviral vectors (FIG. 4, FIG. 6).

[0402] To analyze stem loop modulation in a constitutive setting using lentiviral vectors, a test system was designed with an independent reporter signal as baseline for normalization. In this system, reporter and loop-regulated transgenes are expressed by separate promoters and therefore independent mRNA transcripts (FIG. 7). With this dual-promoter system, stem loop modulated expression can be normalized to constant reporter expression. Murine phosphoglycerate kinase 1 (mPGK) and human elongation factor 1 alpha (hEFla) promoters were combined in a back-to-back orientation and monomeric EGFP (mEGFP) was used as a fluorescent reporter (FIG. 8) (Zacharias, et al., 2002, Science, 296, 913-916). Additionally, whether stem loop regulation is impacted by the WPRE sequence was tested using versions of the test system with and without WPRE downstream of the stem loop (FIG. 8), because WPRE forms a large tertiary mRNA stabilizing structure and is widely used to boost transgene expression (Zufferey, et al., 1999, J Virol, 73, 2886-2892).

[0403] In line with results using electroporated mRNA, stem loops also demonstrated regulatory function in a constitutive setting in both CD4+ and CD8+ primary human T cells (FIGs. 9-11, 13-15). Comparing median fluorescent intensities (MFIs) of CARO 19 normalized to mEGFP (z.e., MFI CAR / MFI mEGFP = normalized expression or “NE”), it was observed that constructs with functional loops, 2Loop and t2Loop, demonstrated dampened NE whereas analogous constructs with dead loops, d2Loop and dt2Loop, retained comparable NE to each other and to the no loop control (FIGs. 9, 13). While not statistically significant, the “tightened” t2Loop showed slightly lower NE compared to 2Loop, suggesting that energetic changes such as replacing wobble base pairing with more energetically favorable Watson-Crick base pairing even in structures not directly required to mediate function (Pl stem) can potentially influence regulation in designed secondary RNA structures (FIGs. 9, 13). Further, the WPRE did not significantly affect stem loop regulation and instead only contributed to an overall increase in NE with the WPRE present (FIGs. 9-11, 13-15). To quantify this, the “regulation ratio”, defined as the ratio between dead loop normalized expression (NE dead) and functional loop normalized expression (NE functional), was compared between constructs with and without the WPRE (FIGs. 12, 16). A greater ratio indicated a larger difference between transgene expression and transgene repression with stem loops. There was no significant difference in the regulation ratios between constructs with or without the WPRE in both the normal and tight loop versions (FIGs. 12, 16).

[0404] However, the tight loop version displayed a greater regulation ratio, indicating that it has a greater functional range between transgene expression and repression. Thus, the tight loop version was used for subsequent experiments (FIGs. 12, 16).

[0405] Confirming the regulatory roles ofRegnase-1 andRoquin-1 on stem loop mediated transgene modulation'. Although stem loop designs were inspired by putative targets of Regl and Roql and dead loop controls gave a degree of confidence that regulation was dependent on Regl and Roql, the roles of Regl and Roql were confirmed by measuring how Regl- deficient and Roql -deficient T cells behaved when transduced with a transgene regulated by these loops. To this end, Regl and Roql double knockout (DKO) primary human T cells were generated and transduced with the t2Loop-regulated CARO 19 alongside no loop and dt2Loop controls. In both CD4+ and CD8+ T cells, t2Loop attenuated CARO 19 expression while dt2Loop maintained similar CARO 19 expression compared to the no loop control in unedited Mock cells, as expected (FIGs. 17-26). However, DKO of Regl and Roql attenuated expression modulation, rescuing CARO 19 expression to levels near that of the no loop control and confirming that regulatory function is mediated through these RBPs (FIGs. 17-26). These observations suggest that modulation of transgene expression by inclusion of stem loops in transcript 3’UTRs in human T cells requires Regl and Roql activity. To determine whether harnessing endogenous Regnase-1 and Roquin-1 to regulate exogenous transgenes would act as a sink to reduce regulation on natively regulated genes, ICOS expression was measured in T cells transduced with CARO 19 regulated by stem loops (FIGs. 27-30). Since ICOS is a putative target of Regnase-1 and Roquin-1, and the ICOS stem loop was used in the present study as part of the t2Loop construct, sink activity could theoretically be detected by measuring any changes in ICOS expression in cells with additional transcripts regulated in part with the ICOS stem loop. In CD4 T cells, no differences were observed in ICOS expression in t2Loop regulated cells compared to cells regulated with No Loop and dt2Loop (FIGs. 27-30).

