WO2025255080A1 - Hypoxia inducible high-fidelity circuit - Google Patents

Abstract

The disclosure provides constructs that permit hypoxia-controlled expression to be selectively active in tumors and tumor microenvironments, thereby reducing off target effects of expressed therapeutic genes, such as CAR T cells.

Description

DESCRIPTION HYPOXIA INDUCIBLE HIGH-FIDELITY CIRCUIT PRIORITY CLAIM [0001] This application claims benefit of priority to U.S. Provisional Application Serial No. 63/655,736 filed June 4, 2024, the entire contents of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING [0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on May 27, 2025, is named CHOPP0093WO.xml and is 33,677 bytes in size. FIELD [0003] The present disclosure relates generally to systems for delivery of agents like CARs and other therapeutic agents to patients using hypoxic responsive elements to drive selective expression are provided. BACKGROUND [0004] Cancer is the second leading cause of death in the world, accounting for one in six deaths. Current treatment options, including surgery, chemotherapy, radiotherapy, and target therapy, can lead to complete remission. However, most of the patients still did not respond or experienced tumor progression after therapy, calling for new therapeutic strategies in cancer treatment. Adoptive chimeric antigen receptor (CAR) T cell therapy has shown durable complete remissions in leukemia and lymphoma, thus revolutionizing clinical guidelines in hematologic malignancies. However, the clinical outcomes of CAR T therapy in solid tumors have been largely disappointing, caused by loss of the antigen targeted by the CAR due to tumor heterogeneity or tumor adaptation to immunotherapy. While targeting a certain tumor antigen may cause escape of non-targetable tumor cells, CAR-T cells recognizing multiple tumor-associated antigens may also lead to on-target off-tumor toxicity. Improved CAR T cell therapies that overcome one of more of these limitations are therefore greatly in need. SUMMARY [0005] In accordance with the present disclosure, there is provided an expression vector comprising an expression vector comprising a nucleic acid encoding a first gene of interest under the control of a hypoxia responsive (HRE) promoter, wherein the gene of interest is linked to a coding region of an oxygen-dependent degradation (ODD). The expression vector may further comprise(s) an origin of replication, and/or wherein the TCF is HIF-1α HRE promoter. The TCF may comprise (i) an orthogonal DNA binding domain, such as Gal4, LexA, tTR, HNF1A, PAX6, ZF10 or a zinc finger protein (e.g., ZFHD1) and (ii) an activation domain, such as VP64, VP16, GAL4, Med2, p65A, rTA, MRTF-A, STAT1 and NRF2. The expression vector may be a viral vector, such as a retroviral expression vector a lentiviral expression vector, an adenoviral expression vector, such as an oncolytic adenovirus, a herpes simplex virus expression vector, such as an oncolytic herpes simplex virus, or a non-integrating adeno-associated viral expression vector, optionally further characterized as a self-inactivation lentiviral expression vector, or a non-viral expression vector. The expression vector may further encode a Lum-IL-12/ODD domain fusion protein construct under the control of an HRE. The first gene of interest may be a first chimeric antigen receptor (CAR). The expression vector may further encode a second gene of interest under the control of a constitutive promoter, such as an EF1α promoter, a CMV promoter, a PGK promoter, an SFFV promoter, or a ubiquitin promoter. The second gene of interest may be a second CAR that binds specifically to a non-endogenous small ligand, such as fluorescein isothiocyanate (FITC), pepvIII, or PNE peptide. [0006] Also provided is an engineered cell carrying the expression vector as described herein. The engineered cell may be a transgenic T cell, a chimeric antigen receptor (CAR) T cell, a macrophage, an NK cell, a neutrophil, a monocyte, an NKT cell, a gamma-delta T cell, an allogeneic T cell, or an immortalized T cell line (e.g., TALL104 cells, NK92). The engineered cell expresses said gene of interest only at less than 5% oxygen, such as wherein said gene of interest is only expressed at 0-4.0%, 0-3.0%, 0-2.0%, 0-1.5%, 0-1.25%, 0-1.0%, or 0-0.5% hypoxic conditions. [0007] In another embodiment, there is provided an expression vector comprising (a) a first nucleic acid encoding a transcriptional control factor (TCF) fused an oxygen-dependent degradation (ODD) domain under the control of a hypoxia responsive (HRE) promoter 2 4930-3431-6359, v.1 element active in eurkaryotic cells; and (b) a second nucleic acid encoding chimeric antigen receptor (CAR) under the control a transcription control element bound and activated by said TCF. The expression vector may further comprise(s) an origin of replication. The TCF may comprise (i) an orthogonal DNA binding domain, such as Gal4, LexA, tTR, HNF1A, PAX6, ZF10 or a zinc finger protein (e.g., ZFHD1) and (ii) an activation domain, such as VP64, VP16, GAL4, Med2, p65A, rTA, MRTF-A, STAT1 and NRF2. The expression vector may be a viral vector, such as a retroviral expression vector a lentiviral expression vector, an adenoviral expression vector, such as an oncolytic adenovirus, a herpes simplex virus expression vector, such as an oncolytic herpes simplex virus, or a non-integrating adeno- associated viral expression vector, optionally further characterized as a self-inactivation lentiviral expression vector, or may be a non-viral expression vector. The expression vector may further encode a Lum-IL-12/ODD domain fusion protein construct under the control of an HRE, such as the same HRE controlling CAR expression; and/or further encoding a second CAR construct targeting a tumor antigen under the control of a constitutive promoter, such as an EF1α promoter, a CMV promoter, a PGK promoter, an SFFV promoter, or a ubiquitin promoter. The second CAR construct may bind specifically to a non-endogenous small ligand, such as fluorescein isothiocyanate (FITC), pepvIII, or PNE peptide. The expression vector may further comprise a nuclear localization signal and a nuclear exportation signal that modulate cytosolic/nuclear distribution. The engineered cell may be transgenic T cell, a chimeric antigen receptor (CAR) T cell, a macrophage, an NK cell, a neutrophil, a monocyte, an NKT cell, a gamma-delta T cell, an allogeneic T cell, or an immortalized T cell line (e.g., TALL104 cell, NK92 cell). [0008] In further embodiments, there provided a method of killing a cancer cell comprising contacting said cancer cell with an engineered cell as described herein, and