Inducible transgenes encoding intracellular molecules for modulating immune cell function

Abstract

Artificial expression constructs including a dnSHP1, caBCL2, dnNRDP1, PCK1, cFLIP.v3, caAKT, survivin, or dnFADD transgene under the control of an inducible promoter are described. The artificial expression constructs can be used to enhance the function of a recombinant receptor-immune cell (e.g., CAR T-cell). The dnSHP1, caBCL2, dnNRDP1, PCK1, cFLIP.v3, caAKT, survivin, or dnFADD transgene disclosed herein enhances the potency of the recombinant receptor-immune cell (e.g., CAR T-cell), resulting in potentiated cell killing ability, proliferation, cytokine output, and / or maintenance of artificial expression construct positivity.

Description

INDUCIBLE TRANSGENES ENCODING INTRACELLULAR MOLECULES FOR MODULATING IMMUNE CELL FUNCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 588,598 filed October 6, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3B85512.XML. The file is 244,714 bytes, was created on October 1 , 2024, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The current disclosure provides artificial expression constructs including transgenes encoding intracellular molecules under the control of an inducible promoter. The artificial expression constructs can be used to modulate the function of an immune cell (e.g., CAR T-cell).BACKGROUND OF THE DISCLOSURE

[0004] According to the World Health Organization, cancer is a leading cause of death globally, and was responsible for nearly 10 million deaths in 2020. Traditional treatments such as surgery, chemotherapy, and / or radiation therapy have been around for more than a century and impact normal as well as cancerous cells. Further, surgery, chemotherapy, and radiation therapy have significant and sometimes permanent side effects. In recent years, more targeted therapies for cancer have been developed.

[0005] Targeted therapies specifically target cancer cells by identifying and exploiting molecular and / or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express markers on their cellular surfaces which may be used as targets for antibody-based therapeutics. Some targeted therapies use genetically engineered cells of the immune system such as chimeric antigen receptor (CAR)-expressing cells (e.g., CAR T-cells) to target and kill unwanted cell types, such as cancer cells.

[0006] CARs are proteins with several distinct subcomponents, that allow genetically modified immune-cells to recognize and kill targeted cell types. The subcomponents of CAR include at least an extracellular component and an intracellular component expressed as a single protein or in a form that assembles into a functional unit. The extracellular component includes a binding domain that binds a marker (e.g., an antigen) that is preferentially present on the surface ofunwanted cells such as cancer cells. When the binding domain binds such markers, the intracellular component signals the immune-cell to destroy the bound cell. CAR can additionally include a transmembrane domain that can link the extracellular component to the intracellular component.

[0007] Other subcomponents of CAR can also be used to increase CAR function. For example, spacers provide CAR with additional conformational flexibility and often increase the binding domain’s ability to bind the targeted cell marker, leading to enhanced cytolytic effects. The appropriate length of a spacer within a particular CAR depends on numerous factors including how close or far a targeted marker is located from the surface of an unwanted cell’s membrane.

[0008] Although CAR T-cells have had substantial success in treating various cancers, challenges remain. For example, CAR T-cells can have suboptimal proliferation, functional anergy or exhaustion, impaired memory differentiation, and / or limited persistence, limiting their ability to create or maintain remission in patients. There may additionally be targeted therapy resistance that needs to be addressed. Thus, strategies to improve CAR T-cell efficacy are needed, especially for scenarios with prolonged antigen exposure that can lead to functional anergy and exhaustion.SUMMARY OF THE DISCLOSURE

[0009] The current disclosure provides artificial expression constructs including a transgene encoding an intracellular molecule under the control of an inducible promoter to modulate the function of an immune cell (e.g., T-cell or chimeric antigen receptor (CAR) T-cell). The transgenes as disclosed herein express a molecule that is not secreted, but remains in the genetically modified cell. Thus, only the activity of the genetically modified cell is directly modified. The inducible intracellular molecules disclosed herein modulate the potency of the immune cell (e.g., T-cell or CAR T-cell) resulting in potentiated cell killing ability, proliferation, cytokine output, and / or artificial expression construct transduction positivity of the genetically modified cell. In particular embodiments, cytokine output includes interleukin (IL)2 secretion, TNFa secretion, and / or IFNy secretion.

[0010] In some aspects, the artificial expression constructs described herein may be used to tailor a treatment to a specific patient or group of patients. For example, different cancers may respond differently to different immune cell enhancements. Further, different immune cell enhancements may be more or less appropriate for different types, stages, or growth rates of various cancers including blood / bone marrow cancers and solid tumor cancers. As described in further detail herein, stimulation of artificial constructs containing dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2),dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), survivin, and dominant negative fas-associating protein with death domain (dnFADD) had varying and unpredictable effects on CD4+ and CD8+ T-cells.

[0011] The varying activity and levels of activity of the artificial constructs may be useful in tailoring a treatment to a specific patient or group of patients as varying effects may be desired in the treatment of different cancers or different populations of individuals suffering from a particular cancer. For example, even if two patients have the same type of cancer, they may have different lifestyle, epigenetic, or genetic variations that may make them more or less responsive to different therapeutic treatments.

[0012] In particular embodiments, the transgene encodes at least one intracellular molecule selected from dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD. Intracellular molecules may enhance or otherwise modify the activity of the CAR T cells in killing, proliferation, cytokine output, and maintenance of artificial expression construct transduction positivity (e.g., EGFRt positivity), a marker for CAR positive T cells. In some aspects, expression of the transgenes may be regulated by one or more inducible promoters such as an iSynPro promoter. In some aspects, the use of an inducible promoter may provide potentiation of immune cell activity regardless of the presence of one or more transgenes. In some aspects, encoding more than one of the intracellular molecules selected from the group including dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD may create synergistic effects.

[0013] Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to modify immune-cell mediated cell killing. Particular embodiments utilize dnNRDPI to enhance immune-cell mediated killing by CD4+ cells. Particular embodiments utilize dnSHPI , caBCL2, and survivin to enhance immune-cell mediated killing by CD8+ cells. Particular embodiments utilize dnSHPI , caBCL2, survivin, or PCK1 to enhance immune-cell mediated killing by CD8+ cells.

[0014] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to enhance proliferation. Particular embodiments utilize inducible expression of dnNRDPI to enhance CD4+ T-cell proliferation. Particular embodiments utilize inducible expression of dnSHPI or caBCL2 to enhance CD8+ T- cell proliferation.

[0015] Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to modify cytokine output. Particular embodimentsutilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to enhance cytokine output.

[0016] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 cFLIP.v3, caAKT, survivin, and dnFADD to modify IL2 secretion. Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnFADD, cFLIP.v3, caAKT, dnSHPI , or caBCL2 to increase IL2 secretion by CD4+ cells. Particular embodiments utilize inducible expression of caAKT, dnSHPI , caBCL2, dnFADD, or dnNRDPI to increase IL2 secretion by CD8+ cells.

[0017] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to modify TNFa secretion. Particular embodiments utilize inducible expression of dnFADD, cFLIP.v3, caBCL2, dnNRDPI , or dnSHPI to increase TNFa secretion in CD4+ cells. Particular embodiments utilize inducible expression of caAKT, dnNRDPI , dnFADD, cFLIP.v3, dnSHPI , caBCL2, PCK1 , or survivin, to increase TNFa secretion in CD8+ cells.

[0018] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to modify IFNy secretion. Particular embodiments utilize inducible expression of dnFADD, caAKT, dnNRDPI , caBCL2, or cFLIP.v3 to increase IFNy secretion in CD4+ cells. Particular embodiments utilize inducible expression of dnNRDPI , dnSHPI , caAKT, surivin, or caBCL2 increases IFNy in CD8+ cells.

[0019] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to maintain artificial expression construct transduction positivity (e.g., truncated epidermal growth factor receptor (EGFRt) positivity) by cells. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, survivin, dnNRDPI , PCK1 , or dnFADD to maintain artificial expression construct transduction positivity by CD4+ T-cells. Particular embodiments utilize caAKT, caBCL2, PCK1 , dnNRDPI , survivin dnSHPI , dnFADD, or cFLIP.v3 to maintain artificial expression construct transduction positivity by CD8+ T-cells.

[0020] In particular embodiments, cells may be stimulated one or more times depending on the desired effect. For example, cells may be stimulated 1 , 2, 3, 4 or more times. Particular embodiments utilize stimulation to potentiate cell killing ability, proliferation, cytokine output, and / or maintenance of artificial expression construct transduction positivity. In particular embodiments, cytokine output includes IL2 secretion, TNFa secretion, and / or IFNy secretion.

[0021] Particular embodiments utilize inducible expression of dnFADD, cFLIP.v3, dnSHPI , caBCL2, or caAKT to enhance CD4+ IL2 secretion after a first stimulation. Particularembodiments utilize inducible expression of PCK1 , dnNRDPI or survivin to enhance CD4+ IL2 secretion after a third stimulation. Particular embodiments utilize inducible expression of caAKT, dnFADD, dnSHPI , or caBCL2 to enhance CD8+ IL2 secretion after a first stimulation. Particular embodiments utilize inducible expression of caAKT, dnFADD, dnNRDPI , dnSHPI , or caBCL2 to enhance CD8+ IL2 secretion after a third stimulation.

[0022] Particular embodiments utilize inducible expression of dnFADD, cFLIP.3, dnNRDPI , dnSHPI , or caBCL2 to enhance CD4+ TNFa secretion after a first stimulation. Particular embodiments utilize inducible expression of dnFADD or dnNRDPI to enhance CD4+ TNFa secretion after a third stimulation. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to enhance CD8+ expression of TNFa after a first stimulation. Particular embodiments utilize inducible expression of dnFADD, caAKT, PCK1 , dnNRDPI , dnSHPI , or caBCL2 to enhance CD8+ expression of TNFa after a third stimulation.

[0023] Particular embodiments utilize inducible expression of dnFADD, dnNRDPI , or caBCL2 to enhance CD4+ expression of IFNy after a first stimulation. Particular embodiments utilize inducible expression of dnFADD, caAKT, dnNRDPI , cFLIP.v3, or caBCL2 to enhance CD4+ expression of IFNy after a third stimulation. Particular embodiments utilize inducible expression of caAKT, dnNRDPI , survivin, dnSHPI , PCK1 , or caBCL2 to enhance CD8+ expression of IFNy after a first stimulation. Particular embodiments utilize inducible expression of dnSHPI or caBCL2 to enhance CD8+ expression of IFNy after a third stimulation

[0024] Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD4+ immune cells after a first stimulation. Particular embodiments utilize inducible expression of dnFADD, PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD4+ immune cells after a third stimulation. Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD8+ immune cells after a first stimulation. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to maintain artificial expression construct transduction positivity by CD8+ immune cells after a third stimulation.

[0025] In particular embodiments, expression of the transgene may be under the control of a promoter. In particular embodiments, the promoter includes an iSynPro promoter. In particular embodiments, the iSynPro promoter includes S1 -61 (SEQ ID NO: 17 or SEQ ID NO: 18). In particular embodiments, the iSynPro promoter includes S1 -61 (SEQ ID NO: 17) operably linkedto an IL2 minimal promoter (SEQ ID NO: 57). In particular embodiments, an artificial expression construct with the transgene encoding an intracellular molecule further includes or encodes a skip sequence and a control feature.

[0026] In particular embodiments, the immune cell includes a T-cell. In particular embodiments, the immune cell can be engineered to express a recombinant receptor. In particular embodiments, an artificial expression construct encoding the recombinant receptor can be included in the same or a different artificial expression construct from the intracellular transgene. In particular embodiments, the artificial expression construct encoding the recombinant receptor includes or encodes a promoter and a recombinant receptor. In particular embodiments, the artificial expression construct encoding the recombinant receptor includes or encodes a promoter, a recombinant receptor, a skip sequence, and a control feature. In particular embodiments, the recombinant receptor includes a CAR or engineered T-cell receptor (eTCR). In particular embodiments, the recombinant receptor includes an extracellular component that binds a target antigen, a transmembrane domain, and an intracellular component.

[0027] Thus, the current disclosure provides systems and methods to modulate the killing, proliferation, cytokine output, and / or maintenance of artificial expression construct transduction positivity of immune cells. The immune cells can include recombinant receptor-expressing immune cells which can be engineered to target different target cells (e.g., cancer cell, virus, bacteria, fungi) expressing a target antigen by engineering the extracellular domain to include a binding domain that binds the target antigen.BRIEF DESCRIPTION OF THE FIGURES

[0028] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0029] FIG. 1 . Example template artificial expression construct used to screen transgenes for chimeric antigen receptor (CAR) T-cell modulation in killing targeted cells, immune cell proliferation, cytokine output, and maintenance of EGFRt levels.

[0030] FIG. 2. Construct architecture to facilitate T-cell production and screening. To validate that T-cells expressing a single intracellular transgene such as dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active-B cell lymphoma 2 (ca- BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-i p-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), and dominant negative fas- associating protein with death domain (dnFADD) are potency modulators for CAR T-cells, therespective intracellular transgene open reading frames (ORFs) were placed under the regulation of a CAR inducible iSynPro promoter and also placed into a dual promoter construct format. Downstream of this cargo is an elongation factor 1 -alpha (EF1 a(L)) promoter that drives expression of a CAR (CD19CAR), double mutant dihydrofolate reductase (DHFRdm), and truncated epidermal growth factor receptor (EGFRt) with a P2A and T2A separating CAR and DHFRdm as well as the DHFRdm and EGFRt, respectively.

[0031] FIG. 3. Schema for Screening Pipeline. Potency modulator screening schema. CD4+ or CD8+ T cells were separately modified with transposable constructs expressing a CD19CAR or a CD19CAR with an inducible form of a putative potency modulator. T-cells were expanded using T cell interface for CAR limited expansion (TICLE) and subjected to a suite of assays designed to screen for potency enhancements. A scoring system was utilized that tracks T cell killing and proliferation ability using live cell imaging. Cytokine output of effector lines was determined using the MSD platform (Pacific Biolabs, San Francisco, CA). Briefly, the TICLE method includes obtaining a CD8+ fraction, a CD4+ fraction, and a negative fraction from a sample (e.g., peripheral blood mononuclear cells); introducing the artificial expression construct to the CD8+ or CD4+ cells using electroporation or viral transduction; coculturing the transduced cells with the CD4+ or CD8+ fraction and negative fraction; and coculturing in a gas-permeable vessel in media supplemented with 7.5 U / mL IL2, 20 ng / mL IL4, 10 ng / mL IL7, and 20 ng / mL IL21.

[0032] FIG. 4. CD4+ and CD8+ Killing Rank Order: CD4+ and CD8+ effector T-cells harboring CD19CAR only and GFP variants, (GFP:ffluc, GFP:ffluc T2A Her2tG, or turboGFP T2A Her2tG) in an iSynPro construct and or iv) CD19CAR and a single iSynPro intracellular transgene were subjected to a recursive stimulation killing assay using the SK-N-Be(2) (ATCC) target cell line (human neuroblastoma, hereafter referred to as Be2) modified to express a truncated form of CD19 (CD19t) and a fluorescent marker mCherry (Be2 CD19t,mCherry). Effectors and targets were set up at multiple effector to target (E:T) ratios and their kill scores were calculated over the course of the assay. This was performed by summing the difference in target object red integrated intensity between CD19CAR and GFP variants, (GFP:ffluc, GFP:ffluc T2A Her2tG, or turboGFP T2A Her2tG) in an iSynPro construct and or iv) CD19CAR and a single iSynPro intracellular transgene at each time point (every three hours) over the course of the 9-day assay. Several transgenes encoding intracellular molecules provided a clear efficacy enhancement to the CD8+ CAR T-cells (closed circles). Only one transgene (dominant negative neuregulin receptor degradation protein-1 , (dnNRDPI )) provided an enhancement to the CD4+ CAR T-cells (closed triangle) compared to CD19CAR only (open circle). Three intracellular transgenes were not beneficial to CD4+ or CD8+ CAR T-cell killing (stars).

[0033] FIG. 5. In the same killing assay as described in relation to FIG. 4, cell tracking software was used to identify and count the number of effector CD4+ or CD8+ cells harboring either CD19CAR (open circle) or CD19CAR + iSynProJntracellular transgenes (filled in shapes). Effectors and targets were set up at multiple E:T ratios and their proliferation scores were calculated over the course of the assay by summing the difference in effectors (as defined by low red intensity) between CD19CAR and GFP variants, (GFP:ffluc, GFP:ffluc T2A Her2tG, or turboGFP T2A Her2tG) in an iSynPro construct and or iv) CD19CAR and a single iSynPro intracellular transgene at each time point (every three hours) over the course of the 9-day assay. The mean score from 1 :1 , 1 :2, and 1 :4 E:T ratios is shown. Transgene lines that did not provide a killing benefit also did not enhance T-cell proliferation (stars) compared to controls (open circles). Two transgenes that enhanced T-cell killing in the CD8+ subset (dominant negative Src homology region 2 (SH-2) domain-containing phosphatase 1 (dnSHPI ) and constitutively active B-cell lymphoma 2(caBCL2)) also enhanced T-cell proliferation. dnNRDPI enhanced CAR T-cell proliferation for the CD4+ T-cell subset.

