Chimeric transcription factor variants with augmented sensitivity to drug ligand induction of transgene expression in mammalian cells

This application is a U.S. National Phase Application of PCT International Application Number PCT/US2017/065597, filed on Dec. 11, 2017, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to U.S. Provisional Application No. 62/433,183, filed on Dec. 12, 2016. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SeqList-SCRI-118NP.txt, created Jun. 4, 2019, which is 294 kb in size. The information provided is the electronic format of the Sequence Listing and is hereby expressly incorporated by reference in its entirety.

Disclosed herein are embodiments relating to genetic systems that facilitate drug-regulated transgene expression in cells, for instance mammalian cells. The present disclosure also concerns the identification of variants of the wild-type HEA3 and wild-type HEA4 chimeric transcription factors, which are useful for the regulation of transgene expression in genetically modified cells including mammalian cells.

Human T lymphocytes can be engineered by gene transfer to express chimeric antigen receptors (CARs), which are specific for surface molecules expressed on tumor cells. Methods of gene transfer useful for this purpose include several types of viral transduction using viral vectors encoding the chimeric antigen receptors. Cells expressing the chimeric antigen receptors can then be adoptively transferred to patients to treat cancer and infectious diseases. Chimeric antigen receptors are synthetic receptors. These synthetic receptors, can include an extracellular ligand binding domain, for example, a single chain variable fragment of a monoclonal antibody (scFv) linked to intracellular signaling components, such as CD3ζ alone or combined with one or more costimulatory domains. Research in the design of chimeric receptors has focused on defining scFvs and other ligand binding elements that target malignant cells without causing severe toxicity to normal tissues, and on defining the optimal composition of intracellular signaling modules to activate T cell effector functions. Additional genetic systems that facilitate expression of chimeric antigen receptors are needed.

Aspects of the present invention concern genetic systems that facilitate drug-regulated transgene expression in cells. In one alternative, regulated transgene expression is targeted to cells, such as lymphocytes, preferably human lymphocytes, which are designed for use in adoptive immunotherapy. This system provides rigorous safety attributes to chimeric antigen receptor (CAR) redirected adoptive therapeutic strategies without sacrificing curative intent that permits real-time clinician control of CAR expression in vivo. By engineering vectors that enable drug responsive transcriptional control of CAR expression, the activity of CARs and other cell mediators can be turned “ON” and “OFF” in vivo, based on a clinician prescribed pharmaceutical drug input that exhibits clinically permissive pharmacokinetics, tissue distribution, and partitioning between the extracellular space and cytosol of lymphocytes. In addition to adenovirus and lentivirus genes, the vector platform can also include genes encoding minicircles or piggy bac transposons. The genetic systems described herein provide for drug regulated transgene expression to enforce a functional “OFF” state in the absence of the drug and a functional “ON” state transgene expression in the presence of the drug.

One alternative of such an approach utilizes the drug tamoxifen. Tamoxifen is an estrogen antagonist/partial agonist that is an FDA-approved and commercially available drug. It is taken orally and can be administered daily and over an extended period. Tamoxifen has a proven safety record, favorable pharmacokinetic profile, excellent tissue distribution and a low partition coefficient between the extracellular space and cytosol. Other drugs useful in the approaches described herein can be selected based on safety record, favorable pharmacokinetic profile, and excellent tissue distribution, a low partition coefficient between the extracellular space and cytosol, and/or low toxicities.

Tamoxifen can have several side effects, however, such as cataracts, changes in menstrual period, constipation, diarrhea, edema (swelling), fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting and weight loss, for example. As such, it is necessary to turn on a gene for expressing the chimeric antigen receptor at a low plasma concentration of tamoxifen for some patients.

One aspect of the disclosure relates to changes that can be made to transcriptional activators so as to modulate the properties of the transcription factors including, without limitation, altering one or more amino acids in the estrogen receptor ligand binding domain so as to increase the affinity of the factor for estrogen analogs and altering one or more amino acids in the p65 transactivating domain and/or the RelA (p65) transactivation domain, in which altering the amino acids in the p65 transactivating domain (RelA) can enhance the transcriptional activity in response to a drug. The RelA mutations enhance transcriptional activity in response to the drug, however, they do not affect drug-to-receptor binding. Only the mutations in the ER-LBD affect drug-to-receptor binding. In some alternatives, the system employs a synthetic transcriptional regulator, which, in the presence of tamoxifen, binds a synthetic promoter upstream of a transgene to induce expression. The tamoxifen regulated transcription factors (“TamR-tf”, also can refer to HEA3/HEA4 transcription factors) is a chimeric transcription factor composed of human subunits including the N-terminal DNA binding domain of Hepatocyte Nuclear Factor 1-alpha (HNF-1α) fused in frame to the mutant tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD), that is in turn fused to the p65 activation domain of NF-κB (p65). HEA4 is a chimeric transcription factor composed of human subunits including the N-terminal DNA binding domain of Hepatocyte Nuclear Factor 1-alpha (HNF-1α) fused in frame to the mutant tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD) containing three point mutations (G400V, M543A, L544A) that abolish estradiol binding but permit binding to tamoxifen metabolites and other estrogen analogues, that is in turn fused to the p65 activation domain of NF-κB (p65). An exemplary amino acid sequence is set forth in SEQ ID NO: 1 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). SEQ ID NO: 1 comprises one point mutation in the ER-LBD that ablates binding to endogenous estrogen, but confers nanomolar specificity to tamoxifen metabolite 4-OHT fulvestrant and other estrogen analogs. The mutant tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD) is found at amino acids 282-595 of the TamR-tf and has a mutation at position 521 (G521R), and affects drug-to-receptor binding. The p65 activation domain of NF-κB (p65) is found at amino acids 596-862 of SEQ ID NO: 1 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS).

In the absence of tamoxifen, TamR-tf is excluded from the nucleus by binding of cytosolic heat-shock protein 90 (HSP90) to the tamoxifen binding active site and transgene expression is in the “OFF” state. Nanomolar concentrations of cytosolic tamoxifen actively out compete HSP90 for ER-LBD binding, resulting in TamR-tf translocation to the nucleus. Upon nuclear translocation, TamR-tf is readily available to bind its restricted synthetic promoter (e.g. 7×HBD/mE1B as set forth in the nucleic acid sequence of SEQ ID NO: 23 (tagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataa tctacaatagttaataatctacaagagctcagggtatataatg). In the presence of tamoxifen, binding of TamR-tf to 7×HBD/mE1B promoter induces the “ON” state of transgene expression. In some alternatives, this transcriptional regulator can be modified to provide for a varying level of control of transgene expression. Amino acid substitutions in the LBD of TamR-tf permit selective responsiveness to tamoxifen and its metabolites, which affect the drug-to-receptor binding, where 4-hydroxy tamoxifen (4-OHT) is the most pharmacologically active metabolite, with respect to TamR-tf activity, while lacking interaction with endogenous estrogen.