a method of treating a subject with a solid tumor comprising administering to said subject an engineered cell as described herein. The cancer cell may be, or said solid tumor may comprise, a lung cancer cell, brain cancer cell, head & neck cancer cell, breast cancer cell, skin cancer cell, liver cancer cell, pancreatic cancer cell, stomach cancer cell, colon cancer cell, rectal cancer cell, uterine cancer cell, cervical cancer cell, ovarian cancer cell, testicular cancer cell, skin cancer cell, or esophageal cancer cell. Th method may further comprise contacting said cancer cell or solid tumor with a second anti-cancer agent or treatment, such as wherein said second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, 3 4930-3431-6359, v.1 immunotherapy, hormonal therapy, or toxin therapy. The second anti-cancer agent or treatment ,au be given at the same time as said engineered cell or given before and/or after said engineered cell. The cancer cell or solid tumor may be a metastatic cancer cell/solid tumor, a multiply drug-resistant cancer cell/solid tumor or a recurrent cancer cell/solid tumor. The method may further comprise giving at least a second administration of said engineered cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 20 additional administrations. The administration may be intratumoral, into tumor vasculature, local to the tumor, regional to the tumor or systemic. [0009] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number. [0010] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. 4 4930-3431-6359, v.1 BRIEF DESCRIPTION OF THE DRAWINGS [0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0012] Figs. 1A-B. Schematic of direct fusion and indirect fusion hypoxic circuit configurations. [0013] Figs.2A-D. Comparison of circuit activity under hypoxic conditions. (Figs.2A- B) CAR expression in Jurkat cells transduced with direct or indirect fusion circuit at 1.5% oxygen and normoxia. (Figs. 2C-D) CAR expression following treatment with 20 µM daproducstat. [0014] Figs.3A-D. Comparison of circuit activity under hypoxic conditions. (Figs.3A- B) GFP expression in Jurkat cells transduced with direct or indirect fusion circuit at 1.5% oxygen and normoxia. (Figs. 4C-D) GFP expression following treatment with 20 µM daprodustat. [0015] Figs. 4A-C. [Optimization of the hypoxia circuit design] (Fig. 4A) Schematic of original design and (Fig. 4B) improved design and induction of CAR at different oxygen percentages in Jurkat cells after 24 hours. (Fig. 4C) CAR expression by combinations of Gal4 variants and nuclear localization sequences at different oxygen levels in Jurkat cells after 24 hours. [0016] Fig. 5. Comparison of new and original hypoxic circuits. Primary T cells 5 days post-bead stimulation. [0017] Fig.6. [Cytotoxicity of primary human T cells bearing various hypoxia circuit]. The cargo in the hypoxia circuit is a chimeric antigen receptor recognizing antigens on A375 tumor cells. [0018] Figs. 7A-C. [Establishment of a dual-luciferase reporter assay for monitoring the hypoxia circuit response in vivo in tumor-bearing mice] (Fig. 7A) Luciferase 5 4930-3431-6359, v.1 emission spectra of transduced Jurkat cells with luciferin or nano substrate. (Fig. 7B) RFP and luciferase activity in A375 cells transduced with hypoxic circuits following 24 hours hypoxia incubation. (Fig.7C) Constitutive and (Fig.7D) luciferase expression by A375 cells (nano) and T cells (luciferase). [0019] Figs.8A-B. [Establishment and characterization of a humanized hypoxia circuit based on HNF1A.] (Fig. 8A) Schematic of original design and humanization design. (Fig. 8B) CAR expression by HNF1A variants and at different oxygen levels in Jurkat cells after 24 hours. [0020] Fig. 9. A375 tumor cells transduced with hypoxia circuit bearing RFP as a reporter. Cells were then subjected to hypoxia (0.1% O2 for 24 hours). Cells were then stained with the hypoxia probe EF5 as a control. [0021] Figs.10A0B. GAP71-NLS-based hypoxia circuit prevents non-hypoxia induced circuit activation. (Fig. 10A) Primary T cell signaling induced by anti-CD3/CD28 triggers CAR expression in the original GAL4-NLS-based hypoxia circuit under normoxia (21% O₂) (left panel). And this circuit induction by TCR signaling is caused by stabilization of HIF1a (right panel). (Fig.10B) GAP71 NLS-based hypoxia circuit has a negligible response to TCR signaling under normoxia while maintaining a response under hypoxia. [0022] Fig.11. Amino acid sequences of exemplary constructs. [0023] Fig.12. DNA sequence of exemplary constructs. 6 4930-3431-6359, v.1 DETAILED DESCRIPTION [0086] The curative potential of chimeric antigen receptor (CAR) T cell-based cancer immunotherapies has been established in several cancer types, but solid tumor applications have been limited by a paucity of known tumor-specific membrane proteins. Though membrane proteins represent up to a quarter of the proteome, only a fraction of these are specifically expressed on tumors cells and not on normal tissues, and a smaller proportion are essential to tumor homeostasis. [0087] The inventors also developed both “direct” and “indirect” control system that can be applied to the CAR as well as to virtually any cell expressing a therapeutic gene. The direct system relies on a hypoxia responsive (HRE) that drives expression of the therapeutic gene which in turn is attached to an oxygen-dependent degradation (ODD) domain. The indirect system relies on two different expression cassettes, the first encoding a transcriptional control factor (TCF) fused an ODD domain under the control of an HRE promoter element active in eurkaryotic cells. A second expression cassette then encodes a CAR under the control a transcription control element bound and activated by said TCF. Improved expression using this two stage regulation has been achieved. [0088] The cell-intrinsic response to non-hypoxia may non-specifically interfere with the hypoxia signal pathway to a low or mild extent. To address this, the inventors also propose use of a combination of affinity tuning (GAL4 mutant-UAS with 10% activity of the WT GAL4) and nuclear-cytosol distribution (NLS, NES) of transcription factor to shift the activation threshold of the hypoxia circuit without compromising the magnitude of the circuit response, enabling a super tight control of the circuit that only responds to authentic hypoxia instead of TCR signaling or low-oxygen (5%) physioxia. [0089] These and other aspects of the disclosure are discussed in detail below. I. Terminology [0090] Unless otherwise defined, scientific and technical terms used herein shall have the meaning that are commonly understood by those of ordinary skill in the art. Further, unless 7 4930-3431-6359, v.1 otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, antibodies and related molecules, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. [0091] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., B. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996); and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). [0092] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991); Bork et al., J Mol. Biol. 242, 309-320 (1994); Chothia and Lesk J. Mol Biol. 196:901-917 (1987), Chothia et al., Nature 342, 877-883 (1989), and/or or Al-Lazikani et al., J Mol Biol 273, 927-948 (1997). [0093] As used herein, “antigen-binding site” shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen, such as an antibody. The antigen-binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain. For example, in a Fv comprising two different polypeptide 8 4930-3431-6359, v.1 chains from an antibody, the antigen-binding site is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always in one or more of the CDRs in each variable region. In some embodiments, the antigen-binding site is an antigen-binding site of an antibody. In such embodiments, the antigen-binding site may comprise one or more complementarity-determining regions or “CDRs”. In some embodiments, the antigen-binding site of an antibody comprises at least part of a VH or a VL or a Fv. [0094] As used herein the phrase “chimeric antigen receptor (CAR)” refers to a recombinant or synthetic molecule which combines antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits cellular immune activity to the specific antigen. [0095] As used herein the phrase “T Cell Receptor” or “TCR” refers to soluble and non- soluble forms of recombinant T-cell receptor. [0096] As used herein, a “T-cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T-cell. [0097] As used herein a “T Cell Receptor-like antibody (TCRL” or “peptide-centric CAR (PC-CAR)” refers to an antibody which binds an MHC displaying an HLA-restricted peptide antigen. Binding of the TCRL to its target typically has an MHC-restricted specificity: the TCRL does not bind the MHC in the absence of the complexed peptide, and the TCRL does not bind the peptide in an absence of the MHC. TCRLs are characterized by affinity sufficient to permit specific binding to a tumor antigen even when the TCRL is provided in a soluble, rather than membrane-bound, form. TCRLs are being developed as a new therapeutic class for targeting tumor cells and mediating their specific killing. In addition, TCRLs are valuable research reagents enabling the study of human class I peptide-MHC ligand presentation and TCR-peptide-MHC interactions. In an embodiment, the binding agent of the present disclosure is a TCRL. 9 4930-3431-6359, v.1 [0098] As used herein the phrase “MHC (or HLA)-restricted peptide” refers to a peptide which is potentially presented on an MHC molecule. Such peptides may be identified by laboratory procedures such as Mass-Spectrometry, reverse-immunology or by in-silico analysis. An MHC (or HLA)-presented peptide refers to a peptide which is confirmed in vitro or in vivo as being presented by an MHC molecule. [0099] The term “cancer” as used herein is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. [00100] The terms “normoxia” and “hypoxia” are generally known to those of skill in the art. In general, oxygen levels in tumors range from about 0.3 to 4.2% but are mostly below 2%, whereas oxygen levels in normal tissues range from about 3.4 to 6.8%, mostly above 4%. See McKeown S., Br. J. Radiol 87(1035), 2014. For the purposes of this application, normoxia is defined as 5.0% or higher oxygen and hypoxia would be less than 5% oxygen, such as 4% oxygen or lower, 3.0% or lower oxygen, 2.0% or lower oxygen, 1.0% or lower oxygen or 0.5% or lower oxygen. [00101] A “compound” refers to any molecule including small molecules, polypeptides, and other macromolecules. In some embodiments, a compound is a small molecular weight compound with a molecular weight of less than about 2000 Daltons. [00102] The term “naturally occurring” (or “native”) as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally occurring. [00103] The term “operably linked” as used herein refers to positions of components so described that are in a relationship permitting them to function in their intended manner. For example, a control sequence “operably linked” to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. 10 4930-3431-6359, v.1 [00104] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical linker or a disulphide bond, for example. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions. [00105] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds. [00106] The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, or RNA-DNA hetero-duplexes. The term includes single and double stranded forms of DNA. [00107] The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence. 11 4930-3431-6359, v.1 [00108] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2^nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). [00109] The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structural characteristics, as well as specific charge characteristics. [00110] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. [00111] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world. [00112] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously. 12 4930-3431-6359, v.1 [00113] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. II. Hypoxia Responsive Control System [00114] In accordance with the disclosure, there are provided expression control systems that permit regulation of gene expression in hypoxic environments. These systems rely on hypoxia responsive elements (HREs) that are situated upstream and in operational relation to a coding regions, such as CAR, or TCR. In one system, an expression vector comprising an HRE that controls expression of a protein of interest, e.g., a CAR or TCR, is provided. The coding region for the protein of interest may optionally be fused to a coding region for an oxygen-dependent degradation (ODD) domain. Under conditions of hypoxia, expression of the protein of interest is induced, while any “leakage” expression in an oxygen rich environment would results in an oxygen-dependent degradation of the protein of interest. See Fig.1A. [00115] A variation on this system uses two distinct expression cassettes located in one or two different vectors. The first cassette/vector would comprising a first nucleic acid encoding a transcriptional control factor (TCF), optionally fused a coding region for an oxygen-dependent degradation (ODD) domain under the control of a hypoxia responsive (HRE) promoter element. The second cassette/vector comprises a second nucleic acid encoding a protein of interest, such as a CAR or TCR, under the control a transcription control element bound and activated by the TCF. The same tightly controlled regulation under hypoxic conditions, along with elimination of any “leaky” expression in an oxygen rich environment is provided. See Fig.1B). [00116] Examples of HREs include those from VEGFA, PGK1, LDHA, EPO, GLUT1, and ALDA genes. Examples of ODDs include those from HIF1a, HIF2a, HIF3a, EPOR, NDRG3, ZHX2 genes. [00117] Examples of nuclear localization signals (NLSs) include SV40 large T-Ag (PKKKRKV, SEQ ID NO: 1), Polyoma large T-Ag (VSRKPRP, SEQ ID NO: 2), NF-kB p65 (EEKRKR, SEQ ID NO: 3) and Human c-myc (PAAKRVKLD, SEQ ID NO: 4). 