[0034] FIGs. 6A, 6B. (FIG. 6A) CD4+ and (FIG. 6B) CD8+ effectors were stimulated with Be2- CD19t-mCherry targets and supernatants were collected 24 hours later. Additionally, T-cell effectors were challenged two additional times (at 72h and 144h) and supernatants were collected 24 hours after the final challenge. Supernatants were run using the MesoScale Discovery platform to determine cytokine levels (pg / mL) of IL2. (FIG. 6A) In CD4+ effectors, dnNRDPI did not produce more cytokine than CD19CAR in the first stimulation but did produce more in the third stimulation. In CD4s, all three non-performing (killing) transgene secreted high levels of IL2 after the first stimulation but lower levels after the third stimulation. PCK1 and survivin were better at killing for CD8s and secreted more IL2 than CD19CAR alone in CD4s in the third stimulation. (FIG. 6B) Compared to CD19CAR only, caBCL2, dnNRDPI and dnSHPI both secreted a similar level of IL2 after the first stimulation, but more IL2 than CD19CAR after the third stimulation in CD8+ effectors. Other transgenes such as phosphoenolpyruvate carboxykinase 1 (PCK1 ) and survivin did not lead to more IL2 secretion when compared to CD19CAR alone.

[0035] FIGs. 7A, 7B. (FIG. 7A) CD4+ and (FIG. 7B) CD8+ effectors were stimulated with Be2- CD19t-mCherry targets and supernatants were collected 24 hours after stimulation. Additionally, T-cell effectors were challenged two additional times (at 72h and 144h) and supernatants were collected 24 hours after the final challenge. Supernatants were run using the MesoScale Discovery platform to determine cytokine levels (pg / mL) of TNFa. (7A) The CD4+ CAR T-cell potency enhancing molecule dnNRDPI did not exhibit appreciably higher levels of cytokines release after the first stimulation but did show elevated secretion of TNFa after the thirdstimulation. caBcl2 produced more cytokine in the first stimulation than in the third stimulation as compared to CD19CAR alone. (7B) in CD8+ effectors, dnNRDPI , dnSHPI and caBCL2 were able to lead to more TNFa secretion when compared to CD19CAR alone after the third stimulation. Two of the negative transgenes (dominant negative Fas associated via death domain (dnFADD) and cellular FLICE (FADD-like IL-1 [3-converting enzyme)-inhibitory protein (cFLIP.v3)) led to production of a high level of TNFa at the first stimulation, with only cFLIP.v3 producing less TNFa than CD19CAR at the third stimulation.

[0036] FIGs. 8A, 8B. (FIG. 8A) CD4+ and (FIG. 8B) CD8+ effectors were stimulated with Be2- CD19t-mCherry targets and supernatants were collected 24 hours later. Additionally, T-cell effectors were challenged two additional times and supernatants were collected 24 hours after the final challenge. Supernatants were run using the MesoScale Discovery platform to determine cytokine levels (pg / mL) of IFN-y. (FIG. 8A) In CD4+ effectors, dnNRDPI and caBCL2 both led to more IFNy secretion when compared to CD19CAR alone after both the first and third stimulation. Two of the negative transgenes (caAKT and dnFADD) led to production of notably higher levels of cytokine after the third stimulation. (FIG. 8B) In CD8+ effectors dnSHPI and caBCL2 both enhanced the cytokine output in both the first and third stimulation. dnNRDPI enhanced the cytokine output from the first stimulation but did not appreciably increase levels of IFNy after the third stimulation.

[0037] FIGs. 9A, 9B. (FIG. 9A) CD4+ and (FIG. 9B) effector lines containing CD19CAR or CD19CAR + iSynPro transgenes encoding an intracellular molecule were produced and added to Be2-mCherry-CD19t target lines. Cells were assessed for EGFRt-positivity 24 hours after the first and third stimulations. (FIG. 9A) Most CD4+ effector lines (including CD19CAR) exhibited a high level of staining after the first stimulation. After the third stimulation, several transgenes including dnNRDPI increased the percent EGFRt positivity compared to CD19CAR alone. Constitutively active Akt (caAKT) displayed clearly lower levels of EGFRt staining after the first and third stimulations while stronger performing transgenes retained higher levels of EGFRt after the first and third stimulation compared to the CD19CAR and alone. (FIG. 9B) Most CD8+ effector lines (including CD19CAR) exhibited a high level of EGFRt staining after the first stimulation. However, following the third stimulation, all lines dropped appreciably in their EGFRt marker staining while several transgenes were able to retain EGFRt expression at a higher level compared to CD19CAR alone (caBCL2, survivin, dnNRDPI , dnSHPI ).

[0038] FIG. 10. Sequences supporting the disclosure dnNRDPI amino acid sequence (SEQ ID NO: 1); dnNRDPI nucleic acid sequence (SEQ ID NO: 2); dnSHPI amino acid sequence (SEQ ID NO: 3); dnSHPI nucleic acid sequence (SEQ ID NO: 4); caBCL2 amino acid sequence (SEQID NO: 5); caBCL2 nucleic acid sequence (SEQ ID NO: 6); PCK1 amino acid sequence (SEQ ID NO: 7); PCK1 nucleic acid sequence (SEQ ID NO: 8); survivin amino acid sequence (SEQ ID NO: 9); Survivin nucleic acid sequence (SEQ ID NO: 10); dnFADD amino acid sequence (SEQ ID NO: 11 ); dnFADD nucleic acid sequence (SEQ ID NO: 12); cFLIP.v3 amino acid sequence (SEQ ID NO: 13); cFLIP.v3 nucleic acid sequence (SEQ ID NO: 14); caAKT amino acid sequence (SEQ ID NO: 15); caAKT nucleic acid sequence (SEQ ID NO: 16); S1 -61 -version 1 (SEQ ID NO: 17); S1 -61- version 2 (SEQ ID NO: 18); S1-17 (SEQ ID NO: 19); S1 -37 (SEQ ID NO: 20); S1 -4 (SEQ ID NO: 21 ); S1 -1 (SEQ ID NO: 22); S1 -3 (SEQ ID NO: 23); S1 -42 (SEQ ID NO: 24); S1 -62 (SEQ ID NO: 25); S1 -15 (SEQ ID NO: 26); S1 -2 (SEQ ID NO: 27); S1 -27 (SEQ ID NO: 28); S1 -8 (SEQ ID NO: 29); S1 -30 (SEQ ID NO: 30); S1 -33 (SEQ ID NO: 31 ); S1 -41 (SEQ ID NO: 32); S1 -59 (SEQ ID NO: 33); S1 -66 (SEQ ID NO: 34); S1 -71 (SEQ ID NO: 35); S1-56 (SEQ ID NO: 36); S1 - 6 (SEQ ID NO: 37); S1 -60 (SEQ ID NO: 38); S1-86 (SEQ ID NO: 39); S1-32 (SEQ ID NO: 40); S1 -10 (SEQ ID NO: 41 ); S1 -18 (SEQ ID NO: 42); S1 -14 (SEQ ID NO: 43); S1 -16 (SEQ ID NO: 44); S1 -19 (SEQ ID NO: 45); S1 -26 (SEQ ID NO: 46); S1 - 65 (SEQ ID NO: 47); S2- n1 (SEQ ID NO: 48); S4-n1 (SEQ ID NO: 49); S6-n1 (SEQ ID NO: 50); S1 -325 (SEQ ID NO: 51 ); S1 - 60 (SEQ ID NO: 52); S2- 274 (SEQ ID NO: 53); S2-310 (SEQ ID NO: 54); S1 -367 (SEQ ID NO: 55); S1 -7 (SEQ ID NO: 56); IL2 minimal promoter (SEQ ID NO: 57); EF1 a promoter (Long; Ef 1 a-HTLV hybrid) (SEQ ID NO: 58); Anti-CD19 VH-VL (G01 S) scFV (SEQ ID NO: 59); Anti-CD19 VH-VL (G01 S) scFV coding sequence (SEQ ID NO: 60); Anti-CD19 VL-VH scFV (SEQ ID NO: 61 ); Anti- CD19 VL-VH scFV coding sequence (SEQ ID NO: 62); Core insulator- version 1 (SEQ ID NO: 63); Core insulator -version 2 (SEQ ID NO: 64); lgG4 hinge-A (SEQ ID NO: 65); lgG4 hinge coding sequence-A (SEQ ID NO: 66); lgG4 hinge coding sequence-B (SEQ ID NO: 67); lgG4 hinge coding sequence-C (SEQ ID NO: 68); lgG4-CH2 domain encoding sequence (SEQ ID NO: 69); lgG4-CH3 encoding sequence (SEQ ID NO: 70); CD28 Transmembrane Domain-A (SEQ ID NO: 71 ); CD28 Transmembrane Domain - B (SEQ ID NO: 72); CD28 Transmembrane Domain - C (SEQ ID NO: 73); CD28TM encoding sequence-A (SEQ ID NO: 74); CD28TM encoding sequence-B (SEQ ID NO: 75); CD28TM encoding sequence-C (SEQ ID NO: 76); CD28TM encoding sequence-D (SEQ ID NO: 77); 4-1 BB costimulatory domain -A (SEQ ID NO: 78); 4-1 BB costimulatory domain-B (SEQ ID NO: 79); 4-1 BB costimulatory domain -C (SEQ ID NO: 80); 4- 1 BB signaling encoding sequence-A (SEQ ID NO: 81); 4-1 BB signaling encoding sequence-B (SEQ ID NO: 82); 4-1 BB signaling coding sequence-C (SEQ ID NO: 83); CD3 signaling domain- A (SEQ ID NO: 84); CD3 signaling domain-B LPPR (SEQ ID NO: 85); CD3 signaling domain - C (SEQ ID NO: 86); CD3 signaling domain encoding sequence-A (SEQ ID NO: 87); CD3zeta signaling domain encoding sequence-B (SEQ ID NO: 88); Thoseaasigna Virus 2A (T2A); Peptide-v1 (SEQ ID NO: 89); Thoseaasigna Virus 2A (T2A); Peptide-v2 (SEQ ID NO: 90); Thoseaasigna Virus 2A (T2A); Peptide-v3 (SEQ ID NO: 91); Porcine Teschovirus-1 2a (P2A); Peptide (SEQ ID NO: 92); Equine Rhinitis A Virus (ERAV); 2A (E2A); Peptide (SEQ ID NO: 93); Foot-And-Mouth Disease Virus 2A (F2A); Peptide (SEQ ID NO: 94); T2A encoding sequence-v1 (SEQ ID NO:95); T2A encoding sequence-v2 (SEQ ID NO: 96); EGFRt-version 1 (SEQ ID NO: 97); EGFRt- version 2 (SEQ ID NO: 98); EGFRt encoding sequence (SEQ ID NO: 99); tCD19 coding sequence (SEQ ID NO: 100); HER2tG (without start codon) (SEQ ID NO: 101 ); DHFRdm (SEQ ID NO: 102); S1 - 61 -IL2mp_dnNRDP1_T2A_Her2tg_EF1 al_huCD19scFvgO1 s-lgG4hinge-CD28tm-41 BB-zeta- P2A-DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 103); S1 -61 -IL2mp_dnSHP1_T2A_Her2tg_ EF1 al_huCD19scFvg01s-lgG4hinge-CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 104); S1 -61 -IL2mp_ caBCL2_T2A_Her2tg_ EF1 al_huCD19scFvgO1s-lgG4hinge- CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt pbnp (SEQ ID NO: 105); S1-61 - IL2mp_PCK1_T2A_Her2tg_EF1 al_huCD19scFvgO1 s-lgG4hinge-CD28tm-41 BB-zeta-P2A- DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 106); S1-61 -IL2mp_Survivin_T2A_Her2tg_RBGpolyA_2xhs-a_Ef1 a7-3_huCD19scFvgO1 s-IgGhinge- CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt_BGHpolyA_pbnp (SEQ ID NO: 107);S1-61 - IL2mp_dnFADD_T2A_Her2tg_ EF1 al_huCD19scFvgO1 s-lgG4hinge-CD28tm-41 BB-zeta-P2A- DHFRdm-T2A-EGFRt pbnp (SEQ ID NO: 108); S1 -61-IL2mp cFLIP T2A Her2tg EF1 al_huCD19scFvg01s-lgG4hinge-CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 109); S1 -61 -IL2mp_caAKT_T2A_Her2tg_ EF1 al_huCD19scFvgO1s-lgG4hinge- CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 1 10); EF1 a- l_huCD19scFvg01 s-lgG4hinge-CD28tm-41 BB-zeta-P2A-DHFRdm-T2A-EGFRt_pbnp (SEQ ID NO: 111 ), S1 -61 -IL2mp_survivin_T2A_Her2tg (SEQ ID NO: 124), S1 -61 -IL2mp_caBCL2_T2A_Her2tg (SEQ ID NO: 125), S1 -61 -IL2mp_cflipv3_T2A_Her2tg (SEQ ID NO: 126), S1-61 -IL2mp_dnRDP1_T2A_Her2tg (SEQ ID NO: 127), S1 -61 -IL2mp_dnshp1_T2A_Her2tg (SEQ ID NO: 128), S1-61 -IL2mp_dnfadd_T2A_Her2tg (SEQ ID NO: 129), S1 -61 -IL2mp_caakt_T2A_Her2tg (SEQ ID NO: 130), and S1 -61 -IL2mp_pck1_T2A_Her2tg (SEQ ID NO: 131 ).DETAILED DESCRIPTION

[0039] A variety of therapies have been developed over the last few decades to engineer immune cells to express a recombinant receptor that binds antigens on unwanted cell types (e.g., cancer cells, virally infected cells (also referred to as targeted cells herein)). When the recombinant receptor binds the target antigen, the immune-cell can be activated to destroy the bound cell type. Chimeric antigen receptor (CAR) cell therapy is one such therapy.

[0040] Chimeric antigen receptor (CAR) therapy involves engineering immune cells to express synthetic receptors designed to target cells such as cancer cells or infected cells. Although CAR therapies have had substantial success in treating various cancers, challenges remain. For example, current CAR T-cells can have suboptimal proliferation, functional anergy or exhaustion, impaired memory differentiation, and / or limited persistence limiting their ability to create or maintain remission in patients. In some aspects, there may be targeted therapy resistance that needs to be overcome. Thus, strategies to improve CAR T-cell efficacy are needed, especially for scenarios with prolonged antigen exposure that can lead to functional anergy and exhaustion.

[0041] The current disclosure provides artificial expression constructs including a transgene encoding an intracellular molecule under the control of an inducible promoter to enhance the function of an immune cell (e.g., T-cell or chimeric antigen receptor (CAR) T-cell). The transgenes as disclosed herein express a molecule that is not secreted, but remains in the genetically modified cell. Thus, only the activity of the genetically modified cell is directly modified. The inducible intracellular transgenes disclosed herein modulate the potency of the immune cell (e.g., T-cell or CAR T-cell) resulting in potentiated cell killing ability, proliferation, cytokine output and / or maintenance of artificial expression construct transduction positivity (i.e., transduction positivity) in the genetically modified cell. In particular embodiments, cytokine output includes IL2 secretion, TNFa secretion, and / or IFNy secretion.

[0042] In some aspects, the artificial expression constructs described herein may be used to tailor a treatment to a specific patient or group of patients. For example, different cancers may respond differently to different immune cell enhancements. Further, different immune cell enhancements may be more or less appropriate for different types, stages, or growth rates of various cancers. As described in further detail herein, stimulation of artificial constructs containing dominant negative Src homology region 2 (SH-2) domain-containing phosphatase 1 (SHP1 ) (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), and dominant negative fas-associating protein with death domain (dnFADD) had varying effects on CD4+ and CD8+ T-cells. The varying activity of the artificial constructs may be useful in tailoring a treatment to a specific patient or group of patients as varying effects may be desired in the treatment of different cancers or different populations of individuals suffering from a particular cancer. For example, even if two patients have the same type of cancer, they may have different lifestyle, epigenetic, or genetic variations that may make them more or less responsive to different therapeutic treatments.

[0043] In particular embodiments, the transgene encodes at least one intracellular molecule selected from dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD. Intracellular molecules may enhance or otherwise modify the activity of the CAR T cells in killing, proliferation, cytokine output, and maintenance of artificial expression construct transduction positivity (e.g., EGFRt positivity), a marker for CAR positive T cells. In some aspects, expression of the transgenes may be regulated by one or more inducible promoters such as an iSynPro promoter. In some aspects, the use of an inducible promoter may provide potentiation of immune cell activity regardless of the presence of one or more transgenes. In some aspects, encoding more than one of the intracellular molecules selected from the group including dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD may create synergistic effects.

[0044] Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to modify immune-cell mediated cell killing. Particular embodiments utilize dnNRDPI to enhance immune-cell mediated killing by CD4+ cells. Particular embodiments utilize dnSHPI , caBCL2, and survivin to enhance immune-cell mediated killing by CD8+ cells. Particular embodiments utilize dnSHPI , caBCL2, survivin, or PCK1 to enhance immune-cell mediated killing by CD8+ cells.