In some alternatives, variants of wild-type HEA3 and wild-type HEA4 are identified with augmented sensitivity to a drug by virtue of substitutions of the human estrogen receptor ligand binding domain. Mutations in the RelA (p65) transactivation domain augment transcriptional activity in response to drug or drug ligand. In some alternatives, these high affinity forms are generated by amino acid substitutions within the human estrogen receptor that confer selective binding to 4-OHT and other estrogen analogs such as fulvestrant. These changes cooperatively enhance transgene expression in response to drug activators when combined with amino acid substitutions in the HEA3 and HEA4 RelA domain that function as phosphomimetics and acetyl-mimetics, for example. Also described herein is a preferred version of the variant housing all three modifications that does not adversely affect the stringency of the “OFF” state in the absence of ligand, but dramatically improves the sensitivity and magnitude of transgene expression to activating estrogen analogue drug inputs. In another aspect, HEA3 is rendered responsive to the drug CMP8 due to mutations in the ER-LBD (L384M, M421G, G521R), not RelA mutations. The RelA mutations may not affect specificity to drug. The RelA mutations may enhance transcriptional activity. These enhanced HEA3 and HEA4 chimeric transcription factors make this regulatory system responsive to input drug levels that can easily be reached with low dose non-toxic dosing of tamoxifen, fulvestrant, or CMP8.

In a first aspect, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, is provided. The system comprises: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from the group consisting of a tumor specific molecule, a viral specific molecule, and any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte; a second polynucleotide, which encodes a polypeptide spacer, preferably an optimized spacer; a third polynucleotide, which encodes a transmembrane domain; and a fourth polynucleotide, which encodes an intracellular signaling domain; and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter, which is inducible by a drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug as compared to a wild type HEA3 chimeric transcription factor. In some alternatives, the transcriptional activator for the first promoter inducible by a drug, is HEA3 or HEA4. In some alternatives, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives, the at least one mutation affects the drug-to-receptor binding. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional expression at a given concentration compared to a system using a wild type HEA3. In some alternatives, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequence set forth in SEQ ID Nos: 1-3, 8-11 and 121. In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or at a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 chimeric transcription factor. In some alternatives, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives, the second promoter is an inducible promoter. In some alternatives, the second promoter is a constitutive promoter. In some alternatives, the second promoter is the EF1αp. In some alternatives, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives, both vectors are packaged in a viral vector. In some alternatives, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives, the selectable marker is EGFRt and/or HER2t. In some alternatives, the selectable marker confers drug resistance. In some alternatives, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand, as compared to a reference chimeric receptor. In some alternatives, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte; a polynucleotide coding for a polypeptide spacer, wherein the spacer is preferably optimized, a polynucleotide coding for a transmembrane domain and a polynucleotide encoding an intracellular signaling domain. In some alternatives, the first promoter is in opposite orientation to the second promoter. In some alternatives, the ligand binding domain is an antibody fragment, such as a binding fragment. In some alternatives, the ligand binding domain is a single chain variable fragment. In some alternatives, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or any combinations thereof. In some alternatives, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, and/or Epstein-Barr virus early antigen protein. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives, the DNA spacer enhances gene expression of the chimeric antigen receptor.