13 4930-3431-6359, v.1 [00118] Examples of nuclear exportation signals (NESs) include HIV-1 (LQLPPLERLTL, SEQ ID NO: 5), PKI (LALKLAGLDI, SEQ ID NO: 6), ADAR1 (RGVDCLSSHFQELSIYQ, SEQ ID NO: 7), or SMAD4 (ERVVSPGIDLSGLTQ, SEQ ID NO: 8). III. Chimeric antigen receptor (CAR) and TCR Fusion Proteins (TFP) [00119] In an embodiment, there is provided a T-cell receptor fusion protein (TFP) comprising: a) a T-cell receptor (TCR) subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and b) a binding agent of the disclosure, wherein the TCR subunit and the binding agent are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T-cell. [00120] As used herein, the term “at least a portion of a TCR extracellular domain” refers to α and β chains of the TCR comprising variable and constant regions or domains. The term "domain" or "region" can be used interchangeably herein. The variable domain consists of a concatenation of variable region and joining region. The term "TCR alpha variable domain" therefore refers to the concatenation of TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Likewise, the term "TCR beta variable domain" refers to the concatenation of TRBV and TRBD/TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBV region or to a C-terminal truncated TRBC sequence. The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12- 441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database. As used herein, the term “wherein the TCR incorporates into a TCR when expressed in a T-cell” refers to the process of a TFP of the disclosure being expressed and fused to a T-cell receptor of a T-cell. 14 4930-3431-6359, v.1 [00121] Chimeric antigen receptors (CARs) are fusion proteins comprising antigen recognition moieties and T cell-activation domains. Exemplary CARs are provided by US Patent No.8,399,645 and US Patent No.7,638,325. Other exemplary recombinant receptors, including CARs, recombinant T-cell receptors (TCRs), TCR fusion proteins (TFPs), as well as methods for engineering and introducing the receptors into cells, include those described in Int’l Pat. Appl. Nos. WO2017/096329, WO2000/14257, WO2013/126726, WO2012/129514, WO2014031687, WO2013/166321, and WO2013/071154, WO2013/123061, and WO/2014055668; U.S. Pat. App. Nos. US2002131960, US2013287748, and US20130149337; U.S. Pat. Nos. 6,451,995, 7,446,190, 7,638,325, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118; European Pat. App. No. EP2537416; and Sadelain et al. Cancer Discov. April 3(4): 388-398 (2013); Davila et al. PLoS ONE 8(4): e61338 (2013); Turtle et al. Curr. Opin. Immunol. October 24(5): 633-39 (2012); and Wu et al. Cancer, March 18(2): 160-75 (2012). In an embodiment, the binding agent is a TFP as described in U.S. Pat. No.15/419,398. IV. Expression Constructs [00122] According to the disclosure there are provided an engineered vectors that facilitate expression of CAR constructs. Such expression vectors contain elements coding for the non-antigen binding portions of CARs along with cis-acting regulatory elements. The expression vector of some embodiments of the disclosure includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., as a shuttle vector). In addition, typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. The nucleic acid construct of some embodiments of the disclosure includes a signal sequence for secretion or presentation of the binding agent from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence. [00123] Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine 15 4930-3431-6359, v.1 the rate at which transcription is initiated. Preferably, the promoter utilized by the expression vector is active in the specific cell population transformed. Examples of cell type-specific and/or tissue- specific promoters include promoters such as albumin that is liver specific (Pinkert et al., Genes Dev. 1:268-277 (1987)), lymphoid specific promoters (Calame et al., Adv. Immunol. 43:235-275 (1988)); in particular promoters of T-cell receptors (Winoto et al., EMBO J. 8:729-733 (1989)) and immunoglobulins; (Banerji et al., Cell 33:729-740 (1983)), neuron-specific promoters such as the neurofilament promoter (Byrne et al., Proc. Natl. Acad. Sci. USA 86:5473-5477 (1989)), pancreas-specific promoters (Edlunch et al., Science 230:912-916 (1985)) or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No.4,873,316 and European Application Publication No. EP0264166). In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art; however, some variation in this distance can be accommodated without loss of promoter function. [00124] Enhancer elements can stimulate transcription up to 1,000-fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the disclosure include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long-term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983. [00125] Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of TCRL mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the disclosure include those derived from SV40. 