[0045] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to enhance proliferation. Particular embodiments utilize inducible expression of dnNRDPI to enhance CD4+ T-cell proliferation. Particular embodiments utilize inducible expression of dnSHPI or caBCL2 to enhance CD8+ T- cell proliferation.

[0046] Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to modify cytokine output. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to enhance cytokine output.

[0047] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 cFLIP.v3, caAKT, survivin, and dnFADD to modify IL2 secretion. Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnFADD, cFLIP.v3, caAKT, dnSHPI , or caBCL2 to increase IL2 secretion by CD4+ cells. Particular embodiments utilize inducible expression of caAKT, dnSHPI , caBCL2, dnFADD, or dnNRDPI to increase IL2 secretion by CD8+ cells.

[0048] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to modify TNFa secretion. Particular embodiments utilize inducible expression of dnFADD, cFLIP.v3, caBCL2, dnNRDPI , or dnSHPIto increase TNFa secretion in CD4+ cells. Particular embodiments utilize inducible expression of caAKT, dnNRDPI , dnFADD, cFLIP.v3, dnSHPI , caBCL2, PCK1 , or survivin, to increase TNFa secretion in CD8+ cells.

[0049] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to modify IFNy secretion. Particular embodiments utilize inducible expression of dnFADD, caAKT, dnNRDPI , caBCL2, or cFLIP.v3 to increase IFNy secretion in CD4+ cells. Particular embodiments utilize inducible expression of dnNRDPI , dnSHPI , caAKT, surivin, or caBCL2 increases IFNy in CD8+ cells.

[0050] Particular embodiments utilize inducible expression of at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD to maintain artificial expression construct transduction positivity (e.g., truncated epidermal growth factor receptor (EGFRt) positivity) by cells. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, survivin, dnNRDPI , PCK1 , or dnFADD to maintain artificial expression construct transduction positivity by CD4+ T-cells. Particular embodiments utilize caAKT, caBCL2, PCK1 , dnNRDPI , survivin, dnSHPI , dnFADD, or cFLIP.v3 to maintain artificial expression construct transduction positivity by CD8+ T-cells.

[0051] In particular embodiments, cells may be stimulated one or more times depending on the desired effect. For example, cells may be stimulated 1 , 2, 3, 4 or more times. Particular embodiments utilize stimulation to potentiate cell killing ability, proliferation, cytokine output, and / or maintenance of artificial expression construct transduction positivity. In particular embodiments, cytokine output includes IL2 secretion, TNFa secretion, and / or IFNy secretion.

[0052] Particular embodiments utilize inducible expression of dnFADD, cFLIP.v3, dnSHPI , caBCL2, or caAKT to enhance CD4+ IL2 secretion after a first stimulation. Particular embodiments utilize inducible expression of PCK1 , dnNRDPI or survivin to enhance CD4+ IL2 secretion after a third stimulation. Particular embodiments utilize inducible expression of caAKT, dnFADD, dnSHPI , or caBCL2 to enhance CD8+ IL2 secretion after a first stimulation. Particular embodiments utilize inducible expression of caAKT, dnFADD, dnNRDPI , dnSHPI , or caBCL2 to enhance CD8+ IL2 secretion after a third stimulation.

[0053] Particular embodiments utilize inducible expression of dnFADD, cFLIP.3, dnNRDPI , dnSHPI , or caBCL2 to enhance CD4+ TNFa secretion after a first stimulation. Particular embodiments utilize inducible expression of dnFADD or dnNRDPI to enhance CD4+ TNFa secretion after a third stimulation. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to enhance CD8+ expression of TNFa after a first stimulation. Particular embodiments utilize inducible expression of dnFADD,caAKT, PCK1 , dnNRDPI , dnSHPI , or caBCL2 to enhance CD8+ expression of TNFa after a third stimulation.

[0054] Particular embodiments utilize inducible expression of dnFADD, dnNRDPI , or caBCL2 to enhance CD4+ expression of IFNy after a first stimulation. Particular embodiments utilize inducible expression of dnFADD, caAKT, dnNRDPI , cFLIP.v3, or caBCL2 to enhance CD4+ expression of IFNy after a third stimulation. Particular embodiments utilize inducible expression of caAKT, dnNRDPI , survivin, dnSHPI , PCK1 , or caBCL2 to enhance CD8+ expression of IFNy after a first stimulation. Particular embodiments utilize inducible expression of dnSHPI or caBCL2 to enhance CD8+ expression of IFNy after a third stimulation

[0055] Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD4+ immune cells after a first stimulation. Particular embodiments utilize inducible expression of dnFADD, PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD4+ immune cells after a third stimulation. Particular embodiments utilize inducible expression of PCK1 , dnNRDPI , survivin, dnSHPI , or caBCL2 to maintain artificial expression construct transduction positivity by CD8+ immune cells after a first stimulation. Particular embodiments utilize inducible expression of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD to maintain artificial expression construct transduction positivity by CD8+ immune cells after a third stimulation.

[0056] In some examples, the intracellular molecule includes the sequence as set forth in SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, or 15. In particular embodiments, the intracellular molecule is encoded by the sequence as set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16.

[0057] In particular embodiments, the artificial expression construct includes a transgene encoding an intracellular molecule. In particular embodiments, the artificial expression construct further includes an iSynPro promoter. In particular embodiments, the artificial expression construct further includes or encodes a skip sequence and a control feature. In particular embodiments, the iSynPro promoter includes a transcription response element (TRE) or a combination of TREs. In certain examples, the TRE includes S1 -61 (SEQ ID NOs: 17 or 18). In particular embodiments, the TRE or combination of TREs is linked to a minimal promoter, such as a minimal IL2 promoter, minimal U7 promoter (U7prom), minBglobin (also referred to as minBGprom), minCMV, minCMV* (minCMV* is minCMV with a Sacl restriction site removed), minRho, minRho* (minRho* is minRho with a Sacl restriction site removed, or the Hsp68 minimal promoter (proHSP68). In particular embodiments, the iSynPro promoter includes S1 -61 (SEQ ID NO: 17) operably linked to an IL2 minimal promoter (SEQ ID NO: 57).

[0058] The current disclosure also provides systems and methods for genetically modifying an immune cell to express a recombinant receptor. In some embodiments, the artificial expression construct including the transgene encoding an intracellular molecule can further include a sequence encoding the recombinant receptor. In another embodiment, the transgene encoding an intracellular molecule and the sequence encoding the recombinant receptor can be on separate artificial expression constructs. In particular embodiments, a recombinant receptor is under the control of a second promoter. In particular embodiments, the artificial expression construct encoding the recombinant receptor includes or encodes a promoter and a recombinant receptor. In particular embodiments, the artificial expression construct encoding the recombinant receptor includes or encodes a promoter, a recombinant receptor, a skip sequence, and a control feature. In particular embodiments, the artificial expression construct includes or encodes a promoter, a recombinant receptor, a first skip sequence, a selection cassette (e.g., dihydrofolate reductase double mutant, DHFRdm), a second skip sequence, and a transduction marker (EGFRt). In particular embodiments, a recombinant receptor includes a CAR or an engineered T- cell receptor (eTCR). In particular embodiments, the recombinant receptor includes an extracellular component that binds a target antigen, a transmembrane domain, and an intracellular component.

[0059] In particular embodiments, the artificial expression construct with the transgene encoding an intracellular molecule and the recombinant receptor includes or encodes an iSynPro promoter, a Her2tG, a first 2A skip sequence, a transgene encoding an intracellular molecule, an EF1 a(L) promoter, a recombinant receptor, a second 2A skip sequence, a DHFRdm, a third 2A skip sequence, and an EGFRt. In particular embodiments, the artificial expression construct with the transgene encoding an intracellular molecule and the recombinant receptor includes or encodes S1 -61 , an IL2 minimal promoter, a Her2tG, a first 2A skip sequence, a transgene encoding an intracellular molecule, an EF1 a(L) promoter, a CAR, a second 2A skip sequence, a DHFRdm, a third 2A skip sequence, and an EGFRt. In particular embodiments, the transgene encoding an intracellular molecule encodes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and / or dnFADD (see FIG. 2 for schematic of exemplary artificial expression constructs).

[0060] Thus, the current disclosure provides systems and methods to improve the killing, proliferation, cytokine secretion, and maintenance of EGFRt levels of recombinant receptorexpressing immune cells. The recombinant receptor-expressing immune cells can be engineered to target different target cells (e.g., cancer cell, virus, bacteria, fungi, parasites, or arthropods) expressing a target antigen by engineering the extracellular domain to include a binding domain that binds the target antigen.

[0061] The various constructs as described herein may be used to tailor a treatment to a specific patient or group of patients. For example, while not being bound by theory, cytokine release syndrome generally develops when an immune system responds too aggressively. In some instances, it may therefore be helpful to administer cells treated with artificial expression constructs that generate a decreased response in comparison to existing CAR-T cell therapeutics such as a CAR targeting CD19 (i.e., CD19CAR), or that have some effect, but are not the best transgenes at potentiating the killing, proliferation, cytokine secretion, and maintenance of EGFRt levels of recombinant receptor-immune cells. That is, while there is an increased response over native cells, it may be a more modulated response. In other aspects, it may be helpful to administer a series or combination of cells treated with a combination of artificial expression constructs to create a graded or tiered response. For example, the stimulation dose or the number of doses during T-cell activation may be modified depending on the needs of the patient or group of patients. In other aspects more than one transgene encoding an intracellular molecule may be used.

[0062] As shown in FIG. 4, inducible expression of survivin, dnSHPI , and caBCL2 all increase CD8+ killing in comparison to CD19CAR alone or CD19CAR coupled to iSynPro. Inducible expression of caBCL2 was greater than inducible expression of dnSHPI or survivin. However, depending on the needs of the patient or the group of patients, it may be desirable to have inducible expression of survivin or dnSHPI instead of caBCL2. That is, it may be desirable to stimulate lower rates of killing by CD8+ T-cells. Additionally, it may be desirable to use an artificial construct that increases one or more of killing, proliferation, cytokine output, and maintaining transduction positive CD4+ and CD8+ T-cells. For example, inducible expression of dnSHPI also increases CD8+ T-cell proliferation, secretion of IL2 by CD8+ T cells after a first stimulation, secretion of TNFa by CD8+ T-cells after a first stimulation, and maintains transduction positivity in CD4+ T cells after a first and third stimulation. Thus, inducible expression of dnSHPI may be helpful where a plurality of different types of actions are sought. Inducible expression of caBCL2 increases CD8+ killing, CD8+ proliferation, CD8+ IL2 after a first stimulation, CD8+ TNFa after a first stimulation and maintenance of CD8+ transduction positivity after a third stimulation. Thus, the use of caBCL2 may be helpful in treating conditions where more CD8+ activity is sought, or where CD4+ activity is already sufficient. Inducible expression of survivin increases CD8+ killing and CD4+ IL2 production after a third stimulation as shown in FIG. 4 and FIG. 6A. Thus, the use of survivin may be helpful in treating conditions where a more focused response is sought. Inducible expression of dnNRDPI increases CD4+ killing, CD4+ proliferation, CD4+ IL2 secretion after a third stimulation, CD8+ TNFa expression after a first and third stimulation, and CD8+ IFNyafter a first stimulation. Thus, the use of dnNRDPI may be helpful in treating conditions such as respiratory syncytial virus (RSV) or Ebola. Inducible expression of PCK1 increases CD4+ IL2 secretion after a third stimulation and CD8+ TNFa secretion after a first stimulation. Thus, the use of PCK1 may be helpful where a more focused response is desired. Inducible expression of cFLIP.v3 increases CD4+ IL2 secretion after a first stimulation, CD4+ TNFa secretion after a first stimulation and CD8+ TNF a expression after a third stimulation. Thus, cFLIP.v3 may be helpful in treating conditions in which increased TNFa is desirable. caAKT increases CD4+ production of IL2 after a first stimulation, CD8+ production of IL2 after a first and third stimulation, CD8+ TNFa secretion after a third stimulation, CD4+ IFNy secretion after a third stimulation and CD8+ transduction positivity after a third stimulation. Thus, caAKT may be useful in treating conditions where an increase in multiple cytokines are sought. Inducible expression of dnFADD increases CD4+ and CD8+ IL2 secretion after a first stimulation, CD4+ TNFa expression after a first stimulation, and CD4 IFNy secretion after a third stimulation. Thus, the use of dnFADD may be helpful in the treatment of conditions where an increase in multiple cytokines are sought.

[0063] Some cancers overexpress certain cytokines. For example, TNF-a has been implicated in mediating drug-resistance in breast cancer. Therefore, in some aspects, inducible expression of PCK1 or survivin may be selected instead of other transgenes as inducible expression of PCK1 or survivin does not increase expression of TNFa in CD4+ or CD8+ cells. IFNy is involved in promoting an outgrowth of tumor cells with immunoevasive phenotype. Therefore in cancers with immunoevasive phenotypes, inducible expression of dnFADD, cFLIP.v3, PCK1 , survivin, dnSHPI , or caBCL2 may be selected over inducible expression of dnFADD or caAKT.

[0064] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Inducible Expression of Transgenes Encoding an Intracellular Molecule; (ii) Immune Cells; (iii) Cell Sample Collection and Cell Enrichment; (iv) Genetic Engineering Techniques; (v) Control Features Including Tag Cassettes, Transduction Markers, Selection Cassettes, and / or Suicide Switches; (vi) Recombinant Receptors; (vi-a) Binding Domains; (vi-b) Transmembrane Domains; (vi-c) Intracellular Effector Domains; (vi-d) Linkers; (vii) Ex Vivo Manufactured Cell Formulations; (viii) Compositions for Targeted Viral Vectors & Nanoparticles for Cell Modification; (ix) Methods of Use; (x) Kits; (xi) Exemplary Embodiments; and (xii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0065] (i) Inducible Expression of Transgenes Encoding an Intracellular Molecule. In particular embodiments, an intracellular molecule is a molecule that is not secreted from the cell that produced it. The intracellular molecule can be in the cytoplasm, in the nucleus, in the cellmembrane, or in any other portion of the cell so long as it is not secreted from the cell. Herein, the nucleus is the membrane-enclosed organelle within a cell that contains the chromosomes. Herein the cytoplasm refers to the material and space within a cell, excluding the nucleus. The cytoplasm includes the space and material external to the nuclear membrane and internal to the cellular membrane. The cellular membrane as used herein is a semipermeable lipid bilayer that separates the cell from the external environment.

[0066] dnNRDPI is the dominant negative form of neuregulin receptor degradation protein-1 (NRDP1 ). NRDP1 , encoded by Ring Finger Protein 41 (RNF41), acts as an E3 ubiquitin-protein ligase and regulates the degradation of target proteins. Target proteins for NRDP1 include members of the epidermal growth factor (EGF) receptor family, for example ErbB3 (or Her3). Other targets of NRDP1 include ErbB4, ubiquitin-specific protease 8 (Usp8), Birc6 and reticulon 4 (Rtn4, also known as NogoA). Mutations in NRDP1 have been linked to tumor diseases. Overexpression of NRDP1 has been shown to decrease ErbB3 and inhibition of breast cancer growth. Decreased levels of NRDP1 are inversely correlated with ErbB3 levels in primary human breast cancer tissue.

[0067] dnSHPI is the dominant negative form of Src homology region 2 (SH-2) domain-containing phosphatase 1 (SHP1). SHP1 is a cytosolic tyrosine phosphatase encoded by the PTPN6 gene. It contributes to multiple cellular functions including proliferation, differentiation, cell cycle maintenance, and migration. SHP1 has two N-terminal Src homology 2 domains (N-SH2 and C- SH2), a catalytic domain (PTP), and a C-terminal tail. Alterations in SHP1 function or expression (e.g., silencing or mutations resulting in failure of SHP1 -mediated homeostatic maintenance) can contribute to several human diseases, including cancer.

[0068] Constitutively active B-cell lymphoma 2 (caBCL2) is a constitutively active form of the BCL2 protein that regulates apoptosis. It can regulate cell death by either inhibiting or inducing apoptosis. Damage to the gene encoding BCL2 has been identified as a cause of a number of cancers including melanoma, breast cancer, prostate cancer, chronic lymphocytic leukemia, and lung cancer.

[0069] Phosphoenolypyruvate carboxykinase 1 (PCK1 ) is an enzyme that catalyzes gluconeogenesis reactions (i.e., the conversion of oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO2). The expression of PCK1 can be regulated by insulin, glucocorticoids, glucagon, cAMP, and diet.

[0070] Survivin is a member of the inhibitor of apoptosis protein family which has a role in regulating cell division and inhibiting apoptosis by blocking caspase activation. Survivin can befound upregulated in several human cancers including lung cancer, pancreatic cancer, and breast cancer compared to normal tissue.

[0071] Cellular FADD-like IL-1 -converting enzyme inhibitory protein (cFLIP.v3) is death effector domain containing protein that restricts caspase-8 activation.

[0072] Constitutively active AkT (caAKT) is a serine / threonine kinase believed to have a key role in the PI3K signaling pathway.

[0073] Dominant negative fas-associating protein with death domain (dnFADD) is an adapter protein that is recruited to the death-inducing signaling complex (DISC) during signaling via death receptors. FADD is a common conduit in both CD95-mediated and TNF-R-mediated apoptosis.