In a second aspect, a system for inducible expression of a gene that regulates a T cell function is provided. The system comprises: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity, and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter, which is inducible by a drug and, wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a wild type HEA3. In some alternatives, the transcriptional activator is HEA3 or HEA4. In some alternatives, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives, HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3 and/or 8-11 and/or 121. In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 and/or 122. In some alternatives, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is preferably optimized, a polynucleotide coding for a transmembrane domain; and a polynucleotide encoding an intracellular signaling domain. In some alternatives, the first promoter is in opposite orientation to the second promoter. In some alternatives, the ligand binding domain is an antibody fragment, such as a binding fragment. In some alternatives, the ligand binding domain is a single chain variable fragment. In some alternatives, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or any combinations thereof. In some alternatives, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives, both vectors are packaged in a viral vector. In some alternatives, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a third aspect, a chimeric receptor polypeptide coded for by the first second and/or second nucleic acid of the system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a polypeptide spacer, preferably an optimized spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 and/or. 122 In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 and/or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is a system for inducible expression of a gene that regulates a T cell function. In some alternatives, the system for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is preferably optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the systems described herein, both vectors are packaged in a viral vector. In some alternatives of the systems described herein, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a fourth aspect, a host cell comprising a system of any one of the alternative systems described herein is provided. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3 8-11 and/or. 121 In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 and/or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the systems described herein, both vectors are packaged in a viral vector. In some alternatives of the systems described herein, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the host cell, the host cell is a mammalian cell, such as a precursor T cell. In some alternatives of the host cell, the precursor T cell is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a fifth aspect, a composition comprising a host cell of any one or more of alternative host cells, such as mammalian cells, provided herein, in a pharmaceutically acceptable excipient, is provided. The host cell, such as a mammalian cell, comprises a system of any one of the alternative systems described herein is provided. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the host cell, the host cell is a precursor T cell. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the systems described herein, both vectors are packaged in a viral vector. In some alternatives of the systems described herein, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the host cell, such as a mammalian cell, the cell is a precursor T cell, which can be a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a sixth aspect, an in vitro method for preparing a host cell of any one of the alternative host cells, such as mammalian cells, described herein is provided. The host cell, such as a mammalian cell, comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is preferably optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment, such as a binding fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the host cell, the host cell is a mammalian cell, such as a precursor T cell. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the systems described herein, both vectors are packaged in a viral vector. In some alternatives of the systems described herein, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the host cell, the precursor T cell is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the in vitro method for preparing a host cell of any one of the alternative host cells comprises a) providing a system of any one of the alternative systems provided herein and b) introducing the system into an isolated T lymphocyte population and expanding each T lymphocyte population in vitro. In some alternatives of the method, the T lymphocytes are expanded, and wherein the method further comprises culturing the cells in the presence of anti-CD3 and/or anti CD28, and at least one homeostatic cytokine until the cells expand sufficiently for use as a cell infusion. In some alternatives of the method, the isolated T lymphocyte population comprises precursor T cells. In some alternatives of the method, the precursor T cells are hematopoietic stem cells. In some alternatives of the method, the lymphocyte is CD8+ or CD4+. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a seventh aspect, a method of treating, inhibiting, or ameliorating a disease in a subject in need thereof is provided. The method comprises administering to the subject the host cell of any one of the alternative host cells, such as mammalian cells, provided herein, or at least one composition or product combination of any one or more of the alternatives provided herein. The composition comprises a host cell of any one or more of alternative host cells, such as a mammalian cell, provided herein, in a pharmaceutically acceptable excipient. The host cell, such as a mammalian cell comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand, as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment, such as a binding fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives of the host cell, such as a mammalian cell, the host cell is a precursor T cell. In some alternatives of the host cell, the precursor T cell is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell, wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives of the method, the disease is cancer or a viral disease. In some alternatives of the method, the subject is identified or selected to receive an anti-cancer therapy. In some alternatives of the method, the subject is identified or selected to receive an anti-viral therapy. In some alternatives of the method, the method further comprises measuring or evaluating an inhibition of the disease. In some alternatives of the method, the method further comprises providing said subject an additional anti-cancer therapy or anti-viral therapy before, during, or after administration of the cells of any one or more of any one of the alternatives provided herein or at least one composition or product combination of any one or more of the alternatives provided herein. In some alternatives of the method, the cells described above or at least one composition or product combination described above are administered to said subject by adoptive cell transfer. In some alternatives of the method, the host cells described above or at least one composition or product combination described above are administered to said subject after said subject has received another form of anti-cancer therapy or anti-viral therapy. In some alternatives of the method, the method further comprises administering a drug that induces expression of a transgene in the host cell or composition for the treatment of cancer or a viral infection. In some alternatives of the method, the drug is tamoxifen and/or its metabolites, fulvestrant and/or other estrogen analogs or CMP8. In some alternatives of the method, tamoxifen is administered at a dose range of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the method, fulvestrant is administered at a dose range of 250, 300, 350, 400, 450 or 500 mg/dose mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the method, the CMP8 is administered at a dose range to provide 30, 40 or 50 nM of drug levels in serum, or within a range defined by any two of the aforementioned values. In some alternatives of the method, the cancer is a solid tumor or hematologic malignancy. In some alternatives of the method, the solid tumor is selected from the group consisting of a breast cancer, brain cancer, lung cancer, colon cancer, renal cancer, pancreatic cancer, prostate cancer, and ovarian cancer. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In an eighth aspect, a method of treating, inhibiting, or ameliorating a disease in a subject in need thereof, is provided. The method comprises administering to the subject the host cell, such as a mammalian cell, of any one of the alternatives provided herein or at least one composition or product combination of any one or more of the alternative compositions provided herein. In some alternatives, the composition comprises a host cell of any one or more of alternative host cells, such as mammalian cells, provided herein, in a pharmaceutically acceptable excipient. In some alternatives, the host cell, such as mammalian cells, comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives of the host cell, the host cell is a precursor T cell. In some alternatives of the host cell, such as a mammalian cell, the cell is a precursor T cell, which is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives of the method, the disease is cancer or a viral disease. In some alternatives of the method, the subject is identified or selected to receive an anti-cancer therapy. In some alternatives of the method, the subject is identified or selected to receive an anti-viral therapy. In some alternatives of the method, the method further comprises measuring or evaluating an inhibition of the disease. In some alternatives of the method, the method further comprises providing said subject an additional anti-cancer therapy or anti-viral therapy before, during, or after administration of the cells of any one or more of the alternative cells described herein or at least one composition or product combination of any one or more of alternatives described herein. In some alternatives of the method, the cells of any one or more of the alternatives described herein or at least one composition or product combination of any one or more of the alternatives described herein are administered to said subject by adoptive cell transfer. In some alternatives of the method, the host cells of any one or more of the alternatives described herein or at least one composition or product combination of any one or more of the alternatives described herein are administered to said subject after said subject has received another form of anti-cancer therapy or anti-viral therapy. In some alternatives of the method, the method further comprises administering a drug that induces expression of a transgene in the host cell or composition for the treatment of cancer or a viral infection. In some alternatives of the method, the drug is tamoxifen and/or its metabolites, fulvestrant and/or other estrogen analogs or CMP8. In some alternatives of the method, tamoxifen is administered at a dose range of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the method, the fulvestrant is administered at a dose range of 250, 300, 350, 400, 450 or 500 mg/dose mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the method, the CMP8 is administered at a dose range to provide 30, 40 or 50 nM of drug levels in serum, or within a range defined by any two of the aforementioned values. In some alternatives of the method, the cancer is a solid tumor or hematologic malignancy. In some alternatives of the method, the solid tumor is selected from the group consisting of a breast cancer, brain cancer, lung cancer, colon cancer, renal cancer, pancreatic cancer, prostate cancer, and ovarian cancer. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a ninth aspect, a use of the host cell, such as a mammalian cell, of any one of or more of the alternatives described herein or at least one composition or product combination of any one or more of the alternatives described herein in combination with a drug that induces expression of a transgene in the host cell or composition for the treatment of cancer or a viral infection is provided. In some alternatives, the composition comprises a host cell, such as a mammalian cell, of any one or more of alternative host cells provided herein, in a pharmaceutically acceptable excipient. In some alternatives, the host cell, such as mammalian cell, comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives of the host cell, the host cell, such as a mammalian cell, is a precursor T cell. In some alternatives of the host cell, the precursor T cell is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives of the use, the drug is tamoxifen and/or its metabolites, fulvestrant and/or other estrogen analogs or CMP8. In some alternatives of the use, tamoxifen is administered at a dose range of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the use, the fulvestrant is administered at a dose range of 250, 300, 350, 400, 450 or 500 mg/dose or within a range defined by any two of the aforementioned values. In some alternatives of the use, the CMP8 is administered at a dose range to provide 30, 40 or 50 nM of drug levels in serum, or within a range defined by any two of the aforementioned values. In some alternatives of the use, the cancer is a solid tumor or hematologic malignancy. In some alternatives of the use, the solid tumor is selected from the group consisting of a breast cancer, brain cancer, lung cancer, colon cancer, renal cancer, pancreatic cancer, prostate cancer, and ovarian cancer. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In a tenth aspect, a method of performing cellular immunotherapy in a subject having cancer or a viral infection is provided. The method comprises administering the host cells, such as mammalian cells, of any one or more of alternatives provided herein or at least one composition or product combination of any one or more of the alternatives provided herein to the subject and administering a drug that induces expression of a transgene in the composition or the host cells. The composition comprises a host cell of any one or more of alternative host cells, such as mammalian cells, provided herein, in a pharmaceutically acceptable excipient. In some alternatives, the host cell, such as a mammalian cell, comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the host cell, such as a mammalian cell, the host cell is a precursor T cell. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives of the host cell, such as a mammalian cell, the cell is a precursor T cell, which is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives of the method, the cancer is selected from a solid tumor or hematologic malignancy. In some alternatives of the method, the solid tumor is selected from the group consisting of a breast cancer, brain cancer, lung cancer, colon cancer, renal cancer, pancreatic cancer, prostate cancer, and ovarian cancer. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC and/or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

In an eleventh aspect, a use of the host cell of any one of or more of the alternatives described herein or at least one composition or product combination of any one or more of the alternatives described herein as a medicament, is provided. The composition comprises a host cell of any one or more of alternative host cells, such as mammalian cells, provided herein, in a pharmaceutically acceptable excipient. In some alternatives, the host cell, such as a mammalian cell, comprises a system of any one of the alternative systems described herein. In some alternatives, the system is for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 23. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces a high expression of the chimeric antigen receptor, such as at a concentration of at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of drug or a concentration that is within a range defined by any two of the aforementioned values, as compared to a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage amount for inducing expression with a wild type HEA3 or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the signaling domain comprises a CD28, 4-1BB and/or CD3ζ domain. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is an inducible promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is a constitutive promoter. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second promoter is the EF1αp. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator comprises at least one or more amino acid sequences set forth in SEQ ID NO: 1-11 or 121. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the transcriptional activator is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12-22 or 122. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, both vectors are packaged in a viral vector. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the viral vector is an adeno-associated virus or a lentivirus. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a fifth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the second nucleic acid further comprises a sixth polynucleotide that encodes a selectable marker. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker is EGFRt and/or HER2t. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the selectable marker confers drug resistance. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the first nucleic acid further comprises a seventh polynucleotide that encodes a self-cleaving peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the self-cleaving peptide comprises a P2A, T2A, E2A or F2A peptide. In some alternatives of the system for inducible expression of a chimeric antigen receptor in cells, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function. In some alternatives, the system is for inducible expression of a gene that regulates a T cell function comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to a third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the transcriptional activator is HEA3 or HEA4. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, and wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that augments transcriptional activity in response to a drug. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives of the systems described herein, the transcriptional regulator is a variant of a HEA3 chimeric transcription factor, and wherein the variant comprises amino acid substitutions that allow selective binding to the drug. In some alternatives of the systems described herein, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8. In some alternatives of the systems described herein, the drug induces a level of expression of the chimeric antigen receptor that is greater than a wild type HEA3 or a wild type HEA4 chimeric transcription factor at a concentration of the drug that is at least 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nM of the drug or at a concentration that is within a range defined by any two of the aforementioned values. In some alternatives of the systems described herein, the drug induces expression of the chimeric antigen receptor at a non-toxic dosing of drug at less than 10 nM drug but not zero. In some alternatives of the systems described herein, the non-toxic dosing of drug decreases, eliminates or prevents drug side effects in comparison to a dosage for inducing expression with a wild type HEA3 chimeric transcription factor. In some alternatives of the systems described herein, the side effects are cataracts, constipation, diarrhea, edema, fatigue, fluid retention, flushing, headache, hot flashes, high blood pressure, muscle pain, nausea, shortness of breath, vomiting or weight loss. In some alternatives of the systems described herein, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain. In some alternatives of the systems described herein, the first promoter is in opposite orientation to the second promoter. In some alternatives of the systems described herein, the ligand binding domain is an antibody fragment. In some alternatives of the systems described herein, the ligand binding domain is a single chain variable fragment. In some alternatives of the systems described herein, the tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives of the systems described herein, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein. In some alternatives of the systems described herein, the first nucleic acid further comprises a DNA spacer, wherein the spacer is operably linked to the first promoter and operably linked to the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer is between the first promoter and the first polynucleotide that encodes the chimeric antigen receptor. In some alternatives of the systems described herein, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120. In some alternatives of the systems described herein, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values. In some alternatives of the systems described herein, the DNA spacer enhances gene expression of the chimeric antigen receptor. In some alternatives of the host cell, such as a mammalian cell, the host cell is a precursor T cell. In some alternatives of the host cell, the precursor T cell is a hematopoietic stem cell. In some alternatives of the host cell, the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives of the host cell, the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells and a host cell, wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives of the composition, the composition comprises a host cell wherein the host cell is a CD8+T cytotoxic lymphocyte cell selected from the group consisting of naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and a host cell wherein the host cell is a precursor T cell or a hematopoietic stem cell. In some alternatives, the ligand binding domain specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC or VAR2CSA. In some alternatives, the spacer comprises IgG4, IgG4-CH2 (L235D, N297Q) or IgG4-CH3. In some alternatives, the polypeptide that regulates apoptosis is Survivin. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124. In some alternatives, Survivin comprises an amino acid sequence set forth in SEQ ID NO: 123.