16 4930-3431-6359, v.1 [00126] In addition to the elements already described the expression vector of some embodiments of the disclosure may contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell. [00127] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid. [00128] Also provided are cells which comprise the polynucleotides/expression vectors as described herein. Such cells are typically selected for high expression of recombinant proteins (e.g., bacterial, plant or eukaryotic cells e.g., CHO, HEK-293 cells), but may also be host cells having a specific immune effector activity (e.g., T cells or NK cells, such as Jurkat cells) when for instance the CDRs of the TCRL are implanted in a T Cell Receptor or CAR transduced in said cells which are used in adoptive cell therapy. [00129] Next, the CAR library is introduced into mammalian host cells that are then cultured under conditions supporting expression of encoded CARs. The host cells expressing the CAR are then contacted with target antigen positive host cells exhibiting CAR activation are then identified using a variety of different approaches. Once these cells are identified, the binding region can be sequenced and further developed. V. Treatment of Cancers [00130] In accordance with the present disclosure, there are provided methods of treating cancers, particularly solid cancers. The methods involve the administration of CAR T cells to a subject such that the cells are brought in proximity with a cancer cell/cancer cell environment and their therapeutic effect is delivered. The administration may be performed multiple times (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a subject as needed. The subject may be 17 4930-3431-6359, v.1 an infant, a pediatric patient, a juvenile, a young adult, and adult or a senior. The subject may be a human, male or female, or may be a non-human mammal. [00131] In certain aspects the cancer tumor is a renal cell cancer, melanoma, prostate cancer, chronic lymphocytic leukemia, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; 18 4930-3431-6359, v.1 leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. [00132] The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. 19 4930-3431-6359, v.1 [00133] The term “therapeutically-effective amount,” as used herein, pertains to that amount of binding agent, or a material such as an antibody-drug conjugate, composition or dosage form comprising an active binding agent, which is effective for producing some desired therapeutic effect when administered in accordance with a desired treatment regimen. [00134] In some embodiments, the treatment reduces or inhibits tumor growth for at least 6, 12, 24, 36, or 48 months. In some embodiments, the treatment enhances an immune response against the tumor. [00135] The subject/patient may be an animal or any species of mammal, including, without limitation, a horse, a dog, a cat, a pig, or a primate. In a preferred embodiment, the subject/patient is a human. A. Pharmaceutical Formulations and Routes of Administration [00136] Pharmaceutical compositions provided herein comprise an effective amount of one or more therapeutic compositions and, optionally, an additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains a therapeutic nucleic acid construct or a therapeutic engineered cell and one or more excipients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. [00137] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations 20 4930-3431-6359, v.1 thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated. [00138] In certain embodiments, the pharmaceutical composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. In certain embodiments, pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). [00139] In certain embodiments, the actual dosage amount of a composition administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. [00140] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 15 21 4930-3431-6359, v.1 microgram/kg/body weight, about 20 microgram/kg/body weight, about 25 microgram/kg/body weight, about 30 microgram/kg/body weight, about 35 microgram/kg/body weight, about 0.04 milligram/kg/body weight, about 0.05 milligram/kg/body weight, about 0.06 milligram/kg/body weight, about 0.07 milligram/kg/body weight, about 0.08 milligram/kg/body weight, about 0.09 milligram/kg/body weight, about 0.1 milligram/kg/body weight, about 0.2 milligram/kg/body weight, to about 0.5 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 0.01 mg/kg/body weight to about 0.1 mg/kg/body weight, about 0.04 microgram/kg/body weight to about 0.08 milligram/kg/body weight, etc., can be administered, based on the numbers described above. [00141] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. [00142] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof. [00143] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present embodiments. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the 22 4930-3431-6359, v.1 aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines. [00144] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area. [00145] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. [00146] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. B. Combination Therapies [00147] In order to increase the effectiveness of a nucleic acid, polypeptide or nanoparticle complex of the present embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest. As a non- 23 4930-3431-6359, v.1 limiting example, the treatment of cancer may be implemented with a CAR/CAR T cell of the present disclosure along with other anti-cancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the CAR/CAR T cell and the other agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the CAR/CAR T cell and the other includes the second agent(s). [00148] Treatment with the CAR/CAR T cell may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the anti-cancer peptide or nanoparticle complex are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the anti-cancer peptide or nanoparticle complex would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations. [00149] Various combinations may be employed, where the CAR/CAR T cell therapy is “A” and the other agent is “B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A 24 4930-3431-6359, v.1 In certain embodiments, administration of the CAR/CAR T cell therapy and/or other agent(s) to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy. [00150] Chemotherapy. Cancer therapies also include a variety of combination therapies. In some aspects, a TUSC2 therapeutic and/or an immune checkpoint inhibitor of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof. [00151] Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, 25 4930-3431-6359, v.1 mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegaI1; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; 26 4930-3431-6359, v.1 mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac (e.g., from about 400 to about 800 mg/day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein. [00152] Radiotherapy. Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. [00153] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. [00154] Immunotherapy. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin 27 4930-3431-6359, v.1 (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. [00155] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with a TUSC2 therapy of the present embodiments. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. [00156] Gene Therapy. In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic composition. Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996). [00157] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. A variety of proteins are encompassed within the present embodiments, some of which are described below. [00158] As noted above, the tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. 28 4930-3431-6359, v.1 [00159] Genes that may be employed as secondary treatment in accordance with the present embodiments include p53, p16, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors), and MCC. Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, Proc. Nat’l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists. [00160] Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri). [00161] Surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. [00162] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery 29 4930-3431-6359, v.1 (Mohs’ surgery). It is further contemplated that the present embodiments may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. [00163] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. [00164] Anti-Inflammatory Agents. In certain aspects TUSC2 therapies and/or an immune checkpoint inhibitor are administered in conjuction with an anti-inflammatory agent. An anti-inflammatory agent is defined herein to refer to an agent that is known or suspected to be of benefit in the treatment or prevention of inflammation in a subject. Corticosteroids are a major class of anti-inflammatory agent. The corticosteroids may be short, medium, or long acting, and may be delivered in a variety of methods. A non-limiting list of corticosteroids contemplated in the present embodiments include the oral corticosteroids such as: cortisone, hydrocortisone, prednisone, and dexamethasone. [00165] Another major class of anti-inflammatory agents are non-steroidal anti- inflammatory agents. Non-steroidal anti-inflammatory agents include a class of drugs used in the treatment of inflammation and pain. The exact mode of action of this class of drugs is unknown. Examples of members of this class of agents include, but are not limited to, ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid, diflunisal, oxaprozin, rofecoxib, and celecoxib. One of ordinary skill in the art would be familiar with these agents. Included in this category are salicylates and derivates of salicylates, such as acetyl salicylic acid, sodium salicylate, choline salicylate, choline magnesium salicylate and diflunisal. [00166] Other anti-inflammatory agents include anti-rheumatic agents, such as gold salts (e.g., gold sodium thiomalate, aurothioglucose, and auranofin), anti-rheumatic agents (e.g., chloroquine, hydroxychloroquine, and penicillamine), antihistamines (e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine, and triprolidine), and 30 4930-3431-6359, v.1 immunosuppressive agents (e.g., methotrexate, mechlorethamine, cyclophosphamide, chlorambucil, cyclosporine, and azathioprine). Other immunosuppressive agents contemplated by the present embodiments is tacrolimus and everolimus. Tacrolimus suppresses interleukin-2 production associated with T-cell activation, inhibits differentiation and proliferation of cytotoxic T cells. Today, it is recognized worldwide as the cornerstone of immunosuppressant therapy. One of ordinary skill in the art would be familiar with these agents, and other members of this class of agents, as well as the mechanism of actions of these agents and indications for use of these agents. [00167] Other agents. It is contemplated that other agents may be used in combination with the compositions provided herein to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the compositions provided herein by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the compositions provided herein to improve the anti- hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present disclosure. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the compositions provided herein to improve the treatment efficacy. [00168] In certain embodiments, hormonal therapy may also be used in conjunction with the present embodiments or in combination with any other cancer therapy previously 31 4930-3431-6359, v.1 described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases. VI. Examples [00169] The following examples are included to demonstrate particular embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Hypoxia Responsive 2.5F Chimeric Antigen Receptor Expression [00170] For the direct fusion approach, the inventors constructed a single lentiviral vector containing a CAR (vector 1; Fig. 1A). The indirect fusion approach required two lentiviral vectors; one with the hypoxia-regulated Gal4-vp64 (vector 2; Fig.1B) and the other with the Gal4-vp64 (via 5x UAS) regulated CAR (vector 3; Fig. 8B). Jurkat cells were transduced with virus produced with vector 1 or 2. Cells with vector 2 were then transduced with vector 3. [00171] To monitor hypoxia responsiveness, the cells were incubated for 24 hours at 1.5% oxygen in a regulated chamber and then re-exposed to normoxia. 2.5F CAR expression was monitored via flow cytometry after hypoxic incubation and again after normoxic incubation. The indirection fusion approach enabled stronger CAR detection in terms of intensity and percentage induction than the direct fusion approach (Figs. 2A-B). Following re-exposure to normoxia, CAR expression quickly decreases within 24 hours, which confirms the tight regulation of the circuit. Additionally, the inventors evaluated the robustness of their circuits with daprodustat, a HIF prolyl hydroxylase chemical inhibitor known to induce a strong hypoxic response. They performed a time course study to monitor the rate of induction. The results were consistent with the inventors’ previous findings using 32 4930-3431-6359, v.1 the hypoxic chamber (Figs. 2C-D). Specifically, the indirect fusion approach was superior to direct fusion in terms of the rate of induction and overall 2.5F CAR expression after 24 hours. [00172] Finally, the inventors validated the modularity of their approach with a membrane bound GFP instead of the CAR to ensure that the results were not case-specific. Following the same experiments conducted with the CAR version, the membrane bound GFP performed similarly to the CAR (Figs.3A-D). Again, they observed stronger induction with indirect fusion TF approach. The indirect fusion approach using the synthetic TF is thus preferred over the direct fusion approach for multiple reasons. First, with a synthetic TF system, the inventors do not need to change the protein sequence of the CAR, which eliminates the possibility of function impairment. Additionally, they can use the same circuit design to deliver therapeutics that are not only membrane-bound (CAR), but also secreted, cytoplasmic, or nuclear. [00173] The inventors further optimized their circuit for primary T cells because they noticed leakiness of their circuit at 5% oxygen (Fig. 4A), the physiological concentration in the human body, as well as following T cell activation in normoxia. To minimize killing activity of the engineered T cells in normoxia, they performed a screen to identify elements to reduce the baseline expression of the CAR in the hypoxic circuit (Fig. 4B). The inventors identified a mutant Gal4 (GAP71, Gap71 carrying three point mutations at S22D, K23Q and K25F in its DNA-binding domain; see Ferdous et al, Mol Biosyst, 2015. 