[0074] Transgenes encoding an intracellular molecule can be placed under the control of an inducible synthetic promoter (iSynPro). The iSynPro promoter can include a sequence including a TRE or a combination of TREs, such as those described in S1 -17 (SEQ ID NO: 19), S1-37 (SEQ ID NO: 20), S1-4 (SEQ ID NO: 21 ), S1 -1 (SEQ ID NO: 22), S1 -3 (SEQ ID NO: 23), S1 -42 (SEQ ID NO: 24), S1-61 (SEQ ID NO: 17 or SEQ ID NO: 18), S1 -62 (SEQ ID NO: 25), S1 -15 (SEQ ID NO: 26), S1 -2 (SEQ ID NO: 27), S1 -27 (SEQ ID NO: 28), S1 -8 (SEQ ID NO: 29), S1 -30 (SEQ ID NO: 30), S1 -33 (SEQ ID NO: 31 ), S1 -41 (SEQ ID NO: 32), S1-59 (SEQ ID NO: 33), S1-66 (SEQ ID NO: 34), S1 -71 (SEQ ID NO: 35), S1 -56 (SEQ ID NO: 36), S1 -6 (SEQ ID NO: 37), S1 -60 (SEQ ID NO: 38), S1 -86 (SEQ ID NO: 39), S1 -32 (SEQ ID NO: 40), S1 -10 (SEQ ID NO: 41 ), S1 -18 (SEQ ID NO: 42), S1 -14 (SEQ ID NO: 43), S1-16 (SEQ ID NO: 44), S1 -19 (SEQ ID NO: 45), S1 - 26 (SEQ ID NO: 46), S1- 65 (SEQ ID NO: 47), S2-n1 (SEQ ID NO: 48), S4-n1 (SEQ ID NO: 49), S6-n1 (SEQ ID NO: 50), S1 -325 (SEQ ID NO: 51 ), S1- 60 (SEQ ID NO: 52), S2- 274 (SEQ ID NO: 53), S2-310 (SEQ ID NO: 54), S1 -367 (SEQ ID NO: 55), or S1 -7 (SEQ ID NO: 56) in FIG. 10. In particular embodiments, the iSynPro promoter includes a TRE or combination of TREs, such as those described in S1 -61 (SEQ ID NO: 17 or SEQ ID NO: 18) in FIG. 10. In particular embodiments, the iSynPro promoter includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to S1 -17 (SEQ ID NO: 19), S1 -37 (SEQ ID NO: 20), S1-4 (SEQ ID NO: 21 ), S1 -1 (SEQ ID NO: 22), S1 -3 (SEQ ID NO: 23), S1 -42 (SEQ ID NO: 24), S1-61 (SEQ ID NO: 17 or SEQ ID NO: 18), S1 -62 (SEQ ID NO: 25), S1 -15 (SEQ ID NO: 26), S1 -2 (SEQ ID NO: 27), S1 -27 (SEQ ID NO: 28), S1 -8 (SEQ ID NO: 29), S1 -30 (SEQ ID NO: 30), S1 -33 (SEQ ID NO: 31 ), S1 -41 (SEQ ID NO: 32), S1-59 (SEQ ID NO: 33), S1-66 (SEQ ID NO: 34), S1 -71 (SEQ ID NO: 35), S1 -56 (SEQ ID NO: 36), S1-6 (SEQ ID NO: 37), S1 -60 (SEQ ID NO: 38), S1 -86 (SEQ ID NO: 39), S1 -32 (SEQ ID NO: 40), S1 -10 (SEQ ID NO: 41 ), S1 -18 (SEQ ID NO: 42), S1 -14 (SEQ ID NO: 43), S1 -16 (SEQ ID NO: 44), S1-19 (SEQ ID NO: 45), S1 -26 (SEQ ID NO: 46), S1 - 65 (SEQ ID NO: 47), S2-n1 (SEQ ID NO: 48), S4-n1 (SEQ ID NO: 49),S6-n1 (SEQ ID NO: 50), S1-325 (SEQ ID NO: 51 ), S1 -60 (SEQ ID NO: 52), S2-274 (SEQ ID NO: 53), S2-310 (SEQ ID NO: 54), S1 -367 (SEQ ID NO: 55), or S1 -7 (SEQ ID NO: 56). In particular embodiments, the iSynPro includes S1 -61 (SEQ ID NO: 17 or SEQ ID NO: 18).

[0075] In particular embodiments, the iSynPro promoter includes S1 -61 (SEQ ID NO: 17 or SEQ ID NO: 18). In particular embodiments, the iSynPro promoter includes a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18.

[0076] Particular embodiments combine a sequence encoding a TRE or combination of TREs described above with a minimal promoter. In particular embodiments, the minimal promoter includes an IL2 minimal promoter. In particular embodiments, the IL2 minimal promoter includes the sequence as set forth in SEQ ID NO: 57.

[0077] In particular embodiments, the iSynPro promoter includes S1-61 operably linked to a minimal promoter.

[0078] Functional truncations and variants of the transgenes encoding an intracellular molecule may also be used. Functional truncations retain intracellular activity while having fewer amino acid residues than a corresponding reference wild-type sequence. Functional variants retain intracellular activity while having one or more amino acid mutations as compared to a corresponding reference wild-type sequence.

[0079] (ii) Immune Cells. The present disclosure describes an immune cell genetically modified to inducibly express a transgene encoding an intracellular molecule (e.g., dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD) that modulates the function of an immune cell. In particular embodiments, the immune cell is also genetically modified to express a recombinant receptor, such as a CAR. The immune cell can be any cell in which the inducible promoter (e.g., iSynPro) can inducibly control expression of the transgene encoding an intracellular molecule. In particular embodiments, immune cells can include lymphocytes, monocytes / macrophages, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC). In particular embodiments, immune cells include lymphocytes. In particular embodiments, lymphocytes include T-cells, B-cells, natural killer (NK) cells, or NK T-cells.

[0080] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCRp) genes and are called a- and p-TCR chains.

[0081] Y§ T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In y5 T-cells, the TCR is made up of one y-chain and one 5-chain. This group of T-cells is much less common (2% of total T-cells) than the a|3 T-cells.

[0082] CD3 is expressed on all mature T-cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.

[0083] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B-cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4+ protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0084] Cytotoxic T-cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8+ glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0085] "Central memory" T-cells (or "TCM") refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.

[0086] "Effector memory" T-cell (or "TEM") refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.

[0087] "Naive" T-cells refers to a non-antigen experienced T-cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.

[0088] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated inresponse to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T-cells that can clear the infection. NK cells express CD8, CD16, and CD56 but do not express CD3.

[0089] NK cells include NK T-cells. NK T-cells are a specialized population of T-cells that express a semi-invariant T-cell receptor (TCR ab) and surface antigens typically associated with natural killer cells. NK T-cells contribute to antibacterial and antiviral immune responses and promote tumor-related immunosurveillance or immunosuppression. Like natural killer cells, NK T-cells can also induce perforin-, Fas-, and TNF-related cytotoxicity. Activated NK T-cells are capable of producing IFN-y and IL-4. In particular embodiments, NK T-cells are CD3+ / CD56+.

[0090] Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components. Monocytes / macrophages express CD1 1b, F4 / 80; CD68; CD11 c; IL-4Ra; and / or CD163.

[0091] Immature dendritic cells (i.e., pre-activation) engulf antigens and other non-selfcomponents in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T-cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC- 205.

[0092] Hematopoietic Stem / Progenitor Cells or HSPC refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0093] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to all other hematopoietic cell types.

[0094] A hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cell types. In certain embodiments, hematopoietic progenitor cells are CD24l0Lin- CD117+hematopoietic progenitor cells. HPC can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.

[0095] HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof. Also, the HSPC can be negative for an expressed marker relative to other types ofhematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. Preferably, the HSPC are CD34+cells.

[0096] A statement that a cell or population of cells is "positive" for or expressing a particular marker refers to the detectable presence on or in the cell of the particular marker. When referring to a surface marker, the term can refer to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0097] A statement that a cell or population of cells is "negative" for a particular marker or lacks expression of a marker refers to the absence of substantial detectable presence on or in the cell of a particular marker. When referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0098] Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or allogeneic when appropriate, and can also be in vivo or ex vivo. In particular embodiments, immune cell is a lymphocyte. In particular embodiments, the lymphocyte is a T-cell, B-cell or NK cell. In particular embodiments, the T-cell is a CD4+ T-cell or CD8+ T-cell.

[0099] (iii) Cell Sample Collection and Cell Enrichment. Methods of sample collection and enrichment are known by those skilled in the art. In particular embodiments, cells are derived from humans, for example a patient to be treated. Cells can be derived from cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, or pig.

[0100] In some embodiments, T-cells are derived or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, 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. In particular embodiments, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in particular embodiments, contain lymphocytes, including T-cells, monocytes, granulocytes, B-cells, other nucleated white blood cells, HSC, HPC, HSPC, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets and further processing is necessary.

[0101] In some embodiments, blood cells collected from a 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 particular embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. Washing can be accomplished using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In particular embodiments, cells can be re-suspended in a variety of biocompatible buffers after washing, such as, Ca++ / Mg++ free PBS.

[0102] The isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and / or simultaneously. In particular embodiments, the isolation, culture, and / or engineering of the different populations is carried out from the same starting material, such as from the same sample.

[0103] In particular embodiments, a sample can be enriched for T-cells by using density-based cell separation methods and related methods. For example, white blood cells can be separated from other cell types in the peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.

[0104] In particular embodiments, a bulk T-cell population can be used that has not been enriched for a particular T-cell type. In particular embodiments, a selected T-cell type can be enriched for and / or isolated based on cell-marker based positive and / or negative selection. In positive selection, cells having bound cellular markers are retained for further use. In negative selection, cells not bound by a capture agent, such as an antibody to a cellular marker are retained for further use. In some examples, both fractions can be retained for a further use. In particular embodiments, CD4+ and / or CD8+ T-cells are enriched from PBMCs.

[0105] The separation need not result in 100% enrichment or removal of a particular cellpopulation or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type 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 refers to decreasing the number or percentage of such cells but need not result in a complete removal of all such cells.

[0106] 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.

[0107] In some embodiments, an antibody or binding domain for a cellular marker 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. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ); see also US 4,452,773; US 4,795,698; US 5,200,084; and EP 452342.

[0108] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0109] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5):355-376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined cell subsets at high purity.

[0110] Cell-markers for different T-cell subpopulations are described above. In particular embodiments, specific subpopulations of T-cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and / or CD45RA T-cells, are isolated by positive or negative selection techniques.

[0111] CD3+, CD28+ T-cells can be positively selected for and expanded using anti-CD3 / anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® (Life Technologies AS, Norway) M-450 CD3 / CD28 T-cell Expander).

[0112] In particular embodiments, a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T-cells. Such CD8+ and CD4+ 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.

[0113] In particular embodiments, a CD8+ and / or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T-cells from a negative fraction. Particular embodiments utilize the TICLE method as described in more detail elsewhere herein.

[0114] Other cell types can be enriched based on known marker profiles and techniques. For example, CD34+ HSC, HSP, and HSPC can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0115] (iv) Genetic Engineering Techniques. Cell populations are genetically modified (or genetically engineered) to express a transgene encoding an intracellular molecule and, optionally a recombinant receptor (e.g., chimeric antigen receptors, CAR) described herein. Desired genes (e.g., dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD transgenes and recombinant receptors) disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.

[0116] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence). The gene can include the transgene encoding an intracellular molecule or encode a recombinant receptor. This definition includes various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not substantially affect the function of the encoded intracellular molecule or encoded recombinant receptor. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the open reading frame(s) within the artificial expression construct. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.

[0117] Gene sequences including the transgene encoding an intracellular molecule and / or encoding recombinant receptors are provided herein and can also be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence. In embodiments, the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.

[0118] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for the synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "gene sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.

[0119] Polynucleotide gene sequences encoding more than one portion of an expressed artificial expression construct can be operably linked to each other and relevant regulatory sequences.For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0120] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or promoters specific for the cytoplasm. Promoters may include strong promoters, weak promoters, constitutive expression promoters (i.e., constitutive promoters), and / or inducible promoters. Constitutive promoters are promoters that allow for the continual transcription of its associated gene or genes. Inducible promoters direct expression in response to certain conditions, signals or cellular events. For example, the promoter may be an inducible promoter that requires a particular ligand, small molecule, transcription factor or hormone protein in order to effect transcription from the promoter. In particular embodiments, the inducible promoter includes the iSynPro described above and a minimal promoter. In particular embodiments, the minimal promoter includes an IL2 minimal promoter.

[0121] Particular examples of constitutive promoters include human elongation factor la promoter (EF1 a, including EF1 a(s) and EF1a(L)), myeloproliferative sarcoma virus (MND), cytomegalovirus (CMV), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In particular embodiments, the recombinant receptor is under the control of EF1 a (SEQ ID NO: 58).

[0122] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids, bacterial artificial chromosomes, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0123] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 , and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0124] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone etal, Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica (United Kingdom), the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. In particular embodiments, cells are genetically engineered to express artificial expression constructs using a lentivirus or lentiviral vector.

[0125] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0126] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581 -599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291 -302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431 -434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351 -359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0127] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors andpackaging cells for transducing mammalian host cells with viral particles including transgenes are described in, e.g., US 8,119,772; Walchli, etal., 2011 , PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Then 14:1155; Frecha, et al., 2010, Mol. Then 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0128] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 andUS8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661 , WO2014 / 093694WO2014 / 093701 , WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351 , WO2015 / 089354WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465WO2015 / 089473 and WO2015 / 089486, WO201620571 1 , WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0129] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, etal. Science 300, 764 (2003); Bibikova, etal. Genetics 161 , 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1 156- 1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0130] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-likeeffector (TALE) DNA binding protein and a DNA cleavage domain. For additional information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Common, 2013, 4: 1762; Scharenberg et al., Corr Gene Ther, 2013, 13(4):291 -303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011 ); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0131] Nanoparticles that result in selective in vivo genetic modification of targeted cell types have been described and can be used within the teachings of the current disclosure. In particular embodiments, the nanoparticles can be those described in WO20141531 14, WO20171811 10, and WO201822672.

[0132] (v) Control Features Including Tag Cassettes, Transduction Markers, Selection Cassettes, and / or Suicide Switches. In particular embodiments, artificial expression constructs can include or encode one or more tag cassettes and / or transduction markers. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate genetically modified cells in vitro, in vivo and / or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of an artificial expression construct, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element (or skip sequence) that separates the transduction marker from the rest of the expressed molecule.

[0133] Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 112), Flag tag (DYKDDDDK; SEQ ID NO: 113), Xpress tag (DLYDDDDK; SEO ID NO: 1 14), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 115), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 116), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 117), Myc tag (EQKLISEEDL; SEQ ID NO: 118), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 119), STREP® tag II (WSHPQFEK SEQ ID NO: 120 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 121 ), Softag 3 (TQDPSRVG; SEQ ID NO: 122), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 123).

[0134] Conjugate binding molecules that bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliersincluding Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0135] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011 ); a cell surface-localizing polypeptide tag based on truncated human HER2, designated Her2tG; an ECD of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 ( 5 Pt 1 ); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). In particular embodiments, cells are genetically modified to express EGFRt. In particular embodiments, cells are genetically modified to express Her2tG.

[0136] In particular embodiments, a selection cassette provides for positive selection or negative selection of a desired cell population. Negative selection is when several cell types are removed, leaving the cell type of interest. Positive selection involves targeting the desired cell population to only retain desired cells.

[0137] A selection cassette can encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cells. In particular embodiments, a positive selection cassette includes resistance genes to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In particular embodiments, a selection cassette includes the DHFR (dihydrofolate reductase) gene or DHFR double mutant (DHFRdm) gene providing resistance to methotrexate (MTX), the MGMT P140K gene responsible for the resistance to O6BG / BCNU, the HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine) or other genes for detoxification with respect to some drugs. In particular embodiments, the selection agent includes neomycin, hygromycin, puromycin,phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O6BG / BCNll, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gin synthetase, or ADA.

[0138] In particular embodiments, the selection cassette includes DHFRdm and the selection agent includes MTX. In particular embodiments, the method does not require a selection cassette to acquire highly purified cell populations.

[0139] In particular embodiments, negative selection cassettes include a gene for transformation of a substrate present in the culture medium into a toxic substance for the cell that expresses the gene. These molecules include detoxification genes of diptheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1 ):77-86, 1993; Yanagawa etal., Transgenic Res. 8(3):215-221 , 1999), the kinase thymidine gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or FIAU. The HPRT gene may also be used as a negative selection by addition of 6-thioguanine (6TG) into the medium, and for all positive and negative selections, a poly A transcription termination sequence from different origins, the most classical being derived from SV40 poly A, or a eukaryotic gene poly A (bovine growth hormone, rabbit -globin, etc.).