FIGS. 1A to 1F show HEA3 (low affinity) RelA single mutant variants and HEA4 (high affinity) RelA single mutant variants and their response when dosed with 4-OHT, when they are transduced into cells. Shown in the figures is a schematic of dual-packaged lentiviral constructs housing the constitutively expressed drug-inducible transcription factors, ( FIG. 1A ) HEA3 or ( FIG. 1B ) HEA4 single mutant RelA variants (S536E or K310Q positions in the RelA protein domain, which correspond to position S846E and K621Q in HEA3 and HEA4, respectively), and a ZsGreen fluorescent reporter. ZsGreen reporter expression in Jurkat cells expressing ( FIG. 1C ) HEA3 or ( FIG. 1D ) HEA4 RelA variants following 24 hours of culture with 500 nM 4-OHT. 4-OHT dose response: Percentage of EGFRt+ZsGreen+ cells and ZsGreen median fluorescent intensity of ( FIG. 1E ) HEA3 or ( FIG. 1F ) HEA4 RelA variant expressing Jurkat cells.

FIGS. 2A to 2E show HEA4 (high affinity) RelA double mutant variants and CMP8-responsive HEA3 RelA variants and their response when dosed with 4-OHT and CMP8, respectively, when they are transduced into cells. Shown in the figures is a schematic of dual-packaged lentiviral constructs housing the constitutively expressed drug-inducible transcription factors, ( FIG. 2A ) HEA4 double mutant RelA variant (S536E/K310Q positions in the RelA protein domain, which correspond to position S846E and K621Q in HEA3 and HEA4, respectively) or ( FIG. 2B ) CMP8-responsive HEA3 single mutant RelA variant (S563E), and a ZsGreen fluorescent reporter. ZsGreen reporter expression in Jurkat cells expressing the ( FIG. 2C ) HEA4 double mutant RelA variant or the ( FIG. 2D ) CMP8-responsive HEA3 RelA variant, following 24 hours of culture with 500 nM 4-OHT and CMP8. 4-OHT and CMP8 dose response: Percentage of EGFRt+ZsGreen+ cells and ZsGreen median fluorescent intensity of Jurkat cells expressing the ( FIG. 2E ) HEA4 double mutant RelA variant or ( FIG. 2F ) CMP8-responsive HEA3 single mutant RelA variant.

FIGS. 3A to 3C shows the results of dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4 (p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc, when they are transduced into cells. Shown in the FIG. 3A , CD4 and CD8 T cells were transduced with dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc. Following purification and expansion of transduced CD4 and CD8 T cells, ( FIG. 3B ) Western blot was performed using anti-p65 antibody to confirm expression of chimeric transcription factor. ( FIG. 3C ) EGFRt expression was confirmed in sorted cells by flow cytometry, and leakiness of eGFP expression in the absence of inducing ligand was determined.

FIGS. 4A to 4B shows the dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc and the results when the lentiviral constructs are transduced into cells. ( FIG. 4A ) CD4 and CD8 T cells were transduced with dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc. Following purification and expansion of transduced CD4 and CD8 T cells, ( FIG. 4B ) T cells were incubated with 0 nM, 30 nM, or 500 nM of 4-OHT in the presence or absence of aCD3/aCD28 microbeads (2:1 bead:cell ratio) or PMA (2 uM)/Ionomycin (20 ng/mL) for 24 hours. eGFP expression was subsequently quantified by flow cytometry.

FIGS. 5A to 5E shows dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc and the results when the lentiviral constructs are transduced into cells. ( FIG. 5A ) CD4 and CD8 T cells were transduced with dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible fluorescent reporter eGFP:ffluc. Following purification and expansion of transduced CD4 and CD8 T cells, T cells were incubated with increasing concentrations of 4-OHT in the presence or absence of aCD3/aCD28 microbeads (2:1 bead:cell ratio) or PMA (2 uM)/Ionomycin (20 ng/mL) for 24 hours. eGFP expression was quantified by flow cytometry. ( FIG. 5B ) % EGFRt+eGFP+CD4 and ( FIG. 5C ) CD8 T cells and ( FIG. 5D ) MFI of eGFP expression in ( FIG. 5D ) CD4 and ( FIG. 5E ) CD8 T cells following 24 hours of treatment with 4-OHT.

FIGS. 6A to 6D shows results of cells transduced with lentiviral constructs. Jurkat cells were transduced with dual-packaged lentiviral constructs encoding drug-regulated transcription factor (HEA4(p65/S536E/K310Q)) and inducible ( FIG. 6A ) chimeric cytokine receptor (CCR(CD122)) or ( FIG. 6B ) Survivin linked to Her2tG via a cleavable 2A linker. Expression of ( FIG. 6C ) CCR(CD122) and ( FIG. 6D ) Survivin following 24 hours of treatment with 500 nM 4-OHT was determined by Her2tG upregulation.

In the description that follows, the terms should be given their plain and ordinary meaning when read in light of the specification. One of skill in the art would understand the terms as used in view of the whole specification.

“About” has its plain and ordinary meaning when read in light of the specification, and may be used, for example, when referring to a measurable value and may be meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value.

“Antigen” or “Ag,” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a molecule that provokes an immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both. It is readily apparent that an antigen can be generated synthesized, produced recombinantly or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid such, for example, blood, plasma or ascites fluid. In some alternatives of the systems provided herein, the ligand recognized by a chimeric antigen receptor is an antigen, capable of provoking an immune response.

“Anti-tumor effect” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a biological effect, which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or a decrease of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by a decrease in recurrence or an increase in the time before recurrence.