[0406] T cell stimulation can trigger upregulation in transgenes regulated by stem loops'. Various studies have established that signaling downstream of the TCR transiently represses Regl and Roql activity through the MALT1 paracaspase (Uehata, et al., 2013, Cell, 153, 1036-1049; Jeltsch, et al., 2014, Nat Immunol, 15, 1079-1089). As a result, transcripts targeted by Regl or Roql are under a layer of post-transcriptional repression when T cells are resting and become derepressed upon activation through TCR stimulation (FIG. 31A) (Uehata, et al., 2013, Cell, 153, 1036-1049; Jeltsch, et al., 2014, Nat Immunol, 15, 1079- 1089). It is contemplated herein that this dynamic could be harnessed to program activationspecific upregulation of transgene expression. In particular, whether activation through an engineered receptor bearing analogous components of TCR intracellular domains, such as a CAR, can facilitate the same effect was explored herein (FIG. 31A). To examine this, the expression dynamics of a stem loop regulated transgene after stimulation through anti- CD3 / CD28 or through antigen specific to the engineered CAR receptor was monitored at 24, 48, 72, and 120 hours. The same constructs were used as previously described with CARO 19 as the stem loop regulated transgene, and changes in CARO 19 expression after stimulation through the CAR itself were observed. Specifically, in the absence of stimulation, CARO 19 expression remained consistent over time in all groups. The dt2Loop demonstrated comparable CARO 19 expression to no loop, whereas t2Loop showed dampened expression (FIGs. 33-39, 84-87). Treatment with anti-CD3 / CD28 stimulation induced similar fold changes of CARO 19 upregulation in both the dt2Loop and no loop groups but the greatest upregulation in the t2Loop group (FIGs. 33-39, 84-87). Upregulation of CARO 19 expression was transient and increased up to about two days before expression began to taper back down toward expression levels in unstimulated cells (FIGs. 33-39, 84-87). Similarly, stimulation through the CAR using CD 19-positive Nalm6 tumor cells also induced the greatest expression upregulation in the t2Loop group and lower, comparable levels of upregulation in the dt2Loop and no loop groups (FIGs. 33-39, 84-87). These dynamics suggest that stem loops can facilitate transgene upregulation in response to both CD3 / CD28 and antigen stimulation.

[0407] Next, whether stimulation-induced upregulation could be engaged more than once was tested. To explore this, stimulated cells were rested for seven days before restimulation with either CD3 / CD28 activator or antigen and CARO 19 expression was measured at the same previous time points relative to when cells were stimulated (FIG. 31B). After resting for seven days, expression of CARO 19 returned to levels similar to those before activation (FIGs. 40-45). Upon stimulation with CD3 / CD28 activator, CARO 19 expression increased again in both t2Loop and no loop groups, with some variability to the degree of upregulation relative to the first stimulation across different donors (FIGs. 40-45). CARO 19 modulated by t2Loop demonstrated a greater MFI fold change compared to CARO 19 with no loop and differences in upregulation between t2Loop and no loop groups were greater in CD4 T cells than CD8 T cells (FIGs. 40-45). Stimulation with antigen produced more nuanced responses, with smaller differences in MFI fold changes between CARO 19 modulated with t2Loop and no loop (FIGs. 40-45). This may be partially due to idiosyncratic dynamics of CAR expression upon engagement with antigen; in one donor, co-culture with CAR-T cells and antigen-bearing Nalm6 tumor cells induced CAR suppression and other CARs have also demonstrated similar antigen-associated downregulation (FIGs. 38-39) (Good, et. al., 2021, Cell, 184(25): 6081-6100). These results suggest that stimulation-induced upregulation mediated by stem loops have the capacity to be re-engaged, particularly with CD3 / CD28 stimulation.

[0408] While CARO 19 tagged with stem loops was initially used in the present study as a model transgenes to gauge whether CAR self regulation could be induced through its own downstream effects on Regnase-1 and Roquin-1 activity, dampened CAR expression typically reduced antitumor activity. To confirm this, T cells expressing CARO 19 regulated with No Loop, t2Loop, or dt2Loop, were co-cultured with CD19-expressing Nalm6 cells to measure target cell killing ability (FIG. 46). As expected, regulation of CARO 19 with t2Loop resulted in lower CAR expression, leading to reduced cell killing activity at 48 hours and 96 hours, compared to CARO 19 regulated with No Loop or dt2Loop (FIG. 46). This suggests that CARO 19, at least functionally, may not be the most ideal transgene to regulate in this setting, and that other transgene candidates may be more optimal candidates for beneficial regulation towards enhancing cellular therapies.