[0140] In particular embodiments, artificial expression constructs can include a polynucleotide or skip sequence that encodes a self-cleaving polypeptide. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the transgene encoding an intracellular molecule and the polynucleotide encoding the control feature. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and a polynucleotide encoding a selection cassette. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the selection cassette and the polynucleotide encoding the transduction marker (e.g., EGFRt). Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011 ). In particular embodiments, cells are genetically modified to include a selfcleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes T2A. In particular embodiments, a 2A sequence does not result in a 2A scar. In particular embodiments, a 2A sequence results in the addition of amino acids to proteins (also referred to as a 2A scar). A 2A sequence is a peptide sequence that induces ribosomal skipping during translation. Cleavage is triggered by ribosomal skipping of a peptide bond between proline and glycine within the 2A sequence. This results in the upstream protein having extra amino acids at the C terminal end, which has unknown impact on protein functionality.

[0141] Control features may be present in multiple copies in an artificial expression construct or can be expressed as distinct molecules with the use of a skip sequence. For example, an artificial expression construct can have one, two, three, four or five tag cassettes and / or one, two, three, four, or five transduction markers could also be expressed. For example, embodiments can include an artificial expression construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13 / 463,247.

[0142] One advantage of including at least one control feature in an artificial expression construct is that cells expressing the artificial expression construct administered to a subject can be increased or depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing an artificial expression construct by using an antibody specific for the tag cassette, using a cognate binding molecule specific for the control feature, or by using a second modified cell expressing an artificial expression construct and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature. For example, if EGFRt is used, then an anti-EGFRt binding domain (e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal) may be used, or an anti-EGFRt / anti-CD3 bispecific scFv, or an anti-EGFRt CAR T-cell may be used. Similarly, if Her2tG is used, then an anti-Her2tG binding domain fused to or conjugated to a cell-toxic reagent may be used.

[0143] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into an artificial expression construct as a suicide switch.

[0144] In certain embodiments, modified cells expressing an artificial expression construct may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, iron-oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI-scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0145] Thus, modified cells expressing at least one control feature within an artificial expression construct can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and / or eliminated as compared to a modified cell without a tag cassette.

[0146] (vi) Recombinant Receptors. In particular embodiments, a recombinant receptor is or includes a binding domain that binds a target antigen, wherein the recombinant receptor isexpressed by a cell following the artificial introduction of a nucleic acid encoding the recombinant receptor into the cell. The recombinant receptor can be, e.g., a CAR, a T-cell receptor (TCR), or a CAR / TCR hybrid.

[0147] CAR, for example, include several distinct subcomponents that allow genetically modified cells (e.g., T-cells) to recognize and kill target cells, such as cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that binds a marker that is preferentially present on the surface of unwanted cells. When the binding domain binds such markers, the intracellular component activates the cell to destroy the bound cell. CAR can additionally include a transmembrane domain that links the extracellular component to the intracellular component, and other subcomponents that can increase the recombinant receptor’s function. For example, the inclusion of a spacer and / or one or more linker sequences can allow the recombinant receptor to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted cell marker.

[0148] TCR are protein complexes found on the surface of T cells that are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. TCR can be engineered to form eTCR and to bind a particular antigen. A TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Va and VP) and a constant region (Ca and CP). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region.

[0149] (vi-a) Binding Domains. In certain examples, recombinant receptors include a binding domain that binds a target antigen that is preferentially present on the surface of unwanted cells. Binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. Examples of binding domains include cellular marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T-cell receptors), or combinations and engineered fragments or formats thereof.

[0150] As is understood by those of ordinary skill in the art, a traditional antibody includes two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. References to “V ” or “VH” refer to the variable region of an immunoglobulin heavy chain. References to “V ” or “VL” refer to the variable region of an immunoglobulin light chain.

[0151] Mammalian heavy chains are classified as a, 6, E, y, and p, and mammalian light chains are classified as A or K. Immunoglobulins including the a, 5, E, y, and p heavy chains are classified as immunoglobulin (lg)A, IgD, IgE, IgG, and IgM. The complete antibody forms a “Y” shape. The stem of the Y consists of the second and third constant regions (and for IgE and IgM, the fourth constant region) of two heavy chains bound together and disulfide bonds (inter-chain) are formed in the hinge. Heavy chains y, a, and 0 have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains p and £ have a constant region composed of four immunoglobulin domains. The second and third constant regions are referred to as “CH2 domain” and “CH3 domain”, respectively. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”.

[0152] CDR sets can be based on, for example, Kabat numbering (Kabat et al. (1991 ) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 (“Chothia” numbering scheme)); Martin (Abinandan et al. (2008) Mol Immunol. 45:3832-3839 (“Martin” numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand etal. (1998) Protein Eng. 11 :1015-1025; Gelfand etal. (1996) Proc Natl Acad Sci USA. 93:3675-3678; Gelfand et al. (1998) J Comput Biol. 5:467- 477 (“Gelfand” numbering scheme)); Contact (MacCallum et al. (1996) J. Mol. Biol. 262:732-745 (Contact numbering scheme)); IMGT (Lefranc et al. (2003) Dev Comp Immunol 27(1 ):55-77 (“IMGT” numbering scheme)); AHo (Honegger and Pluckthun (2001 ) J Mol Biol 309(3):657-670 (“AHo” numbering scheme)); North (North et al. (2011 ) J Mol Biol. 406(2):228-256 (“North” numbering scheme)); or other numbering schemes. Software programs and bioinformatical tools, such as ABodyBuilder (Leem et al. (2016) MAbs 8(7):1259-1268), PIGSPro (Lepore et al. (2017) Nucleic Acids Res 45(W1):W17-W23), Kotai Antibody Builder (Yamashita et al. (2014) Bioinformatics 30(22):3279-3280), Rosetta Antibody (Weitzner et al. (2017) Nature Protocols 12:401-416), Paratome (Kunik etal. (2012) Nucleic Acids Res 40:W521 -W524), Antibody i-Patch (Krawczyk et al. (2013) Protein Eng Des Sei 26(10):621 -629), and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108 can also be used to determine CDR sequences.

[0153] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1 , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the heavy chain of the antibody are referred to as CDRH1 , CDRH2, and CDRH3, whereas the CDRs located in the variable domain of the light chain of the antibody are referred to as CDRL1 , CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0154] Antibodies that bind an antigen can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target antigen. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and / or determined. Many relevant antibodies are also publicly known and commercially available.

[0155] In some alternatives, antibodies bind to a cancer cell or virally-infected cell surface molecule and do not cross react with nonspecific components such as bovine serum albumin or other unrelated antigens.

[0156] “Antibody fragment” refers to at least one portion of an antibody, that retains the ability to bind an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, single chain variable (scFv) antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment including VH and constant CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid variable heavy only (VHH) domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). An antigen binding fragment can also be incorporated into single domainantibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson (2005) Nature Biotechnology 23:1126- 1136).

[0157] In particular embodiments, a binding domain can include humanized forms of non-human (e.g., murine) antibodies or antigen binding fragments thereof. A humanized antibody includes an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g., the CDR, of an animal (non-human) immunoglobulin. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived but avoid an immune reaction against the non-human antibody. In particular embodiments, a binding domain can include a fully human antibody or antibody fragment thereof, where the whole molecule is of human origin or includes an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0158] “scFv” refers to an engineered fusion protein including the VH and VL of an antibody linked via a linker and capable of being expressed as a single chain polypeptide. The scFv retains the specificity of the intact antibody from which it is derived. In particular embodiments, a linker connecting the variable regions can include glycine-serine linkers, for example, SEQ ID NOs: 59- 62 or provided elsewhere herein. In particular embodiments, an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may include VL-linker-VH or may include VH-linker-VL.

[0159] Recombinant receptors also include TCR (including engineered TCR) that can be used independently or as a binding domain within a CAR (e.g., CAR / TCR hybrid). For example, the sequences of numerous TCR that bind particular antigen fragments are known and publicly available.

[0160] TCR can also be identified for use with a particular antigen by, for example, isolating T- cells that bind a particular antigen / MHC complex and sequencing the TCR chains binding the complex. TCR genes encoding TCR can be readily cloned by, for example, the 5' RACE procedure using primers corresponding to the sequences specific to the TCR a-chain gene and the TCR p-chain gene.

[0161] In particular embodiments, it may be necessary to pair TCR chains following sequencing (i.e., to perform paired chain analysis). Various methods can be utilized to pair chains, when necessary. For example, chain pairing may be assisted in silico by computer methods, such as immunology gene alignment software available from IMGT, JOINSOLVER, VDJSolver, SoDA, iHMMune-align, or other similar tools for annotating VDJ gene segments. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0162] In particular embodiments, an engineered TCR includes a single chain T-cell receptor (scTCR) including Va / p and Cct / p chains (e.g., Vo-Ca, Vp-Cp, Va-Vp) or including Va-Ca, Vp-Cp, Va-Vp pair specific for a target of interest (e.g., peptide-MHC complex).

[0163] In particular embodiments, a CAR / TCR hybrid includes components of a CAR and components of a TCR. For example, a CAR / TCR hybrid can include a TCR binding domain and intracellular signaling domains specific to CAR. This allows for antigen recognition similar to that of a TCR but use of intracellular signaling similar to a CAR.

[0164] Cancer antigens are proteins that are produced by cancer cells and viral antigens are proteins produced by virally-infected cells. Binding domains of recombinant receptors disclosed herein can be selected to bind cancer antigens or viral antigens. In some alternatives, cancer or viral antigens are selectively expressed or overexpressed on the cancerous or infected cells as compared to other cells of the same tissue type. In some alternatives, a cancer or viral antigen is a cell surface molecule that is found on cancer cells or virally-infected cells and is not substantially found on normal tissues, or restricted in its expression to non-vital normal tissues.

[0165] In particular embodiments, cancer antigens or viral antigens are preferentially expressed by cancer cells or virally-infected cells, respectively. “Preferentially expressed” means that the antigen is found on the targeted cell type at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than on non-targeted cells.

[0166] Exemplary cancer antigens include carcinoembryonic antigen (CEA), prostate specific antigen, Prostate Stem Cell antigen (PSCA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1 , CE7, ROR1 , mesothelin, c-Met, GD-2, MAGE A3 TCR, EGFR, EGFRvlll, EphA2, IL13Ra2, L1 CAM, oaGD2, GD2, B7H3, CD33, FITC, VAR2CSA, MUC16, PD-L1 , ERBB2, folate receptor (FOLR), CD56; glypican-2, disialoganglioside, EpCam, L1 -CAM, Lewis Y, WT-1 , Tyrosinase related protein 1 (TYRP1 / gp75); GD2, B-cell maturation antigen (BCMA), CD24, SV40 T, carboxy-anhydrase-IX (CAIX); and CD133. Other examples are known to those of ordinary skill in the art. Particular embodiments utilize binding domains that bind CD19.

[0167] In particular embodiments, a binding domain that binds a cancer antigen includes an scFv. In particular embodiments, an scFv includes an huCD19 (G01 S) scFv, a muCD19 (FMC63) scFv, a CD20 (Leu 16) scFv, a CD22 (m971 ) scFv, a B7H3 (hBRCA84D) scFv, an L1 CAM (CE7) scFv, an EGFR scFv, an EGFRVI 11 (806) scFv, an EphA2 (2A4) scFv, an EpHA2 (4H5) scFv, an FITC (E2) scFv, a GD2 (hu3F8) scFv, a Her2 (Herceptin) scFv, an IL13Ra2 (hu08) VIVh scFv, an IL13Ra2 hu08 VhV1 scFv, an IL13Ra2 (hu07) VhV1 scFv, an IL13Ra2 (hu07) VhVI scFv, an oaGD2 (8B6) VIVh, a ROR1 (R12) scFv, a CD33 (h2H 12) VhVI scFv, a CD33 (h2H12) VIVh scFv,a mesothelin (P4) scFv, a VAR2CSA (ID1 -DBL2Xb) scFv, or an IL13Ra2 (IL13 zetakine) amino acid sequence.

[0168] In particular embodiments, the huCD19 (G01 S) scFv with a GMCSFss includes the sequence: MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAP GKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYH YYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTG TTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCS SYAGRYNSVLFGGGTKLTVL (SEQ ID NO: 146).

[0169] In particular embodiments, a huCD19 (G01 S) scFv includes the sequence: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYA DSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYHYYDSAEHAFDIWGQGTVVTV SSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQL MLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL (SEQ ID NO: 59).

[0170] In particular embodiments, the muCD19 (FMC63) scFv includes the sequence: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITG STSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSS (SEQ ID NO: 147).

[0171] In particular embodiments, a CD19 scFv includes the sequence: DIQMTQTTSSLSALGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKL QESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:148).

[0172] In particular embodiments, the CD33 (h2H12) VhVI scFv include the sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNE KFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRL VDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIK (SEQ ID NO:149).

[0173] In particular embodiments, the CD33 (h2H12) VIVh scFv includes the sequence: DIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSG SGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQL VQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKA KATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSS (SEQ ID NO: 150).

[0174] In particular embodiments, the CD33 scFv (e.g., CD33 (h2H12) VhVI scFv and / or CD33 (h2H12) VIVh scFv) include a granulocyte-macrophage colony stimulating factor (GM-CSF) signal sequence. In particular embodiments, the GM-CSF signal sequence includes the sequence: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 151 ).

[0175] In particular embodiments, binding domains that bind CD20 can be based on the binding domains of SP32 (ab64088), EP459Y (ab78237), rlGEL / 773 (ab219329), ocrelizumab, rituximab, ofatumumab, obinutuzumab), ibritumomab, or tositumomab.

[0176] Exemplary viral antigens include coronaviral antigens: the spike (S) protein; cytomegaloviral antigens: envelope glycoprotein B and CMV pp65; Epstein-Barr antigens: EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens: the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3 and HCV NS4; herpes simplex viral antigens: immediate early proteins and glycoprotein D; HIV antigens: gene products of the gag, pol, and env genes such as HIV gp32, HIV gp41 , HIV gp 120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, the Nef protein and reverse transcriptase; influenza antigens: hemagglutinin and neuraminidase; Japanese encephalitis viral antigens: proteins E, M-E, M-E-NS1 , NS1 , NS1 -NS2A and 80% E; measles antigens: the measles virus fusion protein; rabies antigens: rabies glycoprotein and rabies nucleoprotein; respiratory syncytial viral antigens: the RSV fusion protein and the M2 protein; rotaviral antigens: VP7sc; rubella antigens: proteins E1 and E2; and varicella zoster viral antigens: gpl and gpl I. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991 ) for additional examples of viral antigens. In particular embodiments, binding domains that bind viral antigens can be used.

[0177] In particular embodiments, a bacterial antigen includes antigens expressed by bacteria. In particular embodiments, antigens are expressed by cells associated with bacterial infections. Exemplary bacteria include anthrax; gram-negative bacilli, chlamydia, diptheria, haemophilus influenza, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsiae, staphylococcus, streptococcus and tetanus.

[0178] As particular examples of bacterial antigens: anthrax antigens include anthrax protectiveantigen; gram-negative bacilli antigens include lipopolysaccharides; haemophilus influenza antigens include capsular polysaccharides; diptheria antigens include diptheria toxin; Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein and antigen 85A; pertussis toxin antigens include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase; pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharides; rickettsiae antigens include rompA; streptococcal antigens include M proteins; and tetanus antigens include tetanus toxin.

[0179] In particular embodiments, a fungal antigen includes antigens expressed by fungi. In particular embodiments, antigens are expressed by cells associated with fungal infections. Exemplary fungi include Candida, coccidiodes, cryptococcus, histoplasma, leishmania, plasmodium, protozoa, parasites, schistosomae, tinea, toxoplasma, and trypanosoma cruzi.

[0180] As further particular examples of fungal antigens: coccidiodes antigens include spherule antigens; cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heat shock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; plasmodium falciparum antigens include merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, protozoal and other parasitic antigens including the blood-stage antigen pf 155 / RESA; schistosomae antigens include glutathione-S-transferase and paramyosin; tinea fungal antigens include trichophytin; toxoplasma antigens include SAG-1 and p30; and trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa antigen.

[0181] In particular embodiments, an arthropod antigen includes antigens expressed by an arthropod. In particular embodiments, a parasite antigen includes antigens expressed by a parasite.

[0182] (vi-b) Transmembrane Domains. As indicated, transmembrane domains within a recombinant receptor serve to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.

[0183] The transmembrane domain can be derived either from a natural and / or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, or chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 CD154, Toll-like receptor 1 (TLR1 ), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In particular embodiments, a transmembrane domain may include at least the transmembraneregion(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11 a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD160, CD19, IL2R[3, IL2Ry, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, GDI lb, ITGAX, GDI Ic, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9(CD229), , PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB- A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.

[0184] In particular embodiments, a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof.

[0185] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15 amino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the recombinant receptor is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In particular embodiments, the transmembrane domain is a CD28 transmembrane domain (encoded by, for example, SEQ ID NOs: 71 , 72, 73, 74, 75, 76 or 77). In particular embodiments, the CD28 transmembrane domain has the sequence as set forth in SEQ ID NOs: 71 , 72, or 73.

[0186] (vi-c) Intracellular Effector Domains. The intracellular effector domains of a recombinant receptor are responsible for activation of the cell in which the recombinant receptor is expressed. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signalwhen bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as costimulatory domains.

[0187] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T-cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.