“Chimeric receptor” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a synthetically designed receptor comprising a ligand binding domain of an antibody or other protein sequence that binds to a molecule associated with the disease or disorder and is linked via a spacer domain to one or more intracellular signaling domains of a T cell or other receptors, such as a costimulatory domain. Chimeric receptor can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immunoreceptors, and chimeric antigen receptors (CARs). These CARs are engineered receptors that can graft an arbitrary specificity onto an immune receptor cell. The term chimeric antigen receptors or “CARs” are also considered by some investigators to include the antibody or antibody fragment, the spacer, signaling domain, and transmembrane region. Due to the surprising effects of modifying the different components or domains of the CAR described herein, such as the epitope binding region (for example, antibody fragment, scFv, or portion thereof), spacer, transmembrane domain, and/or signaling domain), the components of the CAR are frequently distinguished throughout this disclosure in terms of independent elements. The variation of the different elements of the CAR can, for example, lead to stronger binding affinity for a specific epitope or antigen.

A “single-chain variable fragment (scFv)” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a fusion protein that can have variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The scFv can be specific for an antigen. “Antigen” or “Ag” as used herein refers to a molecule that provokes an immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be generated, synthesized, produced recombinantly or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid such, for example, blood, plasma or ascites fluid. In some alternatives of the CARs provided herein, the CAR comprises an antibody fragment, scFv, or portion thereof e.g., a polypeptide comprising the CDR domains. In some alternatives, the scFv specifically binds to CD19. This particular scFv is referred to as huCD19 scFv (G01S) and is encoded by SEQ ID NO: 40 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagaggtgcagctggtcgaatccgg aggaggactggtccagcccggccggagcctgagactgagctgtgccgcttccggattcacttttgacgattacgcaatgcattgggtg aggcaggcccctggcaaggggctggaatgggtctccggaatctcttggaacagtgggcgcattggatatgccgattctgtgaaggg ccgattcactatctctcgggacaacgctaaaaatagtctgtttctgcagatgaattccctgcgcgccgaggataccgccgtgtactattg cgcccgagaccagggctaccactactatgatagcgccgaacatgcattcgacatttggggacagggaactgtggtcaccgtgagctc cggaggaggaggaagcggaggaggagggtccggaggcgggggatcacagagcgcactgacccagccacggagcgtgagcg gatttcctgggcagtctgtcaccattagttgcacaggcaccacatcagacgatgtgagctggtaccagcagcacccagggaaggctc cccagctgatgctgtatgacgtgtccaaaagaccttctggcgtcccacataggtttagtggaagccggagcggccgggcagccagtc tgatcatttcagggctgcagacagaggacgaagctgattatttctgttctagttacgcaggcagatataactctgtgctgtttggcgggg gaacaaagctgactgtcctg). In some alternatives, the CAR comprises an scFv. In some alternatives, the scFv comprises an huCD19 scFv (G01S) amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 41 (MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMH WVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDT AVYYCARDQGYHYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQ PRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSR SGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41.

In some alternatives, the scFv is encoded by the muCD19 (FMC63) scFv nucleotide sequence. In some alternatives, the scFv specifically binds to CD19. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 42 (Atgctgctgctggtgaccagcctgctgtgcgagctgccccaccccgcctttctgctgatccccgacatccagatgacccagac cacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactg gtatcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagc ggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaa cacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcga gggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgca ccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtga tctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgtt cctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatgg actactggggccagggcaccagcgtgaccgtgagcagc). In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 43 (MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWY QQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 43.

In some alternatives, the scFv is encoded by a CD20 (CD20 (Leu16)) nucleic acid sequence. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 44 (atggagacagacacactcctgctatgggtgctgctgctctgggttccaggttccacaggtgacattgtgctgacccaatctccagctat cctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaaattacatggactggtaccagaagaagcc aggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacct cttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaatccacccacgttcggag gggggaccaagctggaaataaaaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgaggtg cagctgcagcagtctggggctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagtt acaatatgcactgggtaaagcagacacctggacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaat cagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgagga ctctgcggactattactgtgcaagatctaattattacggtagtactggttcttcgatgtctggggcgcagggaccacggtcaccgt ctcctca). In some alternatives, the CD20 (Leu 16) scFv comprises an amino acid sequence set forth in SEQ ID NO: 45 (METDTLLLWVLLLWVPGSTGDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWY QKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSF NPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKAS GYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY MQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 45.

In some alternatives, the scFv is encoded by a CD22 (m971) scFv nucleotide sequence. In some alternatives, the scFv specifically binds to CD22. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 46 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggtacagctgcagcagtcagg tccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctgcttggaac tggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagtatctgt gaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgt gtattactgtgcaagagaagtgactggggatctcgaggatgcttttgatatctggggccaagggacaatggtcaccgtctcctcaggc ggagggggctctggcggcggaggatctgggggagggggcagcgacatccagatgacccagtctccatcgtccctgtctgcatctgt aggagacagagtcaccatcacttgccgggcaagccagaccatttggagctacttaaattggtatcagcagagaccagggaaagccc ctaacctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcaggggatctgggacagatttcactctca ccatcagcagtctgcaagctgaagattttgcaacttactactgtcaacagagttacagtatccctcagacttttggccaggggaccaagc tggagatcaaacgaact). In some alternatives, the scFv comprises a CD22 (m971) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 47 (MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGS GTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRT). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 47.

In some alternatives, the scFv comprises a B7H3 (hBRCA84D) scFv nucleotide sequence. In some alternatives, the scFv binds specifically to the B7-H3 receptor. The B7-H3 receptor is known to be expressed on a variety of cancer cells. Without being limiting, these cancer cells can included gastric, ovarian and/or non-small cell lung cancers. In some alternatives, the CAR is used in the treatment or amelioration of cancer and/or inflammation. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 48 (atgctgcgtcggcggggcagccctggcatgggtgtgcatgtgggtgcagccctgggagcactgtggttctgcctcacaggagccc tggaggtccaggtccctgaagacccagtggtggcactggtgggcaccgatgccaccctgtgctgctccttctcccctgagcctggctt cagcctggcacagctcaacctcatctggcagctgacagataccaaacagctggtgcacagctttgctgagggccaggaccagggca gcgcctatgccaaccgcacggccctcttcccggacctgctggcacagggcaacgcatccctgaggctgcagcgcgtgcgtgtggc ggacgagggcagcttcacctgcttcgtgagcatccgggatttcggcagcgctgccgtcagcctgcaggtggccgctccctactcgaa gcccagcatgaccctggagcccaacaaggacctgcggccaggggacacggtgaccatcacgtgctccagctaccagggctaccct gaggctgaggtgttctggcaggatgggcagggtgtgcccctgactggcaacgtgaccacgtcgcagatggccaacgagcagggct tgtttgatgtgcacagcatcctgcgggtggtgctgggtgcaaatggcacctacagctgcctggtgcgcaaccccgtgctgcagcagg atgcgcacagctctgtcaccatcacagggcagcctatgacattccccccagaggccctgtgggtgaccgtggggctgtctgtctgtct cattgcactgctggtggccctggctttcgtgtgctggagaaagatcaaacagagctgtgaggaggagaatgcaggagctgaggacc aggatggggagggagaaggctccaagacagccctgcagcctctgaaacactctgacagcaaagaagatgatggacaagaaatag cc). In some alternatives, the scFv comprises a B7H3 (hBRCA84D) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 49 (MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPE PGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQR VRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQ GYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRN PVLQQDAHSSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEE NAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49.