4(11):1116–1125) that has a reduced binding affinity to 5x UAS target sites, and therefore requires a lower level of oxygen to induce strong CAR expression (Fig. 4C). They then validated this new circuit in 3 different T cell donors after 5 days of bead stimulation. All three donors had lower baseline circuit activity with the new circuit in comparison to the original circuit (Fig.5). [00174] Finally, the inventors confirmed that the CAR expression in normoxia substantially impacts the degree of killing of A375 melanoma cells (Fig. 6). The original circuit kills similarly in normoxia, hypoxia, and following daprodustat treatment. The new, improved circuit only demonstrates killing under hypoxia and daprodustat. Furthermore, the degree of killing in hypoxia for both circuits is comparable. Therefore, the new circuit has 33 4930-3431-6359, v.1 an improved safety profile in comparison to the original circuit while maintaining the same efficacy. [00175] The inventors developed a dual imaging system such that they could simultaneously monitor hypoxia in T cells and tumor cells via IVIS with the circuit. The dual system was constructed with firefly and nano luciferase, which have separate emission spectra and substrates that do not cross react (Fig.7A). The CAR in the hypoxic circuit was replaced with a luciferase and a fluorescent protein to monitor hypoxic induction in A375 cells (Fig. 7B). Luciferase activity and RFP expression was only observed in hypoxia and not normoxia. NSG mice were then engrafted with A375 cells that were transduced with constitutive nano luciferase and then injected with T cells transduced with constitutive firefly luciferase. Luminescence was measured via IVIS, and signal was detected primarily in the spleen for T cells and the tumor. NSG mice were next engrafted with A375 cells that were transduced with hypoxic circuits that express nano luciferase and then injected with T cells transduced with firefly luciferase hypoxic circuit. Upon imaging, signal was only localized to the tumor region. These data support the stringent activity of the circuit with minimal off- target expression in vivo. [00176] The inventors also developed a humanized circuit with similar response dynamics as the Gal4 circuit. While the HREs and ODDs are derived from human sequences, Gal4 is a yeast transcription factor. To maximize the clinical translatability of the approach, one must consider human analogs of Gal4 as there is a risk of immunogenicity. As a result, the inventors generated circuits with the liver-specific protein hepatocyte-nuclear factor 1- alpha (HNF1A), which has minimal activity in T cells, instead of Gal4 (Fig. 8A). HNF1A binds to specific response elements (RE) to initiate gene transcription, which can be used in place of Gal4 and UAS to drive the hypoxic circuit. Naturally-occurring DNA binding domain variants of HNF1A (such as A98V, Q100K, P112L variants; see Najmi et al., Diabetes 66(2):335-346, 2016) in addition to wild-type HNF1A were screened. All circuits were shown to respond similarly to the optimized Gal4-based circuit. [00177] The inventors benchmarked the inventors’ GAP71-NLS-based hypoxia circuit to clinically tested EF5 for its sensitivity. They found that their GAP71-NLS-based hypoxia circuit demonstrated comparable sensitivity to EF5 (if not more stringent) (Fig.9). 34 4930-3431-6359, v.1 This result suggested safety (i.e., minimal circuit leakage) when this circuit is to be eventually used for generating T cell or other immune cell-based immunotherapies. [00178] GAP71-NLS-based hypoxia circuit prevents non-hypoxia induced circuit activation. The inventors discovered an unexpected phenomenon in which primary T cell activation itself triggers the original GAL4-NLS-based hypoxia circuit, and this appears to be caused by the stabilization of HIF-1α upon primary T cell activation (Fig. 10A). Interestingly, this only happens in primary T cells, but not in Jurkat T cell line. Since most T cell-based therapy uses primary T cells, and this will be a critical issue to address to further reduce off-tumor activities. The inventors confirmed that their new GAP71-NLS based hypoxia circuit is super tight and could completely prevent circuit activation triggered by primary T cell TCR signaling (Fig.10B). [00179] Advantages and Improvement Over Existing Devices and Methods. Treatment options for advanced solid tumors have been limited and unsuccessful. As a novel therapeutic approach in cancer treatment, CAR-T therapy has achieved great success in hematological malignancies while solid tumor is still a big challenge. Tumor heterogeneity, antigen loss, and an immunosuppressive microenvironment are considered major hurdles leading to poor responsiveness. To overcome these issues, therapies that are efficacious but can have unwanted off-target effects can be locally delivered to the tumor to prevent systemic toxicity. By introducing the CAR into a sophisticatedly designed hypoxic circuit, CAR-T functions were restrained only in hypoxic environments, reducing possible toxicity to normal tissues. Furthermore, the inventors improved the indirect design by specifically limiting CAR expression to oxygen conditions lower than physioxia and eliminating responsiveness to TCR stimulation. This method will allow for use of CARs to target tumors with aberrant expression, but limit targeting of healthy tissue with lower expression. Additionally, this system allows for combination of T cell vaccination to boost anti-tumor response without stimulating the hypoxic circuit in lymphoid tissue. Furthermore, the indirect method allows introduction of other therapies, such as enzyme pro-drugs or cytokines, without changing the protein sequences themselves and therefore potentially impacting anti-tumor activity. * * * * * * * * * * * * * * * * * 35 4930-3431-6359, v.1 [00180] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. 36 4930-3431-6359, v.1