[0409] Tuning of stem loop mediated transgene modulation'. In the systems described and tested thus far herein, the relatively strong hEFla promoter was used to drive expression of the regulated transgene. As a result, while expression regulation with the t2Loop was observed compared to controls, transgene expression is still present compared to untransduced cells. This may or may not be desirable depending on the application. With a CAR as the model transgene, which can be used to induce modulation of itself, some level must be present at baseline to be able to trigger regulation. However, with other transgenes, it may be unnecessary or undesirable to have baseline expression. Thus, the next experiments were designed to explore functionally tuning regulated expression levels to make them very low or absent at baseline.

[0410] To achieve this, the hEFla promoter was replaced with weaker promoters including a previously reported weaker variant of hEFla, hEFla-3 (Zheng, el al.. 2014, IntJMed Sci, 11, 404-408), and the ubiquitin C (UbC) promoter, which is reported to be one of the weakest across different cell types (FIG. 47) (Qin, et al., 2010, PLoS One, 5, el0611). With the CAR as the regulated transgene, exchanging the hEFla with hEFla-3 led to no differences in CARO 19 expression in either t2Loop or no loop versions (FIGs. 48, 49, 88-90 ). In contrast, exchanging the hEFla promoter for the UbC promoter decreased CARO 19 expression in both t2Loop and no loop versions, with the t2Loop version nearing UTD levels of expression (FIGs. 48, 49, 88-90). To confirm that these expression level differences have functional effects, the tumor killing abilities of each of these versions was evaluated. With the hEFla promoter, CARO 19 with no loop had the greatest killing, whereas CARO 19 with t2Loop had slightly weaker killing (FIGs. 50A, 50B). Switching hEFla for hEFla-3 did not alter killing function, whereas switching it for UbC led to decreased killing in both t2Loop and no loop groups, with the t2Loop group decreasing to killing function comparable to that of UTD cells (FIGs. 50A, 50B). These results indicate that varying the promoter strength provides another layer of control to functionally tune the desired regulated expression levels without disrupting the regulatory function of stem loops.

[0411] Application of stem loop mediated regulation to other transgenes in CAR-T cells'. With evidence that expression of transgenes regulated by stem loops could be tuned without affecting stem loop function, this regulation was next applied to other transgenes in T cells. Of particular interest in T cells are therapeutic payloads, such as effector cytokines or factors that promote survival or mitigate exhaustion (Koneru, et al., 2015, Oncoimmunology, 4, e994446; Lynn, et al., 2019, Nature, 576, 293-300). To apply and test stem loop modulation on other transgenes in CAR-T cells, new constructs were made by swapping out CARO 19 from the previous construct architecture with other transgenes and moving it under the mPGK promoter (FIGs. 51, 55). The cytokine IL 18 and the pro-survival factor Bcl-xL were tested as examples of secreted and intracellular payloads, respectively (FIGs. 51, 55). The stronger hEFla promoter was used to test regulation of IL 18 (FIG. 51) because baseline expression of IL 18 has not been shown to be toxic and may be beneficial (Jaspers, et al., 2023, J Clin Invest, 133). On the other hand, expression of the pro-survival factor Bcl-xL is implicated in some malignancies (Trisciuoglio, et al., 2017, Cell Death Dis, 8, 3216; Scherr, et al., 2020, Cell Death Dis, 11, 875), motivating stricter control of baseline expression levels and use of the weaker UbC promoter (FIG. 55).

[0412] Application of stem loop regulation to IL18 in CAR-T cells dampened IL18 secretion across different stimulation conditions (FIGs. 52, 53, 54). Interestingly, although the strong hEFla promoter was used to drive IL18 expression, below background levels of secreted IL18 were observed in resting cells (FIGs. 52, 53, 54). Stimulation with both anti-CD3 / CD28 and antigen ligation of the CAR led to high IL18 secretion, but lower IL18 secretion when regulated with t2Loop compared to no loop (FIGs. 52, 53, 54). Application to Bcl-xL in CAR-T cells led to expression dynamics more consistent with activation-specific upregulation.