In some alternatives, the scFv comprises the L1CAM (CE7) scFv nucleotide sequence. In some alternatives, the scFv is specific for the epitope, CE7 epitope, of L1CAM. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 50 (atgctgctgctggtgaccagcctgctgctgtgcgagctgccccaccccgcctttctgctgatcccccaggtgcagctgcagcagcct ggcgccgagctggtgaagccaggcgccagcgtgaagctgtcctgcaaggccagcggctacaccttcaccggctactggatgcact gggtgaagcagagacccggccacggcctggaatggatcggcgagatcaaccccagcaacggccggaccaactacaacgagcgg ttcaagagcaaggccaccctgaccgtggacaagagcagcaccaccgccttcatgcagctgtccggcctgaccagcgaggacagcg ccgtgtacttctgcgccagggactactacggcaccagctacaacttcgactactggggccagggcaccacactgaccgtgagcagc ggcggagggggctctggcggcggaggatctgggggagggggcagcgacatccagatgacccagagcagcagcagcttcagcg tgagcctgggcgaccgggtgaccatcacctgtaaggccaacgaggacatcaacaaccggctggcctggtatcagcagacccccgg caacagccccaggctgctgatcagcggcgccaccaacctggtgaccggcgtgcccagccggtttagcggcagcggctccggcaa ggactacaccctgaccatcacaagcctgcaggccgaggacttcgccacctactactgccagcagtactggtccacccccttcaccttc ggcagcggcaccgagctggaaatcaaa). In some alternatives, the scFv comprises the L1CAM (CE7) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 51 (MLLLVTSLLLCELPHPAFLLIPQVQLQQPGAELVKPGASVKLSCKASGYTFTGYWM HWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDS AVYFCARDYYGTSYNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSSSSFS VSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGK DYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 51.

In some alternatives, the scFv is encoded by the EGFR (cetuximab) scFv nucleotide sequence. In some alternatives, the scFv is specific for EGFR. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 52 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggtgcagctgaaacagagcg gcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgg gtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttacca gccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcagagcaacgataccgcgatttattat tgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcgggcggcggcgg cagcggcggcggcggcagcggcggcggcggcagcgatattctgctgacccagagcccggtgattctgagcgtgagcccgggcg aacgcgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgc ctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagc attaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaa ctggaactgaaacgcacc). In some alternatives, the scFv comprises the EGFR scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 53 (MLLLVTSLLLCELPHPAFLLIPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHW VRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIY YCARALTYYDYEFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDILLTQSPVILSVSP GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI NSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 53.

In some alternatives, the scFv comprises the EGFRVIII (806) scFv nucleotide sequence. In some alternatives, the scFv is specific for EGFR. In some alternatives, the scFv is encoded by the nucleic acid sequence set forth in SEQ ID NO: 54 (atgctgctgctggtgacaagcctgctgctgtgcgagctgccacaccctgctttcctgctgatccctgatgtgcagctgcaggaatcag gcccaagcctggtcaaaccctcccagtctctgagtctgacctgtacagtgactgggtactccatcacatctgatttcgcatggaactgg attaggcagtttccaggcaataagctggagtggatgggctacatctcatatagcgggaacactcgctataatcccagtctgaaatcacg gatcagcattactagagacaccagcaagaaccagttctttctgcagctgaattccgtgaccattgaggataccgccacatactattgcgt cacagctggcagaggctttccatactggggacagggcacactggtgactgtcagcgccggctccacctctgggagtggaaaacctg gctccggggaaggatctacaaagggagacatcctgatgactcagtccccaagctccatgtcagtgagcctgggcgacaccgtctcta ttacatgtcactctagtcaggatatcaacagtaatattggctggctgcagcagcgacccggcaagtctttcaaagggctgatctatcatg gaactaacctggacgatgaagtgcctagcagattttccggctctgggagtggagctgattacagtctgaccatttcaagcctggagtca gaagacttcgcagattactattgcgtccagtatgcccagttcccctggacttttggcgggggaaccaagctggagatcaaacgg). In some alternatives, the scFv comprises the EGFRVIII (806) scFv amino acid sequence. In some alternatives, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 55 (MLLLVTSLLLCELPHPAFLLIPDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWN WIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATY YCVTAGRGFPYWGQGTLVTVSAGSTSGSGKPGSGEGSTKGDILMTQSPSSMSVSLG DTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTI SSLESEDFADYYCVQYAQFPWTFGGGTKLEIKR). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 55.

In some alternatives, the scFv is encoded by the EphA2 (2A4) scFv nucleotide sequence. In some alternatives, the scFv is encoded by the nucleic acid sequence set forth in SEQ ID NO: 56 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggtgcagctgttggagtctggg ggaggcttggtacagcctggggtccctgagactctcctgtgcagcctctggattcacctttagagactataccatgtcttgggtgcg acaggcccctggacaagcgcttgagtggatgggaaccattagtagtcgtggtacttacacctactatccagacagtgtgaagggccg attcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgt gcgagagaagctatctttactcactggggccgtggcaccctggtcaccgtctcctcaggtggtggtggttctggcggcggcggctcc ggtggtggtggttctgacatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcaaggc gagtcaggacattaataactatcacagctggtaccagcagaaacctggccaggctcccaggctcctcatctatcgtgcaaacagattg gtcgatggggtcccagacaggttcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctg catattacttctgtctgaaatataatgtgtttccgtacacgttcggccaagggaccaaggtggagatcaaa). In some alternatives, the scFv comprises the EphA2 (2A4) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 57 (MLLLVTSLLLCELPHPAFLLIPQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMS WVRQAPGQALEWMGTISSRGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCAREAIFTHWGRGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDR VTITCKASQDINNYHSWYQQKPGQAPRLLIYRANRLVDGVPDRFSGSGYGTDFTLTI NNIESEDAAYYFCLKYNVFPYTFGQGTKVEIK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 57.

In some alternatives, the scFv is encoded by the EpHA2 (4H5) scFv nucleotide sequence. In some alternatives, the scFv is encoded by the nucleic acid sequence set forth in SEQ ID NO: 58 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggtgcagctgttggagtctggg ggaggcttggtacagcctggggtccctgagactctcctgtgcagcctctggattcacctttgagagctataccatgtcttgggtgcg acaggcccctggacaagcgcttgagtggatgggaaccattagtagtggtggtacttacacctactatccagacagtgtgaagggccg attcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgt gcgagagaagctatctttacttactggggccgtggcaccctggtcaccgtctcctcaggtggtggtggttctggcggcggcggctccg gtggtggtggttctgacatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcaaggcg agtcaggacattaataactatttaagctggtaccagcagaaacctggccaggctcccaggctcctcatctatcgtgcaaacagattggt agatggggtcccagacaggttcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcat attacttctgtctgaaatatgatgtgtttccgtacacgttcggccaagggaccaaggtggagatcaaa). In some alternatives, the scFv comprises the EpHA2 (4H5) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 59 (MLLLVTSLLLCELPHPAFLLIPQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMS WVRQAPGQALEWMGTISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCAREAIFTYWGRGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDR VTITCKASQDINNYLSWYQQKPGQAPRLLIYRANRLVDGVPDRFSGSGYGTDFTLTI NNIESEDAAYYFCLKYDVFPYTFGQGTKVEIK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 59.