[0188] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11 , CD3y, CD35, CD3E, CD3 , CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRp (FceRI b), FcRy, Fyn, HVEM (LIGHTR), IGOS, LAG3, LAT, Lek, LRP, NKG2D, NOTCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, TCR , TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7- H3, a ligand that binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1 ), CD127, CD160, CD19, CD4, CD8a, CD8[3, IL2F?p, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 a, ITGAM, CD11b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In particular embodiments, the effector domain includes a CD3 signaling domain.

[0189] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of ITAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3E, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRlla, FcRp (Fee Rib), DAP10, and DAP12. In particular embodiments, variants ofCD3 retain at least one, two, three, or all ITAM regions.

[0190] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a costimulatory domain, or any combination thereof.

[0191] Additional examples of intracellular signaling components include the cytoplasmic sequences of the CD3£ chain, and / or co- receptors that act in concert to initiate signal transduction following binding domain engagement.

[0192] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance proliferation, effector function, and survival of human CAR T-cells in vitro and augments human T-cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8 , IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In particular embodiments, the co- stimulatory domain includes a 4-1 BB signaling domain.

[0193] In particular embodiments, the the intracellular signaling components include CD3 (encoded by, for example, SEQ ID NOs: 84, 85, 86, 87, or 88) and a variant of 4-1 BB (encoded by, for example, SEQ ID NOs: 78, 79, 80, 81 , 82 or 83). In particular embodiments, CD3 has the sequence as set forth in SEQ ID NOs: 84, 85, or 86 and 4-1 BB has the sequence as set forth in SEQ ID NO: 78, 79, or 80.

[0194] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor(FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1 R, IL2R, IL7R, or IL15R).

[0195] (vi-d) Linkers. A linker within a recombinant receptor can be any portion of a recombinant receptor that serves to connect two subcomponents or domains of the recombinant receptor. In particular embodiments, linkers can provide flexibility for different components of the recombinant receptor. Linkers can also include spacer regions and junction amino acids. In certain examples, when a more rigid linker is required, proline-rich linkers can be used. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose, such as a multimerization domain.

[0196] Spacers are used to create appropriate distances and / or flexibility from other recombinant receptor sub-components. As indicated, in particular embodiments, the length of a spacer is customized for binding targeted cells and mediating destruction. In particular embodiments, a spacer length can be selected based upon the location of a cellular marker epitope, affinity of a binding domain for the epitope, and / or the ability of the binding domain to mediate cell destruction following target binding. Spacers typically include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.

[0197] In particular embodiments, a spacer is 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, or 75 amino acids. These lengths qualify as short spacers.

[0198] In particular embodiments, a spacer is 76 amino acids, 90 amino acids, 100 amino acids, 1 10 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, or 179 amino acids. These lengths qualify as intermediate spacers.

[0199] In particular embodiments, a spacer is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids,220 amino acids, 228 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, or 270 amino acids. These lengths qualify as long spacers.

[0200] Exemplary spacers include all or a portion of an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wildtype immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is a human immunoglobulin hinge region. As used herein, a “wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.

[0201] An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an IgG 1 , lgG2, lgG3, or lgG4 hinge region. Sequences from IgG 1 , lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region. In particular embodiments, the lgG4 hinge region includes the sequence as set forth in SEQ ID NO: 65. Other examples of hinge regions that can be used in a recombinant receptor described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.

[0202] In particular embodiments, a spacer includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. A “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the ECD of human CD94 (GenBank Accession No. AAC50291.1 ) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'L Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11 , 2002). These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71 -93 and an ECD ranging from amino acids 94-233. The CTLD includes amino acids 1 19-231 and the stalk region includes amino acids 99-1 16, which may be flanked by additional junction amino acids. Other type II C-lectin orCD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., Gen Bank Accession Nos. NP 001993.2; AAH07037.1 ; NP 001773.1 ; AAL65234.1 ; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively). Linkers can, for example, link VL and VH of antibody derived binding domains of scFvs and serve as junction amino acids between subcomponent portions of a recombinant receptor.

[0203] Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker. Linkers can include junction amino acids. For example, in particular embodiments, linkers provide flexibility and room for conformational movement between different components of the recombinant receptor. Commonly used flexible linkers include Gly-Ser linkers. In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 152), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 153), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 132), or (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 133). In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 134), (Gly4Ser)3(SEQ ID NO: 135), (Gly4Ser)2(SEQ ID NO: 136), (Gly4Ser)i (SEQ ID NO: 137), (Gly3Ser)2(SEQ ID NO: 138), (Gly3Ser)i (SEQ ID NO: 139), (Gly2Ser)2(SEQ ID NO: 140) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 141 ), GGSGGGSGSG (SEQ ID NO: 142), or GGSGGGSG (SEQ ID NO: 143).

[0204] In particular embodiments, a linker region is (GGGGS)n (SEQ ID NO: 152) wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the linker is (EAAAK)n (SEQ ID NO: 144) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the linker includes a Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 145).

[0205] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of recombinant receptor needed for a particular use. In these instances, rigid or semirigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0206] Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfidebond cleavage. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0207] Junction amino acids can be a linker which can be used to connect sequences when the distance provided by a spacer is not needed and / or wanted. For example, junction amino acids can be short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components. In particular embodiments, junction amino acids are 9 amino acids or less (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). In particular embodiments, a glycine-serine doublet can be used as a suitable junction amino acid linker. In particular embodiments, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable junction amino acid.

[0208] In particular embodiments, the recombinant receptor can optionally include a multimerization domain. Protein biological activities depend upon their tertiary and quaternary structure. The quaternary structure requires the physical and chemical interaction of different protein subunits or polypeptides. A “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0209] (vii) Ex Vivo Manufactured Cell Formulations. In particular embodiments, genetically modified cells can be harvested from a culture medium and washed and concentrated into a carrier in a therapeutically-effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), and combinations thereof.

[0210] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0211] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0212] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.

[0213] Where necessary or beneficial, formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0214] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0215] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011.

[0216] In formulations disclosed herein, cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater than 104cells / m I, 107cells / ml or 108cells / ml.

[0217] In particular embodiments, formulations can include one or more genetically modified cell types e.g., modified T-cells, NK cells, or stem cells). Formulations can include different types of genetically-modified cells (e.g., T-cells, NK cells, and / or stem cells in combination).

[0218] Different types of genetically-modified cells or cell subsets (e.g., modified T-cells, NK cells, and / or stem cells) or cells genetically modified with different artificial expression constructs can be provided in different ratios e.g., a 1 :1 :1 ratio, 2:1 :1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5:1 :1 ratio, 1 :5:1 ratio, 1 :1 :5 ratio, 10:1 :1 ratio, 1 :10:1 ratio, 1 :1 :10 ratio, 2:2:1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1 :5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1 :10:10 ratio, 10:1 :10 ratio, etc. These ratios can also apply to numbers of cells expressing the same or different transgenes encoding intracellular molecules and / or recombinant receptors. If only two of the cell types are combined or only 2 combinations of expressed transgene encoding an intracellular molecule components are included within a formulation, the ratio can include any 2-number combination that can be createdfrom the 3 number combinations provided above. In embodiments, the combined cell populations are tested for efficacy and / or cell proliferation in vitro, in vivo and / or ex vivo, and the ratio of cells that provides for efficacy and / or proliferation of cells is selected. Particular embodiments include a 1 :1 ratio of CD4+ T-cells and CD8+ T-cells.

[0219] The cell-based formulations disclosed herein can be prepared for administration by, e.g., injection, infusion, perfusion, or lavage. The formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous injection.

[0220] (viii) Compositions for Targeted Viral Vectors & Nanoparticles for Cell Modification. Targeted viral vectors and / or nanoparticles can also be used to genetically-modify immune cells in vivo or ex vivo. Viral vectors that can be used to deliver artificial expression constructs to cells are described elsewhere herein, and numerous targeted (e.g., pseudotyped) viral vectors are known in the art.

[0221] Exemplary cell-targeted nanoparticles include a cell targeting ligand (e.g., CD3, CD4, CD8, CD34) on the surface of the nanoparticle wherein the cell targeting ligand results in selective uptake of the nanoparticle by a selected cell type. The nanoparticle then delivers gene modifying components that result in expression of the transgene encoding an intracellular molecule and transgene encoding a recombinant receptor.

[0222] Exemplary nanoparticles include liposomes (microscopic vesicles including at least one concentric lipid bilayer surrounding an aqueous core), liposomal nanoparticles (a liposome structure used to encapsulate another smaller nanoparticle within its core); and lipid nanoparticles (liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles can also be used as well as porous nanoparticles constructed from any material capable of forming a porous network. Exemplary materials include metals, transition metals and metalloids (e.g., lithium, magnesium, zinc, aluminum and silica).

[0223] For delivery and cellular uptake (e.g., in vivo), nanoparticles can have a neutral or negatively-charged coating and a size of 130 nm or less. Dimensions of the nanoparticles can be determined using, e.g., conventional techniques, such as dynamic light scattering and / or electron microscopy. In particular embodiments, the nanoparticles can be those described in WO2014153114, WO20171811 10, and WO201822672.

[0224] Therapeutically effective amounts of vectors and / or nanoparticles within formulations can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 30 pig / kg, 90 |_ig / kg, 150 pig / kg, 500 pg / kg, 750 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. Inother examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0225] (ix) Methods of Use. Methods disclosed herein include treating subjects (humans, nonhuman primates, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)) with formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0226] An "effective amount" is the amount of a formulation necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an enhanced immunogenic anti-cancer or anti-infection effect (e.g., infection with virus, bacteria, fungi, parasite, or arthropod). Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a cancer or infection’s development or progression. An immunogenic formulation can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0227] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer or infection or displays only early signs or symptoms of a cancer or infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer or infection further. Thus, a prophylactic treatment functions as a preventative treatment against a cancer or infection. In particular embodiments, prophylactic treatments reduce, delay, or prevent metastasis from a primary cancer tumor site from occurring. In particular embodiments, prophylactic treatments reduce, delay, or prevent infection from a bacteria, virus, fungi, parasite, or arthropod.

[0228] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a cancer or infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the cancer or infection. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer or infection and / or reduce control or eliminate side effects of the cancer or infection.

[0229] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0230] In particular embodiments, therapeutically effective amounts potentiate cell killing ability, proliferation, and / or cytokine output by immune cells. In particular embodiments, therapeuticallyeffective amounts potentiate cell killing ability, proliferation, IL2 secretion TNFa secretion, IFNy secretion and / or transduction positivity by immune cells. These effects can provide anti-cancer effects and / or anti-infection effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment. Anti-infection effects include a decrease in the amount or level of infective pathogen, fatigue, loss of appetite, weight loss, fevers, night sweats, chills, aches and pains, diarrhea, bloating, abdominal pain, skin rashes, coughing, and / or a runny nose.

[0231] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.

[0232] Cancers that can be treated by modified or modifying formulations and drug compositions disclosed herein include: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. Other exemplary cancers that can be treated according to the disclosure include hematopoietic tumors of lymphoid lineage, for example T cell and B cell tumors, including: T cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) of the T cell type; Sezary syndrome (SS); adult T cell leukemia lymphoma (ATLL); hepatosplenic Tcell lymphoma; peripheral / post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T cell lymphoma; angiocentric (nasal) T cell lymphoma; anaplastic (Ki 1 +) large cell lymphoma; intestinal T cell lymphoma; and T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL).

[0233] In particular embodiments, a subject’s immune cells are screened for responsiveness to inducible transgenes encoding an intracellular molecule. Screening methods include introducing an artificial expression construct including a sequence encoding an inducible intracellular molecule transgene as described elsewhere herein to a plurality of immune cells from a subject; and measuring the modulated function of the immune cells. In particular embodiments, potentiated function of an immune cell includes potentiated cell killing ability, proliferation, cytokine output, and / or maintenance of transduction positivity. In particular embodiments, potentiated cytokine output includes potentiated IL2, IFNY and / or TNFa output. Methods for screening a subject’s immune cells for responsiveness to inducible intracellular molecule expression can include cell killing assays and proliferation assays such as an MTT cell proliferation assay, trypan blue assay, carboxyfluorescein succinimidyl ester (CFSE) assay, Ki67, XTT assay, BrdU assay, flow cytometry, or DNA quantitation; or cytokine output assays such as quantitative PCR or immunodetection (e.g., enzyme-linked immunosorbent assay). Methods for screening a subject’s immune cells for responsiveness to inducible intracellular molecule expression can be performed in vitro or in vivo.

[0234] In particular embodiments, a transgene encoding an intracellular molecule that elicits increased function of immune cells compared to a baseline can be selected for administration to the subject. In particular embodiments, the baseline includes immune cell function before introduction of the artificial expression construct or immune cell function after introduction of a control (non-intracellular molecule transgene) artificial expression construct.

[0235] In particular embodiments, a combination of intracellular molecule transgenes can be chosen for administration to a subject based on the screening results. For example, if a first intracellular molecule transgene increases cell killing ability and proliferation and a second intracellular molecule transgene increases cytokine output in a first subject, then the first intracellular molecule transgene and second intracellular molecule transgene may be chosen for administration to the first subject. Alternatively, if a third intracellular molecule transgene increases cell killing ability, a fourth intracellular molecule transgene increases proliferation, and a fifth intracellular molecule transgene increases cytokine output in a second subject, then the third intracellular molecule transgene, the fourth intracellular molecule transgene, and the fifth intracellular molecule transgene may be chosen for administration to the second subject.

[0236] In particular embodiments, any combination of transgenes encoding an intracellular molecule can be administered. In particular embodiments, the combination of transgenes encoding an intracellular molecule that potentiates cell killing ability, proliferation, cytokine output, and / or maintenance of transduction positivity includes selecting at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD; and at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD, wherein the same transgene is not chosen. In particular embodiments, the combination of transgenes encoding an intracellular molecule that potentiates cell killing ability , proliferation, cytokine output, and maintenance of transduction positivity includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD and at least one of dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD. In particular embodiments, the combination of transgenes encoding an intracellular molecule that potentiates cell killing ability, proliferation, cytokine output, and / or maintenance of transduction positivity includes dnSHPI and at least one of caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD.

[0237] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer or infection, stage of cancer or infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0238] Therapeutically effective amounts of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1 O10cells, or greater than 1011.

[0239] Therapeutically effective amounts of vectors and / or nanoparticles within formulations can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 30 pg / kg, 90 pg / kg, 150 pg / kg, 500 pg / kg, 750 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0240] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0241] Therapeutically effective amounts can be administered by, e.g., injection, infusion, perfusion, or lavage. Routes of administration can include bolus intravenous, intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous administration.

[0242] In certain embodiments, formulations and / or compositions are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities. In particular embodiments, cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.

[0243] In certain embodiments, formulations and / or compositions may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents; alkyl sulfonates; aziridines; ethylenimines and methylamelamines; nitrogen mustards; nitrosureas; antibiotics; anti-metabolites; folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti-adrenals; folic acid replenisher; platinum analogs; retinoic acid derivatives; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and anti-androgens; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, CHOP, i.e., Cyclophosphamide (Cytoxan®, Ingenus Pharmaceuticals, LLC, Tampa, FL), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and Prednisone.

[0244] In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the formulation or composition. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In other embodiments, thechemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further include administering two or more chemotherapeutic agents.

[0245] A variety of additional therapeutic agents may be used in conjunction with the formulations described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®, Bristol-Myers Squibb Company, New York, NY), pembrolizumab (Keytruda®, Merck Sharp & Dohme LLC, Rahway, NJ), pembrolizumab, pidilizumab, and atezolizumab, and CTLA-4 inhibitors, such as ipilimumab (Yervoy®, Bristol- Myers Squibb Company).

[0246] Additional therapeutic agents suitable for use in combination with the disclosure include abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, casodex (bicalutamide), degarelix, docetaxel, enzalutamide, Erleada® (apalutamide), flutamide, goserelin acetate, Jevtana® (cabazitaxel), leuprolide acetate, Lupron® (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), mitoxantrone hydrochloride, Nilandron® (nilutamide), nilutamide, Provenge® (Sipuleucel-T), radium 223 di chloride, sipuleucel-T, taxotere (docetaxel), Viadur (leuprolide acetate), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate), or Zytiga (abiraterone acetate).

[0247] In additional embodiments, the formulations and / or compositions can be administered with an analgesic. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. The formulations and / or compositions can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include steroids and glucocorticoids, and nonsteroidal anti-inflammatory drugs (NSAIDS). In further embodiments, the formulations and / or compositions can be administered with biologic response modifiers. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®, Immunex Corporation, Seattle, WA), adalimumab (HUMIRA®, AbbVie Biotechnology, Ltd, North Chicago, IL) and infliximab (REMICADE®, Janssen Biotech, Inc., Horsham Township, PA), chemokine inhibitors and adhesion molecule inhibitors. In some aspects, the biological response modifiers may include monoclonal antibodies as well as recombinant forms of molecules. The formulations and / or compositions may be administered with disease-modifying antirheumatic drugs. Exemplary disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline. In additional embodiments, the formulations and / or compositions can be administered with an anti-infectiveagent (e.g., antiviral). Exemplary anti-infective agents include ribavirin, 4'-fluorouridine, small molecule TMC353121 , AVG-388, valcyclovir, acyclovir, cidofovir, foscarnet, ganciclovir, valganciclovir, penciclovir, famciclovir, idoxuridine, trifluorothymidine, vidarabine, fomivirsen, amantadine, remintadine, zanamivir, oseltamivir, lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, podophyllotoxin, imiquimod, interferons, pleconaril, maraviroc, enfuvirtide, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, emtricitabine, tenofovir disoproxil fumarate, nevirapine, delavirdine, defavirenz, raltegravir, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, fosamprenavir, lopinavir, atazanavir, tipranavir, and darunavir.