[0413] Next, Bcl-xL expression was measured across different stimulation conditions using truncated human low-affinity nerve growth factor receptor (tLNGFR) as a control transgene. Without stimulation, use of the t2Loop dampened Bcl-xL expression compared to no loop. However, greater Bcl-xL expression was observed compared to that of tLNGFR and UTD controls using either the t2Loop regulation or no loop regulation (FIGs. 56 - 61). Stimulation of the TCR with anti-CD3 / CD28 increased Bcl-xL expression in all groups to comparable expression levels (FIGs. 56 - 61). Compared to no stimulation, CD3 / CD28 stimulation only slightly increased Bcl-xL expression in CAR-T cells with no loop regulation on Bcl-xL, but approximately doubled the Bcl-xL MFI in CAR-T cells with Bcl-xL under t2Loop regulation (FIGs. 56 - 61)

[0414] These dynamics indicate that these stem loops have the capacity to serve as expression dampeners in resting human CAR-T cells, which may be useful for controlling beneficial but potentially risky transgenes. Stimulation with antigen also equalized Bcl-xL expression levels between CAR-T cells with either t2Loop regulation or no loop regulation of Bcl-xL, which still displayed slightly greater Bcl-xL MFIs compared to CAR-T cells with tLNGFR controls and UTD cells (FIGs. 56 - 61). While antigen stimulation equalized Bcl- xL expression in Bcl-xL expressing CAR-T cells, this was due to a decrease in Bcl-xL MFI in the CAR-T cells having no loop regulation, highlighting potential differences in downstream CAR signaling versus signaling through CD3 and CD28. Overall, these studies indicate that stem loops can be used to independently modulate expression of other transgenes in human CAR-T cells.

[0415] To assess the regulatory dynamics of stem loops on other receptors, t2Loop regulation of a mesothelin-targeting CAR (CARM5 or M5 CAR) was tested (FIGs. 62-72), as well as that of a BCMA-targeting VHH-based CAR (BCMA VHH CAR) (FIGs. 73-83). Using the M5 CAR, stimulation with CD3 / CD28 activator induced greater CAR MFI fold change in t2Loop regulated versus unregulated M5 CAR (FIGs. 63-68). However, stimulation with antigen induced CAR downregulation in both the t2Loop regulated and unregulated M5 CAR (FIGs. 63-68). Similarly, when an additional stimulation was performed after 7 days of rest, stimulation through CD3 / CD28 induced a greater CAR MFI fold change in t2Loop regulated versus unregulated M5 CAR, though the magnitude of this upregulation was lower than in the first round of stimulation (FIGs. 69-72). Rest and restimulation with antigen did not recover M5 CAR expression (FIGs. 69-72). Using the BCMA VHH-based CAR, general upregulation under both CD3 / CD28 stimulation and antigen stimulation was observed; however, the VHH-based CAR MFI fold change in t2Loop regulated VHH-CAR T cells was similar to or lower than unregulated VHH-CAR T cells in both stimulation conditions (FIGs. 74-79). Restimulation after rest yielded similar dynamics to the original stimulation, with t2Loop regulated VHH-CAR T cells displaying similar or lower MFI fold changes in response to stimulation compared to unregulated VHH-CAR T cells (FIGs. 80-83). These results indicate that specific expression dynamics may be dependent on the transgene or receptor used.

[0416] Enumerated Embodiments

[0417] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance. Embodiment 1 : A vector comprising: (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and

[0418] (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg-1) and / or Roquin-1 (Roq-1) when Reg-1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0419] Embodiment 2: The vector of embodiment 1, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

[0420] Embodiment 3: The vector of embodiment 1 or embodiment 2, wherein the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

[0421] Embodiment 4: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop.

[0422] Embodiment 5: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0423] Embodiment 6: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0424] Embodiment 7: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO. Embodiment 8: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0425] Embodiment 9: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

[0426] Embodiment 10: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a Pl-Ll region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly- A linker.

[0427] Embodiment 11 : The vector of any one of the preceding embodiments, wherein G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson- Crick G-C base pairing.

[0428] Embodiment 12: The vector of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

[0429] Embodiment 13: The vector of any one of the preceding embodiments, further comprising a WPRE.

[0430] Embodiment 14: The vector of any one of the preceding embodiments, wherein the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus). Embodiment 15: The vector of any one of the preceding embodiments, wherein the exogenous receptor is a CAR, further wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen.

[0431] Embodiment 16: The vector of any one of the preceding embodiments, wherein the exogenous receptor is a CAAR, further wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody-binding fragment thereof.