In some alternatives, the scFv is encoded by a FITC (E2) scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 60 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaagcgtgctgacacagcctagctc cgtgtctgccgcccctggccagaaagtgaccatcagctgtagcggcagcaccagcaacatcggcaacaactacgtgtcctggtatca gcagcaccccggcaaggcccccaagctgatgatctacgacgtgtccaagcggcccagcggcgtgcccgatagattttccggcagc aagagcggcaacagcgccagcctggatatcagcggcctgcagtctgaggacgaggccgactactattgcgccgcctgggacgata gcctgagcgagttcctgtttggcaccggcaccaagctgacagtgctgggcggaggcggaggatctggcggcggaggaagtggcg gagggggatctcaggtgcagctggtggaaagcggcggcaacctggtgcagcctggcggatctctgagactgagctgtgccgccag cggcttcaccttcggcagcttcagcatgagctgggtgcgccaggctcctgggggaggactggaatgggtggcaggactgagcgcc agaagcagcctgacccactacgccgatagcgtgaagggccggttcaccatcagccgggacaacgccaagaacagcgtgtacctgc agatgaacagcctgcgggtggaagataccgccgtgtactactgcgccagacggtcctacgacagcagcggctactggggccacttc tacagctacatggacgtgtggggccagggcaccctcgtgacagtgtct). In some alternatives, the scFv comprises the FITC (E2) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequences set forth in SEQ ID NO: 61 (MLLLVTSLLLCELPHPAFLLIPSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWY QQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWD DSLSEFLFGTGTKLTVLGGGGGSGGGGSGGGGSQVQLVESGGNLVQPGGSLRLSCA ASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSV YLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 61.

In some alternatives, the scFv is encoded by the GD2 (hu3F8) scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 62 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaagcatcgtgatgacccagacccc caagttcctgctggtgagcgccggcgacagggtgaccatcacctgcaaggccagccagagcgtgagcaacgacgtgacctggtac cagcagaaggccggccagagccccccaacaggtacagcggcgtgcccgacaggttcaccggc agcggctacggcaccgccttcaccatcagcaccgtgcaggccaactggccgtgtacttctgccagcaggactacag cagcttcggcggcggcaccaagctggagatcaagagg). In some alternatives, the scFv comprises the GD2 (hu3F8) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 63 (MLLLVTSLLLCELPHPAFLLIPSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVTWY QQKAGQSPKLLIYSASNRYSGVPDRFTGSGYGTAFTFTISTVQAEDLAVYFCQQDYS SFGGGTKLEIKR). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 63.

In some alternatives, the scFv is encoded by the Her2 (Herceptin) scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 64 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagatatccagatgacccagtccccg agctccctgtccgcctctgtgggcgatagggtcaccatcacctgccgtgccagtcaggatgtgaatactgctgtagcctggtatcaaca gaaaccaggaaaagctccgaaactactgatttactcggcatccttcctctactctggagtcccttctcgcttctctggttccagatctggg acggatttcactctgaccatcagcagtctgcagccggaagacttcgcaacttattactgtcagcaacattatactactcctcccacgttcg gacagggtaccaaggtggagatcaaaggcagtactagcggcggtggctccgggggcggatccggtgggggcggcagcagcga ggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgtttgtcctgtgcagcttctggcttcaacattaaag acacctatatacactgggtgcgtcaggccccgggtaagggcctggaatgggttgcaaggatttatcctacgaatggttatactagatat gccgatagcgtcaagggccgtttcactataagcgcagacacatccaaaaacacagcctacctgcagatgaacagcctgcgtgctga ggacactgccgtctattattgttctagatggggaggggacggcttctatgctatggactactggggtcaaggaaccctggtcaccgtct cgagt). In some alternatives, the scFv comprises the Her2 (Herceptin) scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 65 (MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASG FNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 65.

In some alternatives, the scFv is encoded by the IL13R2a (hu08) VlVh scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 66 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagacatccagatgacccagtcccc ctcttctctgtctgcctctgtgggcgacagagtgaccatcacctgtaaggccagtcaggatgtaggtactgctgtagcctggtatcagca gaagcctggcaaggctcccaagctgctgatctactcggcatcctaccggtccactggcgtgccttccagattctccggctctggctctg gcaccgatttcaccctgaccatctcctccctccagcctgaggatttcgccacctactactgccagcaccattatagtgctccgtggacgt ttggcggcggaacaaaggtggagatcaagggtggtggtggttctggcggcggcggctccggtggtggtggttctgaggtgcagctg gtggagtctggcggcggactggtgcagcctggcggctctctgagactgtcttgtgccgcctccggcttcaccttcagtaggaatggca tgtcttgggtgaggcaggcccctggcaagggcctggagtgggtggccaccgttagtagtggtggtagttacatctactatgcagaca gtgtgaaggggcggttcaccatctccagggacaacgccaagaactccctgtacctccagatgaactccctgagggccgaggatacc gccgtgtactactgtgc). In some alternatives, the scFv comprises the IL13Ra2 (hu08) VlVh scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 67 (MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAW YQQKPGKAPKLLIYSASYRSTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYS APWTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGF TFSRNGMSWVRQAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYC). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 67.

In some alternatives, the scFv is encoded by an IL13Ra2 (hu08) VhV1 scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 68 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagaggtgcagctggtggagtctgg cggcggactggtgcagcctggcggctctctgagactgtcttgtgccgcctccggcttcaccttcagtaggaatggcatgtcttgggtga ggcaggcccctggcaagggcctggagtgggtggccaccgttagtagtggtggtagttacatctactatgcagacagtgtgaagggg cggttcaccatctccagggacaacgccaagaactccctgtacctccagatgaactccctgagggccgaggataccgccgtgtactac tgtgccagacaagggactacggcactagctacgaggttcttcgatgtctggggccagggcaccctggtgaccgtgtcctctggtggt ggtggttctggcggcggcggctccggtggtggtggttctgacatccagatgacccagtccccctcttctctgtctgcctctgtgggcga cagagtgaccatcacctgtaaggccagtcaggatgtaggtactgctgtagcctggtatcagcagaagcctggcaaggctcccaagct gctgatctactcggcatcctaccggtccactggcgtgccttccagattctccggctctggctctggcaccgatttcaccctgaccatctc ctccctccagcctgaggatttcgccacctactactgccagcaccattatagtgctccgtggacgtttggcggcggaacaaaggtggag atcaaggaatctaagta). In some alternatives, the scFv comprises an IL13Ra2 hu08 VhV1 scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 69 (MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMS WVRQAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCARQGTTALATRFFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYSASYRSTGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQHHYSAPWTFGGGTKVEIKESK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 69.

In some alternatives, the scFv is encoded by an IL13Ra2 (hu07) VlVh scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 70 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagatattcagatgacccagagccc gagcagcctgagcgcgagcgtgggcgatccgcgtgagcagcacctatctgcattggt atcagcagaaaccgggcaaagcgccgaaactgctgatttatagcaccagcaacctggcgagcggcgtgccgagccgctttagcgg cagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgccatcagtatcatcgca gcccgctgacctttggcggcggcaccaaagtggaaattaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctg aagtgcagctggtggaaagcggcggcggcctggtgcagcggcggcgggcctggtgcgcggcgagcggctttacct ttaccaaatatggcgtgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtggcggtgaaatgggcgggcggcagca ccgattataacagcgcgctgatgagccgctttaccattagccgcgataacgcgaaaaacagcctgtatctgcagatgaacagcctgc gcgcggaagataccgcggtgtattattgcgcgcgcgatcatcgcgatgcgatggattattggggccagggcaccctggtgaccgtg agcagcgaatctaagta). In some alternatives, the scFv comprises an IL13Ra2 (hu07) VhV1 scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 71 (MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCTASLSVSSTYLHWY QQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYHRSP LTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFT KYGVHWVRQAPGKGLEWVAVKWAGGSTDYNSALMSRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARDHRDAMDYWGQGTLVTVSSESK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 71.