[0248] In certain embodiments, the formulations and / or compositions are administered in conjunction with a cytokine. Cytokines that could be co-administered with the formulations and / or composition disclosed herein include, for example, interferons such as interferon-alpha, beta, and - gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL1 , IL1 a, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11 , IL12, sclL12, IL15, IL18, DR-IL18, IL21 , IL36y, a tumor necrosis factor such as TNF-aor TNF- , or TGF[32-7m; and other polypeptide factors including LIF and kit ligand (KL). In relation to these combination treatments, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0249] (x) Kits. The current disclosure also includes kits. Kits can include various components to practice methods disclosed herein. For example, depending on the aspect of the methods practiced, kits could include one or more of nucleic acids encoding an intracellular molecule (i.e., intracellular molecule transgene) as disclosed herein under the control of an iSynPro promoter, an IL2 minimal promoter, and / or a constitutive promoter; nucleic acids encoding a recombinant receptor disclosed herein; a nucleic acid encoding Her2tg; a skip sequence; a nucleic acid encoding an scFv; a nucleic acid encoding a VL; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular effector domain; a nucleic acid encoding EGFRt; a nucleic acid encoding selection cassettes (e.g. DHFRdm); methotrexate; cells (e.g., immune cells, T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, natural killer (NK) cells, NK T-cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC), untransduced T-cells, T-cells transduced with artificial expression constructs describe herein); cell lines; tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, 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); genetic expression components (e.g., genes for expression provided by vectors (e.g., lentiviral vector, retroviral vector), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or nanoparticles); cell formulation or activation components (e.g., saline, buffered saline, phosphate buffered saline (PBS); biocompatible buffers such as, Ca++ / Mg++ free PBS; physiological saline, water, Hanks' solution, Ringer's solution, T-cell stimulating epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads; OKT3, TGN1412), cultureinitiating compositions, RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, non-dividing EBV-transformed lymphoblastoid cells (LCL), IL21 , human serum albumin (HSA) or other human serum components or fetal bovine serum, dextrose, stabilizers, preservatives); combination therapy components (e.g., local anesthetics, chemotherapeutic agents, immunosuppressive agents, anti-inflammatory agents, anti-infective agents); an antibody tagged with a fluorescent molecule; PCR amplification sequences; transgene encoding an intracellular molecule (e.g., dnNRDPI , dnSHPI , survivin, PCK1 , cFLIP.v3, caAKT, dnFADD, caBCL2); culture vessels; reference levels, transgenic animals; primer pairs; GAPDH; IFN-y enzyme-linked immunosorbent assay (ELISA); culture plates; etc.

[0250] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0251] (xi) Exemplary Embodiments.1 . An artificial expression construct including a transgene encoding an intracellular molecule, wherein expression of the transgene is under the regulatory control of a promoter including a transcription response element (TRE) having at least 95% sequence identity to any one of SEQ ID NOs: 17-56.2. The artificial expression construct of embodiment 1 , wherein the TRE has at least 98% sequence identity to any one of SEQ ID NOs: 17-56.3. The artificial expression construct of embodiments 1 or 2, wherein the TRE has at least 99% sequence identity to any one of SEQ ID Nos SEQ ID NOs: 17-56.4. The artificial expression construct of any of embodiments 1-3, wherein the TRE has the sequence as set forth in any one of SEQ ID NOs: SEQ ID NOs: 17-56.5. The artificial expression construct of any of embodiments 1 -4, wherein the promoter further includes a minimal promoter.6. The artificial expression construct of embodiment 5, wherein the minimal promoterincludes an IL2 minimal promoter. The artificial expression construct of embodiment 6, wherein the minimal IL2 promoter includes the sequence as set forth in SEQ ID NO: 57. The artificial expression construct of any of embodiments 1 -7, wherein the intracellular molecule includes dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2), Survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), or dominant negative fas-associating protein with death domain (dnFADD). The artificial expression construct of any of embodiments 1 -8, wherein the intracellular transgene has the sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16. The artificial expression construct of any of embodiments 1 -9, wherein the intracellular molecule includes the sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, or SEQ ID NO: 15 or a sequence having at least 90% sequence identity to the sequence set SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, or SEQ ID NO: 15. The artificial expression construct of any of embodiments 1 -10, further including a control feature. The artificial expression construct of embodiment 11 , wherein the control feature includes a transduction marker. The artificial expression construct of embodiment 12, wherein the transduction marker includes Her2tG. The artificial expression construct of any of embodiments 1 -13, further including a first skip sequence. The artificial expression construct of embodiment 14, wherein the first skip sequence encodes a 2A self-cleaving polypeptide. The artificial expression construct of embodiment 15, wherein the 2A self-cleaving polypeptide includes T2A, P2A, E2A, or F2A.The artificial expression construct of any of embodiments 1 -16, wherein the artificial expression construct has at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131. The artificial expression construct of any of embodiments 1 -17, wherein the artificial expression construct has at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:131. The artificial expression construct of any of embodiments 1 -18, wherein the artificial expression construct has at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131. The artificial expression construct of any of embodiments 1 -19, wherein the artificial expression construct has the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131. The artificial expression construct of any of embodiments 1 -19, further including a sequence encoding a recombinant receptor, wherein the recombinant receptor includes an extracellular component linked to an intracellular component through a transmembrane domain. The artificial expression construct of embodiment 21 , wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. The artificial expression construct of embodiment 22, wherein the second promoter includes an EF1 a(L) promoter, EF1 a(s) promoter, myeloproliferative sarcoma virus (MND) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, or a creatine kinase promoter. The artificial expression construct of any of embodiments 21-23, wherein the extracellular component comprises a binding domain that binds an antigen expressed on the surface of targeted cells. The artificial expression construct of any of embodiments 21 -24, wherein the targeted cells include cancer cells or cells infected with bacteria, virus, fungi, parasites, or arthropods.The artificial expression construct of any of embodiments 21 -25, wherein the intracellular component includes a CD3 signaling domain and / or a 4-1 BB signaling domain. The artificial expression construct of any of embodiments 21 -26, wherein the transmembrane domain includes a CD28 transmembrane domain. The artificial expression construct of any of embodiments 21 -27, further including a second control feature. The artificial expression construct of embodiment 28, wherein the second control feature includes a selection cassette. The artificial expression construct of embodiment 29, wherein the selection cassette includes dihydrofolate reductase double mutant (DHFRdm). The artificial expression construct of any of embodiments 21 -30, further including a third control feature. The artificial expression construct of embodiment 31 , wherein the third control feature includes a transduction marker. The artificial expression construct of embodiment 32, wherein the transduction marker includes epidermal growth factor receptor (EGFRt) or truncated CD19 (tCD19). The artificial expression construct of any of embodiments 21 -33, further including a second skip sequence. The artificial expression construct of embodiment 34, wherein the second skip sequence encodes a 2A self-cleaving polypeptide. The artificial expression construct of embodiment 35, wherein the 2A skip self-cleaving polypeptide includes T2A, P2A, E2A, or F2A. The artificial expression construct of any of embodiments 14-36, wherein the artificial expression construct includes a second skip sequence at the 5’ end of the selection cassette and a third skip sequence at the 5’ end of the transduction marker. The artificial expression construct of any of embodiments 1 -37, wherein the artificial expression construct has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111. The artificial expression construct of any of embodiments 1 -38, wherein the artificial expression construct has the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104,SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109,SEQ ID NO: 110, or SEQ ID NO: 111 . The artificial expression construct of any of embodiments 1 -39, wherein the artificialexpression construct has at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEO ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111. A system for modulating immune cell function including a first artificial expression construct including a transgene encoding an intracellular molecule under the regulatory control of a first promoter having a transcription response element (TRE) having at least 95% sequence identity to any of SEQ ID NOs: 17-56; and a second artificial expression construct including a sequence encoding a recombinant receptor, wherein the recombinant receptor comprises and extracellular component linked to an intracellular component through a transmembrane domain. The system of embodiment 41 , wherein the TRE has at least 98% sequence identity to any one of SEQ ID NOs: 17-56 The system of embodiments 41 or 42, wherein the TRE has at least 99% sequence identity to any one of SEQ ID NOs: 17-56 The system of any of embodiments 41 -43, wherein the TRE has the sequence as set forth in any one of SEQ ID NOs: 17-56. The system of any of embodiments 41 -45, wherein the first promoter further includes a minimal promoter. The system of embodiment 45, wherein the minimal promoter includes a minimal IL2 promoter. The system of embodiment 46, wherein the minimal IL2 promoter includes the sequence as set forth in SEQ ID NO: 57. The system of any of embodiments 41-47, wherein the intracellular molecule includes dominant negative Src homology region 2 (SH-2) domain-containing phosphatase 1 (SHP1 ) (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2), Survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), or dominant negative fas-associating protein with death domain (dnFADD. The system of any of embodiments 41 -47, wherein the intracellular molecule is encoded by the sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO : 16or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 orSEQ ID NO : 16. The system of any of embodiments 41 -49, wherein the intracellular molecule has the sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, or SEQ ID NO: 15 or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, or SEQ ID NO: 15. The system of any of embodiments 41 -50, wherein the first artificial expression construct further includes a control feature. The system of embodiment 51 , wherein the control feature includes a transduction marker. The system of embodiment 50, wherein the transduction marker includes Her2tG. The system of any of embodiments 41 -53, wherein the first artificial expression construct further includes a first skip sequence. The system of embodiment 54, wherein the first skip sequence includes a 2A skip sequence. The system of embodiment 55, wherein the 2A skip sequence encodes a T2A, P2A, E2A, or F2A self-cleaving polypeptide. The system of any of embodiments 41 -56, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. The system of embodiment 57, wherein the second promoter includes an EF1 a(L) promoter, EF1 a(s) promoter, MND promoter, CMV promoter, SV40 early promoter, MMTV promoter, HIV LTR promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. The system of any of embodiments 41-58, wherein the second artificial expression construct further includes a second control feature. The system of embodiment 59, wherein the second control feature includes a selection cassette. The system of embodiment 60, wherein the selection cassette includes dihydrofolate reductase double mutant (DHFRdm). The system of any of embodiments 41-61 , wherein the second artificial expression construct further includes a third control feature. The system of embodiment 62, wherein the third control feature includes a transduction marker.The system of embodiment 63, wherein the transduction marker includes truncated epidermal growth factor receptor (EGFRt) or tCD19. The system of any of embodiments 41-64, wherein the second artificial expression construct further includes a second skip sequence. The system of embodiment 65, wherein the second skip sequence includes a 2A skip sequence. The system of embodiment 66, wherein the 2A skip sequence encodes a T2A, P2A, E2A, or F2A self-cleaving polypeptide. The system of any of embodiments 41-67, wherein the second artificial expression construct includes a second skip sequence at the 5’ end of the selection cassette and a third skip sequence at the 5’ end of the transduction marker. The system of any of embodiments 41 -68, wherein the extracellular component includes a binding domain that binds an antigen expressed on the surface of targeted cells. The system of any of embodiments 41-69, wherein the targeted cells include cancer cells or cells infected with bacteria, virus, fungi, parasites, or arthropods. The system of embodiment 41 -70, wherein the intracellular component includes a CD3 signaling domain and / or a 4-1 BB signaling domain. The system of embodiment 41 -71 , wherein the transmembrane domain includes a CD28 transmembrane domain. The system of any of embodiments 41 -72, wherein the first artificial expression construct and the second artificial expression construct are on the same artificial expression construct. The system of any of embodiments 41 -72, wherein the first artificial expression construct and the second artificial expression construct are on different artificial expression constructs. A nanoparticle encapsulating the artificial expression construct of any of embodiments 1 - 40 or the system of any of embodiments 41-74. A cell genetically modified to express the artificial expression construct of any of embodiments 1 -40 or the system of any of embodiments 41 -74. The cell of embodiment 76, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject. The cell of embodiments 76 or 77, wherein the cell is in vivo or ex vivo. The cell of any of embodiments 76-77, wherein the cell is an immune cell. The cell of embodiment 77, wherein the immune cell is a lymphocyte.The cell of embodiment 78, wherein the lymphocyte includes a T-cell, B-cell, natural killer (NK) cell, or NK T-cell. The cell of any of embodiments 76-81 , wherein the cell is a T-cell selected from a CD3+ T-cell, a CD4+ T-cell, a CD8+ T-cell, a central memory T-cell, an effector memory T-cell, and / or a naive T-cell. The cell of any of embodiments 76-81 , wherein the cell is a CD8+ T-cell. The cell of any of embodiments 76-81 , wherein the cell is a CD4+ T-cell. A population of cells genetically modified to express the artificial expression construct of any of embodiments 1-40 or the system of any of embodiments 41 -74. The population of cells of embodiment 85, wherein the population of cells includes autologous cells or allogeneic cells in reference to a subject. The population of cells of embodiments 85 or 86, wherein the population is in vivo or ex vivo. The population of cells of any of embodiments 85-87, wherein the cell is an immune cell. The population of cells of embodiment 88, wherein the immune cell is a lymphocyte. The population of cells of embodiment 89, wherein the lymphocyte includes a T-cell, 13- cell, natural killer (NK) cell, or NK T-cell. The population of cells of any of embodiments 85-90, wherein the population includes CD4+ T-cells and / or CD8+ T-cells. A formulation including (i) cells genetically modified to express the artificial expression construct of any of embodiments 1-41 or the system of any of embodiments 39-71 and (ii) a pharmaceutically acceptable carrier. A method of genetically modifying an immune cell to have potentiated function including: contacting the cell with the artificial expression construct of any of embodiments 1 -40 or the nanoparticle of embodiment 75. The method of embodiment 93, wherein the potentiated function includes potentiated cell killing ability. The method of embodiment 93, wherein the artificial construct has a sequence encoding a transgene encoding an intracellular molecule, wherein the intracellular molecule includes dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), and dominant negative fas-associatingprotein with death domain (dnFADD). The method of embodiments 94 or 95, wherein the intracellular molecule includes dnNRDPI , survivin, dnSHPI or caBCL2. The method of any of embodiments 93-96, wherein the potentiated function includes potentiated proliferation. The method of embodiment 93, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD. The method of embodiments 93 or 94, wherein the intracellular molecule includes dnSHPI , caBCLI , or dnNRDPI . . The method of any of embodiments 93-99, wherein the potentiated function includes potentiated cytokine output. . The method of embodiment 100, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD. . The method of embodiment 100, wherein the potentiated cytokine output includes IL2 output. . The method of embodiment 102, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, 68urviving, or dnFADD. . The method of any of embodiments 100-103, wherein the potentiated cytokine output includes TNFa output. . The method of embodiment 104, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, 68urviving, or dnFADD. . The method of any of embodiments 100-105, wherein the potentiated cytokine output includes IFNy. . The method of embodiment 106, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , cFLIP.v3, caAKT, survivin, or dnFADD. . The method of embodiment 93-107, wherein the potentiated function includes maintained transduction positivity. . The method of embodiment 108, wherein the intracellular molecule includes dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD. . The method of any of embodiments 93-109, further comprising stimulating the genetically modified immune cell. . The method of embodiment 110, wherein the stimulating comprises a first stimulation, a second stimulation, or a third stimulation. . The method of any of embodiments 102-111 , wherein the intracellular moleculecomprises dnSHPI , caBCL2, cFLIP.v3, caAKT, or dnFADD after a first stimulation.. The method of any of embodiments 102-111 , wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , caAKT, survivin, or dnFADD after a third stimulation. . The method of any of embodiments 104-113, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD after a first stimulation. . The method of any of embodiments 104-113, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , caAKT, or dnFADD after a third stimulation. . The method of any of embodiments 106-115, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , caAKT, survivin, or dnFADD after a first stimulation. . The method of any of embodiments 106-115, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , cFLIP.v3, caAKT, or dnFADD after a third stimulation. . The method of embodiment 108, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , or survivin after a first stimulation. . The method of embodiment 108, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD after a third stimulation. . The method of any of embodiments 93-1 19, wherein the artificial expression construct further includes a sequence encoding a recombinant receptor including an extracellular component linked to an intracellular component though a transmembrane domain. . The method of embodiment 120, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. . The method of embodiment 121 , wherein the second promoter includes an EF1 a(L) promoter, EF1 a(s) promoter, myeloproliferative sarcoma virus (MND) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, or a creatine kinase promoter.123. The method of any of embodiments 93-122, wherein the artificial expression construct is a first artificial expression construct and the method further includes contacting the cell with a second artificial expression construct including a sequence encoding a recombinant receptor including an extracellular component linked to an intracellular component through a transmembrane domain.124. The method of embodiment 123, wherein the contacting the cell with the first artificial expression construct and the second artificial expression construct occurs simultaneously.125. The method of embodiment 123, wherein the contacting the cell with the first artificial expression construct and the second artificial expression construct occurs at different times.126. The method of any of embodiments 93-125, wherein the artificial expression construct includes the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, SEQ ID NO: 1 11 , SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131 or a sequence having 90% sequence identity to a sequence as set forth in SEQ ID NO: 103,SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108,SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 11 1 , SEQ ID NO: 124, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131.127. A method of treating a subject in need thereof including administering a therapeutically effective amount of the artificial expression construct of any of embodiments 1 -40, the system of any of embodiments 41-74, the nanoparticle of embodiment 75, or the formulation of embodiment 92 to the subject thereby treating the subject in need thereof.128. The method of embodiment 127, wherein the subject in need thereof includes cancer or an infection.129. The method of embodiments 127 or 128, wherein the administering a therapeutically effective amount includes administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.