[0432] Embodiment 17: The vector of any one of the preceding embodiments, wherein the exogenous receptor is a TCR.

[0433] Embodiment 18: The vector of any one of the preceding embodiments, wherein the exogenous receptor is a switch receptor (e.g., PD1-CD28).

[0434] Embodiment 19: The vector of any one of the preceding embodiments, wherein the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

[0435] Embodiment 20: The vector of any one of the preceding embodiments, wherein the vector is a viral vector, (e.g., a lentiviral vector).

[0436] Embodiment 21 : A modified cell comprising the vector of any one of the preceding embodiments.

[0437] Embodiment 22: A modified cell comprising a vector, wherein the modified cell is selected from the group consisting of a T cell, a CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell; and wherein the vector comprises:

[0438] (a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and (b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

[0439] Embodiment 23: The modified cell of embodiment 22, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

[0440] Embodiment 24: The modified cell of embodiment 22 or embodiment 23, wherein the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

[0441] Embodiment 25: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop.

[0442] Embodiment 26: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0443] Embodiment 27: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

[0444] Embodiment 28: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10. Embodiment 29: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

[0445] Embodiment 30: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

[0446] Embodiment 31 : The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a Pl-Ll region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

[0447] Embodiment 32: The modified cell of any one of the preceding embodiments, wherein G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson-Crick G-C base pairing.

[0448] Embodiment 33: The modified cell of any one of the preceding embodiments, wherein the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs l, 2, 5, and 6.

[0449] Embodiment 34: The modified cell of any one of the preceding embodiments, wherein the vector further comprises a WPRE.

[0450] Embodiment 35: The modified cell of any one of the preceding embodiments, wherein the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

[0451] Embodiment 36: The modified cell of any one of the preceding embodiments, wherein the exogenous receptor is a CAR, further wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen.

[0452] Embodiment 37: The modified cell of any one of the preceding embodiments, wherein the exogenous receptor is a CAAR, further wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody-binding fragment thereof.

[0453] Embodiment 38: The modified cell of any one of the preceding embodiments, wherein the exogenous receptor is a TCR.

[0454] Embodiment 39: The modified cell of any one of the preceding embodiments, wherein the exogenous receptor is a switch receptor (e.g., PD1-CD28).

[0455] Embodiment 40: The modified cell of any one of the preceding embodiments, wherein the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

[0456] Embodiment 41 : The modified cell of any one of the preceding embodiments, wherein the vector is a viral vector, (e.g., a lentiviral vector).

[0457] Embodiment 42: The modified cell of any one of the preceding embodiments, wherein the modified cell is a human cell.

[0458] Embodiment 43: A pharmaceutical composition comprising a population of the modified cell of any one of embodiments 22-42 and at least one pharmaceutically acceptable carrier.

[0459] Embodiment 44: A method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the modified cell of any one of embodiments 22-42 or the pharmaceutical composition of embodiment 43 to the subject. Embodiment 45: The method of embodiment 44, wherein the disease, disorder, or condition is selected from a cancer, an autoimmune disease, and a neurodegenerative disorder or condition. Embodiment 46: The method of embodiment 44 or embodiment 45, wherein the subject is a human.

[0460] Other Embodiments

[0461] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed:

1. A vector compri sing :(a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and(b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

2. The vector of claim 1, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

3. The vector of claim 1 or claim 2, wherein the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

4. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop.

5. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

6. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

7. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

8. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

9. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

10. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a PILI region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

11. The vector of any one of the preceding claims, wherein G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson-Crick G- C base pairing.

12. The vector of any one of the preceding claims, wherein the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

13. The vector of any one of the preceding claims, further comprising a WPRE.

14. The vector of any one of the preceding claims, wherein the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, aconstitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

15. The vector of any one of the preceding claims, wherein the exogenous receptor is a CAR, further wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen.

16. The vector of any one of the preceding claims, wherein the exogenous receptor is a CAAR, further wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody-binding fragment thereof.