Claims

WHAT IS CLAIMED: 1. An expression vector comprising a nucleic acid encoding a first gene of interest under the control of a hypoxia responsive (HRE) promoter, wherein the gene of interest is linked to a coding region of an oxygen-dependent degradation (ODD).

2. The expression vector of claim 1, wherein the expression vector further comprises an origin of replication, and/or wherein the TCF is HIF-1α HRE promoter.

3. The expression vector of claim 1 or claim 2, wherein the TCF comprises (i) an orthogonal DNA binding domain, such as Gal4, LexA, tTR, HNF1A, PAX6, ZF10 or a zinc finger protein (e.g., ZFHD1) and (ii) an activation domain, such as VP64, VP16, GAL4, Med2, p65A, rTA, MRTF-A, STAT1 or NRF2.

4. The expression vector of any one of claims 1-3, wherein the expression vector is a viral vector, such as a retroviral expression vector a lentiviral expression vector, an adenoviral expression vector, such as an oncolytic adenovirus, a herpes simplex virus expression vector, such as an oncolytic herpes simplex virus, or a non-integrating adeno- associated viral expression vector, optionally further characterized as a self-inactivation lentiviral expression vector.

5. The expression vector of any one of claims 1-4, wherein the expression vector is a non-viral expression vector.

6. The expression vector of any one of claims 1-5, further encoding a Lum-IL-12/ODD domain fusion protein construct under the control of an HRE.

7. The expression vector of any one of claims 1-6, wherein the first gene of interest is a first chimeric antigen receptor (CAR).

8. The expression vector of any one of claims 1-7, further encoding a second gene of interest under the control of a constitutive promoter, such as an EF1α promoter, a CMV promoter, a PGK promoter, an SFFV promoter, or a ubiquitin promoter.

9. The expression vector(s) of claim 8, wherein said second gene of interest is a second CAR that binds specifically to a non-endogenous small ligand. 37 4930-3431-6359, v.1

10. The expression vector of claim 9, wherein the non-endogenous small ligand is fluorescein isothiocyanate (FITC), pepvIII, or PNE peptide.

11. An engineered cell carrying the expression vector of any one of claims 1-10.

12. The engineered cell of claim 11, wherein the engineered cell is a transgenic T cell, a chimeric antigen receptor (CAR) T cell, a macrophage, an NK cell, a neutrophil, a monocyte, an NKT cell, a gamma-delta T cell, an allogeneic T cell, or an immortalized T cell line (e.g., TALL104 cells, NK92).

13. The engineered cell of claim 11, wherein the engineered cell is a CAR T cell.

14. The engineered cell of any one of claims 11-13, wherein said gene of interest is only expressed at less than 5% oxygen.

15. The engineered cell of claim 14, wherein said gene of interest is only expressed at 0- 4.0%, 0-3.0%, 0-2.0%, 0-1.5%, 0-1.25%, 0-1.0%, or 0-0.5% hypoxic conditions.

16. An expression vector comprising: (a) a first nucleic acid encoding a transcriptional control factor (TCF) fused an oxygen-dependent degradation (ODD) domain under the control of a hypoxia responsive (HRE) promoter element active in eurkaryotic cells; and (b) a second nucleic acid encoding chimeric antigen receptor (CAR) under the control a transcription control element bound and activated by said TCF.

17. The expression vector(s) of claim 16, wherein the expression vector) further comprise an origin of replication.

18. The expression vector of claim 16 or claim 17, wherein the TCF comprises (i) an orthogonal DNA binding domain, such as Gal4, LexA, tTR, HNF1A, PAX6, ZF10 or a zinc finger protein (e.g., ZFHD1) and (ii) an activation domain, such as VP64, VP16, GAL4, Med2, p65A, rTA, MRTF-A, STAT1 or NRF2. 38 4930-3431-6359, v.1

19. The expression vector of any one of claims 16-18, wherein the expression vector is a viral vector, such as a retroviral expression vector a lentiviral expression vector, an adenoviral expression vector, such as an oncolytic adenovirus, a herpes simplex virus expression vector, such as an oncolytic herpes simplex virus, or a non-integrating adeno- associated viral expression vector, optionally further characterized as a self-inactivation lentiviral expression vector.

20. The expression vector of any one of claims 16-18, wherein the expression vector is a non-viral expression vector.

21. The expression vector of any one of claims 16-20, further encoding a Lum-IL- 12/ODD domain fusion protein construct under the control of an HRE, such as the same HRE controlling CAR expression; and/or further encoding a second CAR construct targeting a tumor antigen under the control of a constitutive promoter, such as an EF1α promoter, a CMV promoter, a PGK promoter, an SFFV promoter, or a ubiquitin promoter.

22. The expression vector of claim 21, wherein said second CAR construct binds specifically to a non-endogenous small ligand, such as fluorescein isothiocyanate (FITC), pepvIII, or PNE peptide.

23. The expression vector of any one of claim 16-22, further comprising a nuclear localization signal and a nuclear exportation signal that modulate cytosolic/nuclear distribution.

24. An engineered cell carrying the expression vectors of any one of claims 16-22, such as a transgenic T cell, a chimeric antigen receptor (CAR) T cell, a macrophage, an NK cell, a neutrophil, a monocyte, an NKT cell, a gamma-delta T cell, an allogeneic T cell, or an immortalized T cell line (e.g., TALL104 cell, NK92 cell).

25. A method of killing a cancer cell comprising contacting said cancer cell with an engineered cell of claim 23 or claim 24.

26. A method of treating a subject with a solid tumor comprising administering to said subject an engineered cell of claim 23 or claim 24. 39 4930-3431-6359, v.1

27. The method of claim 25 or claim 26, wherein said cancer cell is, or said solid tumor comprises, a lung cancer cell, brain cancer cell, head & neck cancer cell, breast cancer cell, skin cancer cell, liver cancer cell, pancreatic cancer cell, stomach cancer cell, colon cancer cell, rectal cancer cell, uterine cancer cell, cervical cancer cell, ovarian cancer cell, testicular cancer cell, skin cancer cell, or esophageal cancer cell.

28. The method of any one of claims 25-32, further comprising contacting said cancer cell or solid tumor with a second anti-cancer agent or treatment, such as wherein said second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or toxin therapy.

29. The method of claim 28, wherein said second anti-cancer agent or treatment is given at the same time as said engineered cell.

30. The method of claim 28, wherein said second anti-cancer agent or treatment is given before and/or after said engineered cell.

31. The method of any one of claims 26-30, wherein said cancer cell or solid tumor is a metastatic cancer cell/solid tumor, a multiply drug-resistant cancer cell/solid tumor or a recurrent cancer cell/solid tumor.

32. The method of any one of claims 25-31, further comprising giving at least a second administration of said engineered cell, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 20 additional administrations.

33. The method of any one of claims 25-32, wherein administration is intratumoral, into tumor vasculature, local to the tumor, regional to the tumor or systemic. 40 4930-3431-6359, v.1