[0252] (xii) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein areshown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1 .831 -1 .835 and set forth in WIPO Standard ST.26 (implemented on July 1 , 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0253] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0254] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0255] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1 ), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: He (+4.5); Vai (+4.2); Leu(+3.8); Phe (+2.8); Cys (+2.5); Met (+1 .9); Ala (+1 .8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0256] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0257] As detailed in US Patent No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1 ); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0258] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-sig nificant degree.

[0259] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0260] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151 -153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, etal., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the PASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, H I - 20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0261] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 gg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher saltconcentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0262] "Binds" refers to an association of a binding domain (of, for example, a binding domain) to its cognate binding molecule with an affinity or Ka( / .e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Kaof at least 107M'1, at least 108M’1, at least 109M’1, at least 1010M-1, at least 1011M'1, at least 1012M-1, or at least 1013M’1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M’1, up to 106M-1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 105M to 1013M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd(dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (KOff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 57:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0263] The terms “enhance” and “potentiate” are used synonymously herein.

[0264] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed.Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney’s Culture of Animal Cells 8,hEdition (2021).

[0265] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in recombinant receptor-immune cell potentiated cell killing ability, proliferation, cytokine output, and / or maintenance of transduction (e.g., EGFRt) positivity, as described herein.

[0266] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0267] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in their respective testing measurements.

[0268] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0269] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0270] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0271] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0272] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0273] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0274] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMSWhat is claimed is:1 . An artificial expression construct comprising: an inducible promoter comprising SEQ ID NO: 17 and SEQ ID NO: 57; and a transgene comprising a sequence as set forth in SEQ ID NO: 16.

2. An artificial expression construct comprising a transgene encoding an intracellular molecule, wherein the transgene is under regulatory control of a promoter, and wherein the promoter comprises a minimal promoter and transcriptional response element (TRE) having at least 98% sequence identity to any one of SEQ ID NOs: 17-56.

3. The artificial expression construct of claim 2, wherein the TRE has at least 99% sequence identity to any one of SEQ ID NOs: 17-56.

4. The artificial expression construct of claim 2, wherein the TRE has the sequence as set forth in any one of SEQ ID NOs: 17-56.

5. The artificial expression construct of claim 2, wherein the promoter further comprises a minimal promoter.

6. The artificial expression construct of claim 5, wherein the minimal promoter is a minimal IL2 promoter.

7. The artificial expression construct of claim 6, wherein the minimal IL2 promoter comprises the sequence as set forth in SEQ ID NO: 57.

8. The artificial expression construct of claim 2, wherein the intracellular molecule comprises dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active-B cell lymphoma 2 (ca-BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-i p-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), or dominant negative fas-associating protein with death domain (dnFADD).

9. The artificial expression construct of claim 2, wherein the transgene encoding the intracellular molecule has the sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence having at least 90% sequence identity to the sequence set forth SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

10. The artificial expression construct of claim 2, wherein the intracellular molecule has the sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, or SEQ ID NO: 15 or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, or SEQ ID NO: 15.11 . The artificial expression construct of claim 2, further comprising a control feature.

12. The artificial expression construct of claim 11 , wherein the control feature comprises a transduction marker.

13. The artificial expression construct of claim 12, wherein the transduction marker comprises Her2tG.

14. The artificial expression construct of claim 2, further comprising a first skip sequence.

15. The artificial expression construct of claim 14, wherein the first skip sequence encodes a 2A self-cleaving polypeptide.

16. The artificial expression construct of claim 15, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

17. The artificial expression construct of claim 2, wherein the artificial expression construct comprises at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131.

18. The artificial expression construct of claim 2, wherein the artificial expression construct comprises at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131.

19. The artificial expression construct of claim 2, wherein the artificial expression construct comprises at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131.

20. The artificial expression construct of claim 2, wherein the artificial expression construct has the sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131.

21. The artificial expression construct of claim 2, further comprising a sequence encoding a recombinant receptor, wherein the recombinant receptor comprises an extracellular component linked to an intracellular component through a transmembrane domain.

22. The artificial expression construct of claim 21 , wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

23. The artificial expression construct of claim 22, wherein the second promoter comprises an EF1 a(L) promoter, EF1 a(s) promoter, myeloproliferative sarcoma virus (MND) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, or a creatine kinase promoter.

24. The artificial expression construct of claim 21 , wherein the extracellular component comprises a binding domain that binds an antigen expressed on a surface of targeted cells.

25. The artificial expression construct of claim 24, wherein the targeted cells comprise cancer cells or cells infected with bacteria, virus, fungi, parasites, or arthropods.

26. The artificial expression construct of claim 21 , wherein the intracellular component comprises a CD3 signaling domain and / or a 4-1 BB signaling domain.

27. The artificial expression construct of claim 21 , wherein the transmembrane domain comprises a CD28 transmembrane domain.

28. The artificial expression construct of claim 11 , further comprising a second control feature.

29. The artificial expression construct of claim 28, wherein the second control feature comprises a selection cassette.

30. The artificial expression construct of claim 29, wherein the selection cassette comprises dihydrofolate reductase double mutant (DHFRdm).31 . The artificial expression construct of claim 28, further comprising a third control feature.

32. The artificial expression construct of claim 31 , wherein the third control feature comprises a transduction marker.

33. The artificial expression construct of claim 32, wherein the transduction marker comprises epidermal growth factor receptor (EGFRt) or truncated CD19 (tCD19).

34. The artificial expression construct of claim 14, further comprising a second skip sequence.

35. The artificial expression construct of claim 34, wherein the second skip sequence encodes a 2A self-cleaving polypeptide.

36. The artificial expression construct of claim 35, wherein the 2A skip self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

37. The artificial expression construct of claim 32, wherein the artificial expression construct comprises a second skip sequence at the 5' end of the selection cassette and a third skip sequence at the 5' end of the transduction marker.

38. The artificial expression construct of claim 2, wherein the artificial expression construct has at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, or SEQ ID NO: 111.

39. The artificial expression construct of claim 2, wherein the artificial expression construct has at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, or SEQ ID NO: 111.

40. The artificial expression construct of claim 2, wherein the artificial expression construct has the sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, or SEQ ID NO: 111.41 . A system for potentiating immune cell function comprising: a first artificial expression construct comprising a transgene encoding an intracellular molecule under regulatory control of a first promoter, wherein the first promoter comprises a minimal promoter and a transcription response element (TRE) having at least 98% sequence identity to any of SEQ ID NOs: 17-56; and a second artificial expression construct comprising a sequence encoding a recombinant receptor, wherein the recombinant receptor comprises and extracellular component linked to an intracellular component through a transmembrane domain.

42. The system of claim 41 , wherein the TRE has at least 99% sequence identity to any one of SEQ ID NOs: 17-56.

43. The system of claim 41 , wherein the TRE comprises a sequence as set forth in any one of SEQ ID NOs: 17-56.

44. The system of claim 41 , wherein the minimal promoter is a minimal IL2 promoter.

45. The system of claim 44, wherein the minimal IL2 promoter comprises the sequence as set forth in SEQ ID NO: 57.

46. The system of claim 41 , wherein the intracellular molecule comprises dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active- 13 cell lymphoma 2 (ca-BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 ^-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), or dominant negative fas-associating protein with death domain(dnFADD).

47. The system of claim 41 , wherein the transgene encoding the intracellular molecule comprises the sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO : 16.

48. The system of claim 41 , wherein the intracellular molecule comprises the sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, or SEQ ID NO: 15 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, or SEQ ID NO : 15.

49. The system of claim 41 , wherein the first artificial expression construct further comprises a control feature.

50. The system of claim 49, wherein the control feature comprises a transduction marker.51 . The system of claim 50, wherein the transduction marker comprises Her2tG.

52. The system of claim 41 , wherein the first artificial expression construct further comprises a first skip sequence.

53. The system of claim 52, wherein the first skip sequence comprises a 2A skip sequence.

54. The system of claim 53, wherein the 2A skip sequence encodes a T2A, P2A, E2A, or F2A self-cleaving polypeptide.

55. The system of claim 41 , wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

56. The system of claim 55, wherein the second promoter comprises an EF1a(L) promoter, EF1 a(s) promoter, MND promoter, CMV promoter, SV40 early promoter, MMTV promoter, HIV LTR promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

57. The system of claim 41 , wherein the second artificial expression construct further comprises a second control feature.

58. The system of claim 57, wherein the second control feature comprises a selection cassette.

59. The system of claim 58, wherein the selection cassette comprises dihydrofolate reductase double mutant (DHFRdm).

60. The system of claim 41 , wherein the second artificial expression construct further comprises a third control feature.61 . The system of claim 60, wherein the third control feature comprises a transduction marker.

62. The system of claim 61 , wherein the transduction marker comprises truncated epidermal growth factor receptor (EGFRt) or truncated CD19 (tCD19).

63. The system of claim 41 , wherein the second artificial expression construct further comprises a second skip sequence.

64. The system of claim 63, wherein the second skip sequence comprises a 2A skip sequence.

65. The system of claim 64, wherein the 2A skip sequence encodes a T2A, P2A, E2A, or F2A self-cleaving polypeptide.

66. The system of claim 61 , wherein the second artificial expression construct comprises a second skip sequence at the 5’ end of the selection cassette and a third skip sequence at the 5’ end of the transduction marker.

67. The system of claim 41 , wherein the extracellular component comprises a binding domain that binds an antigen expressed on a surface of targeted cells.

68. The system of claim 67, wherein the targeted cells comprise cancer cells or cells infected with bacteria, virus, fungi, parasites, or arthropods.

69. The system of claim 41 , wherein the intracellular component comprises a CD3£ signaling domain and / or a 4-1 BB signaling domain.

70. The system of claim 41 , wherein the transmembrane domain comprises a CD28 transmembrane domain.

71. The system of claim 41 , wherein the first artificial expression construct and the second artificial expression construct are on a same artificial expression construct.

72. The system of claim 41 , wherein the first artificial expression construct and the second artificial expression construct are on different artificial expression constructs.

73. A nanoparticle encapsulating the artificial expression construct of claim 1 or the system of claim 41 .

74. A cell genetically modified to express the artificial expression construct of claim 1 or the system of claim 41 .

75. The cell of claim 74, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject.

76. The cell of claim 74, wherein the cell is in vivo or ex vivo.

77. The cell of claim 74, wherein the cell is an immune cell.

78. The cell of claim 77, wherein the immune cell is a lymphocyte.

79. The cell of claim 78, wherein the lymphocyte comprises a T-cell, B-cell, natural killer (NK) cell, or NK T-cell.

80. The cell of claim 79, wherein the cell is a T-cell selected from a CD3+ T-cell, a CD4+ T- cell, a CD8+ T-cell, a central memory T-cell, an effector memory T-cell, and / or a naive T- cell.81 . The cell of claim 74, wherein the cell is a CD8+ T-cell.

82. The cell of claim 74, wherein the cell is a CD4+ T-cell.

83. A population of cells genetically modified to express the artificial expression construct of claim 1 or the system of claim 41 .

84. The population of cells of claim 83, wherein the population of cells comprises autologous cells or allogeneic cells in reference to a subject.

85. The population of cells of claim 83, wherein the population is in vivo or ex vivo.

86. The population of cells of claim 83, wherein the cell is an immune cell.

87. The population of cells of claim 86, wherein the immune cell is a lymphocyte.

88. The population of cells of claim 87, wherein the lymphocyte comprises a T-cell, B-cell, natural killer (NK) cell, or NK T-cell.

89. The population of cells of claim 83, wherein the population comprises CD4+ T-cells and / or CD8+ T-cells.

90. A formulation comprising (i) cells genetically modified to express the artificial expression construct of claim 1 or the system of claim 41 and (ii) a pharmaceutically acceptable carrier.91 . A method of genetically modifying an immune cell to have potentiated function comprising: contacting the cell with the artificial expression construct of claim 1 or the nanoparticle of claim 73.

92. The method of claim 91 , wherein the potentiated function comprises potentiated cell killing ability.

93. The method of claim 91 , wherein the intracellular molecule comprises dominant negative Src homology region 2 domain-containing phosphatase 1 (dnSHPI ), constitutively active- B cell lymphoma 2 (ca-BCL2), survivin, dominant negative neuregulin receptor degradation protein-1 (dnNRDPI ), phosphoenolpyruvate carboxykinase 1 (PCK1 ), cellular FADD-like IL-1 [3-converting enzyme inhibitory protein (cFLIP.v3), constitutively active AkT (caAKT), or dominant negative fas-associating protein with death domain (dnFADD).

94. The method of claim 92, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , or survivin.

95. The method of claim 91 , wherein the potentiated function comprises potentiated proliferation.

96. The method of claim 95, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD.

97. The method of claim 95, wherein the intracellular molecule comprises dnSHPI , caBCLI , or dnNRDPI .

98. The method of claim 91 , wherein the potentiated function comprises potentiated cytokine output.

99. The method of claim 98, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, and dnFADD.

100. The method of claim 98, wherein the potentiated cytokine output comprises IL2 output.

101. The method of claim 100, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD.

102. The method of claim 98, wherein the potentiated cytokine output comprises TNFa output.

103. The method of claim 102, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD.

104. The method of claim 98, wherein the potentiated cytokine output comprises IFNy.

105. The method of claim 104, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , cFLIP.v3, caAKT, survivin, or dnFADD.

106. The method of claim 91 , wherein the potentiated function comprises maintained transduction positivity.

107. The method of claim 106, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD.

108. The method of claim 91 , further comprising stimulating the genetically modified immune cell.

109. The method of claim 108, wherein the stimulating comprises a first stimulation, a second stimulation, or a third stimulation.

110. The method of claim 100, wherein the intracellular molecule comprises dnSHPI , caBCL2, cFLIP.v3, caAKT, or dnFADD after a first stimulation.

111. The method of claim 100, wherein the intracellular molecule comprises dnSHPI ,caBCL2, dnNRDPI , PCK1 , caAKT, survivin, or dnFADD after a third stimulation.

112. The method of claim 102, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD after a first stimulation.

113. The method of claim 102, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , caAKT, or dnFADD after a third stimulation.

114. The method of claim 104, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , caAKT, survivin, or dnFADD after a first stimulation.

115. The method of claim 104, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , cFLIP.v3, caAKT, or dnFADD after a third stimulation.

116. The method of claim 106, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , or survivin after a first stimulation.

117. The method of claim 106, wherein the intracellular molecule comprises dnSHPI , caBCL2, dnNRDPI , PCK1 , cFLIP.v3, caAKT, survivin, or dnFADD after a third stimulation.1 18. The method of claim 91 , wherein the artificial expression construct further comprises a sequence encoding a recombinant receptor comprising an extracellular component linked to an intracellular component though a transmembrane domain.1 19. The method of claim 118, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

120. The method of claim 119, wherein the second promoter comprises an EF1a(L) promoter, EF1 a(s) promoter, myeloproliferative sarcoma virus (MND) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, or a creatine kinase promoter.121 . The method of claim 91 , wherein the artificial expression construct is a first artificial expression construct and the method further comprises contacting the cell with a second artificial expression construct comprising a sequence encoding a recombinant receptor comprising an extracellular component linked to an intracellular component through a transmembrane domain.

122. The method of claim 121 , wherein the contacting the cell with the first artificial expression construct and the second artificial expression construct occurs simultaneously.

123. The method of claim 121 , wherein contacting the cell with the first artificial expression construct and the second artificial expression construct occurs at differenttimes.

124. The method of claim 91 , wherein the artificial expression construct comprises the sequence as set forth in SEQ ID NOs: SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:1 10, SEQ ID NO: 111 , SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO:131 or a sequence having 90% sequence identity to a sequence as set forth in SEQ ID NOs: SEQID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, SEQ ID NO: 111 , SEQ ID NO: 124, SEQID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQID NO: 130, or SEQ ID NO:131.

125. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the artificial expression construct of claim 1 , the system of claim 41 , the nanoparticle of claim 73, or the formulation of claim 90 to the subject thereby treating the subject in need thereof.

126. The method of claim 125, wherein the subject in need thereof has cancer or an infection.

127. The method of claim 125, wherein the administering a therapeutically effective amount comprises administering intravesically, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.

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