17. The vector of any one of the preceding claims, wherein the exogenous receptor is a TCR.

18. The vector of any one of the preceding claims, wherein the exogenous receptor is a switch receptor (e.g., PD1-CD28).

19. The vector of any one of the preceding claims, wherein the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

20. The vector of any one of the preceding claims, wherein the vector is a viral vector, (e.g., a lentiviral vector).

21. A modified cell comprising the vector of any one of the preceding claims.

22. A modified cell comprising a vector, wherein the modified cell is selected from the group consisting of a T cell, a CD4 T cell, a CD8 T cell, a natural killer (NK) cell, an NKT cell, and a stem cell which has been differentiated to a T cell; and wherein the vector comprises:(a) a first nucleotide sequence encoding an exogenous receptor under control of a first promoter; and(b) a second nucleotide sequence encoding a transgene and a 3’ UTR stem loop under control of a second promoter, wherein the 3’ UTR stem loop is bound by Regnase-1 (Reg- 1) and / or Roquin-1 (Roq-1) when Reg- 1 and / or Roq-1 are present, further wherein binding of the 3’ UTR stem loop by Reg-1 and / or Roq-1 targets the transgene transcript for sequestration and / or degradation, thereby inhibiting expression of the transgene.

23. The modified cell of claim 22, wherein the exogenous receptor is selected from a chimeric antigen receptor (CAR), a chimeric autoantibody receptor (CAAR), a T cell receptor (TCR), a switch receptor, and a dominant negative receptor.

24. The modified cell of claim 22 or claim 23, wherein the 3’ UTR stem loop comprises at least a single stem loop or at least a double stem loop.

25. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a double stem loop comprising a first stem loop and a second stem loop, wherein the first stem loop is linked via a poly-A linker to the second stem loop.

26. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a portion of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

27. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

28. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a single stem loop comprising at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPPIRlO.

29. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a double stem loop, wherein a second stem loop of the double stem loop comprises at least a Pl-Ll region of a 3’ UTR stem loop from a gene selected from the group consisting of TNFa, ICOS, IER3, NFKBID, and PPP1R10.

30. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa.

31. The modified cell of any one of the preceding claims, wherein the 3 ’ UTR stem loop comprises at least a P1-L1-P2-L2 region of a 3’ UTR stem loop from TNFa and a PILI region of a 3’ UTR stem loop from ICOS, wherein the P1-L1-P2-L2 region is linked to the Pl-Ll region via a poly-A linker.

32. The modified cell of any one of the preceding claims, wherein G-U wobble base pairing within a Pl stem of the 3’ UTR stem loop is converted to canonical Watson- Crick G-C base pairing.

33. The modified cell of any one of the preceding claims, wherein the 3’ UTR stem loop comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 1, 2, 5, and 6.

34. The modified cell of any one of the preceding claims, wherein the vector further comprises a WPRE.

35. The modified cell of any one of the preceding claims, wherein the transgene is selected from the group consisting of a cytokine, a chemokine, a switch receptor, a dominant negative receptor, a dominant negative intracellular protein, a kinase, a phosphatase, a transcription factor, an antibody or fragment thereof, a toxin, a superantigen, an enzyme, a cell penetrating peptide, a regulatory RNA, an RNA aptamer, a constitutively active signaling moiety, and an oncolytic virus genome (e.g., a small RNA virus).

36. The modified cell of any one of the preceding claims, wherein the exogenous receptor is a CAR, further wherein the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, preferably wherein the antigen binding domain binds a tumor antigen.

37. The modified cell of any one of the preceding claims, wherein the exogenous receptor is a CAAR, further wherein the CAAR comprises an extracellular domain comprising an autoantigen domain, a transmembrane domain, and an intracellular domain, wherein the autoantigen domain comprises an autoantigen or autoantibody-binding fragment thereof.

38. The modified cell of any one of the preceding claims, wherein the exogenous receptor is a TCR.

39. The modified cell of any one of the preceding claims, wherein the exogenous receptor is a switch receptor (e.g., PD1-CD28).

40. The modified cell of any one of the preceding claims, wherein the exogenous receptor is a dominant negative receptor (e.g., a dominant negative TGFbRII).

41. The modified cell of any one of the preceding claims, wherein the vector is a viral vector, (e.g., a lentiviral vector).

42. The modified cell of any one of the preceding claims, wherein the modified cell is a human cell.

43. A pharmaceutical composition comprising a population of the modified cell of any one of claims 22-42 and at least one pharmaceutically acceptable carrier.

44. A method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering an effective amount of the modified cell of any one of claims 22-42 or the pharmaceutical composition of claim 43 to the subject.

45. The method of claim 44, wherein the disease, disorder, or condition is selected from a cancer, an autoimmune disease, and a neurodegenerative disorder or condition.

46. The method of claim 44 or claim 45, wherein the subject is a human.

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