In some alternatives, the scFv is encoded by the IL13Ra2 (hu07) VhV1 scFv nucleotide sequence. In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 72 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagaagtgcagctggtggaaagcg gcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttaccaaatatggcgtgcattgg gtgcgccaggcgccgggcaaaggcctggaatgggtggcggtgaaatgggcgggcggcagcaccgattataacagcgcgctgat gagccgctttaccattagccgcgataacgcgaaaaacagcctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgt attattgcgcgcgcgatcatcgcgatgcgatggattattggggccagggcaccctggtgaccgtgagcagcggtggtggtggttctg gcggcggcggctccggtggtggtggttctgatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgt gaccattacctgcaccgcgagcctgagcgtgagcagcacctatctgcattggtatcagcagaaaccgggcaaagcgccgaaactgc tgatttatagcaccagcaacctggcgagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccatta gcagcctgcagccggaagattttgcgacctattattgccatcagtatcatcgcagcccgctgacctttggcggcggcaccaaagtgga aattaaagaatctaagta). In some alternatives, the scFv comprises an IL13Ra2 (hu07) VhVl scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 73 (MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGFTFTKYGVH WVRQAPGKGLEWVAVKWAGGSTDYNSALMSRFTISRDNAKNSLYLQMNSLRAED TAVYYCARDHRDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSA SVGDRVTITCTASLSVSSTYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCHQYHRSPLTFGGGTKVEIKESK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 73.

In some alternatives, the antibody or portion thereof or scFv is encoded by the Anti-IL13Ra2 (IL13 zetakine) nucleotide sequence. In some alternatives, the antibody or portion thereof or scFv is encoded by a sequence set forth in SEQ ID NO: 74 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaggccctgtgcctccctctacagcc ctcaggtacctcattgaggagctggtcaacatcacccagaaccagaaggctccgctctgcaatggcagcatggtatggagcatcaac ctgacagctggcatgtactgtgcagccctggaatccctgatcaacgtgtcaggctgcagtgccatcgagaagacccagaggatgctg agcggattctgcccgcacaaggtctcagctgggcagttttccagcttgcatgtccgagacaccaaaatcgaggtggcccagtttgtaa aggacctgctcttacatttaaagaaactttttcgcgagggacggttcaa). In some alternatives, the antibody or portion thereof or scFv comprises an Anti-IL13Ra2 (IL13 zetakine) amino acid sequence. In some alternatives, the antibody or portion thereof or scFv comprises an amino acid sequence set forth in SEQ ID NO: 75 (MLLLVTSLLLCELPHPAFLLIPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSI NLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQ FVKDLLLHLKKLFREGRF). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75.

In some alternatives, the scFv is encoded by an oaGD2 (8B6) VlVh scFv nucleotide sequence. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 76 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagatgttgtgatgacccaaactcca ctctccctgcctgtcagtcttggagatcaagcctcaatctcttgcagatctagtcagagccttctaaaaaatggaaacacctttttaca ttggtacctgcagaagtcaggccagtctccaaagctccttatctacaaagtttccaaccgactttctggggtcccagacaggttcagtgg cagtggatcagggacatatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatat tccgtacacattcggaggggggaccaagctcgagctgaaacgaggcggagggggctctggcggcggaggatctgggggagggg gcagcgaggtgaaactggtggagtctggaggaggcttggtgctgcctggggattctctgagactctcctgtgcaacttctgagttcac cttcactgattactacatgacttgggtccgccagcctccaagaaaggcacttgagtggttgggttttattagaaacagagctaatggttac acaacagagtacaatccatctgtgaagggtcggttcaccatttccagagataattcccaaagcatcctctatcttcaaatgaacaccctg agaactgaggacagtgccacttattactgtgcaagagtctctaactgggcctttgactactggggccaaggcaccactctcacagtctc ctca). In some alternatives, the scFv comprises the oaGD2 (8B6) VlVh scFv amino acid sequence. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 77 (MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNT FLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCS QSTHIPYTFGGGTKLELKRGGGGSGGGGSGGGGSEVKLVESGGGLVLPGDSLRLSCA TSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSIL YLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 77.

In some alternatives, the scFv is encoded by an ROR1 (R12) scFv nucleotide sequence. In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 78 (atgctgctgctggtgacaagcctgctgctgtgcgagctgccccaccccgcctttctgctgatcccccaggaacagctcgtcgaaagc ggcggcagactggtgacacctggcggcagcctgaccctgagctgcaaggccagcggcttcgacttcagcgcctactacatgagctg ggtccgccaggcccctggcaagggactggaatggatcgccaccatctaccccagcagcggcaagacctactacgccacctgggtg aacggacggttcaccatctccagcgacaacgcccagaacaccgtggacctgcagatgaacagcctgacagccgccgaccgggcc acctacttttgcgccagagacagctacgccgacgacggcgccctgttcaacatctggggccctggcaccctggtgacaatctctagc ggcggaggcggatctggtggcggaggaagtggcggcggaggatctgagctggtgctgacccagagcccctctgtgtctgctgccc tgggaagccctgccaagatcacctgtaccctgagcagcgcccacaagaccgacaccatcgactggtatcagcagctgcagggcga ggcccccagatacctgatgcaggtgcagagcgacggcagctacaccaagaggccaggcgtgcccgaccggttcagcggatctag ctctggcgccgaccgctacctgatcatccccagcgtgcaggccgatgacgaggccgattactactgtggcgccgactacatcggcg gctacgtgttcggcggaggcacccagctgaccgtgaccggc). In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 79 (ROR1 (R12) scFv amino acid sequence; MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSW VRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRA TYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAA LGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSS SGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 79.

In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 80 (CD33 (h2H12) VhVl scFv nucleotide sequence (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggttcagctggtgcagtctgga gctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccaattatgatataaattgggtgag acaggcccctggacaagggcttgagtggattggatggatttatcctggagatggtagtaccaaatataatgagaaattcaaggccaag gctaccctgacagctgacacatccaccagcacagcctacatggagctgaggagcctgagatctgatgacacagctgtgtattactgtg cttctggatatgaagatgctatggactactgggggcaagggaccacagtcacagtctcctca). In some alternatives, the scFv is specific for CD33. In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 81 (CD33 (h2H12) VhVl scFv amino acid sequence; MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIN WVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDT AVYYCASGYEDAMDYWGQGTTVTVSS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 81.

In some alternatives, the scFv is encoded by a sequence set forth in SEQ ID NO: 82 (CD33 (h2H12) VlVh scFv nucleotide sequence: atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagacatccagatgacccagtctccat cctcactgtctgcatctgtaggagacagagtcaccatcaattgtaaggctagtcaggacattaatagctatttgagctggtttcagcaga aaccagggaaagcccctaagaccctgatctatagagcaaatagattggtagatggggtcccatcaaggttctctggcagtggatctgg gcaagattatactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgcttgcagtatgatgagtttcctctcacatttgg aggagggaccaaggtggagatcaaa). In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 83 (CD33 (h2H12) VlVh scFv amino acid sequence: MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQ QKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFP LTFGGGTKVEIK). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 83.

In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 84 (Mesothelin (P4) scFv nucleotide sequence: atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacaggtacagctgcagcagtcaggt ccaggactcgtgacgccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaact ggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataacgactatgcagtatctg tgaaaagtcgaatgagcatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctg tgtattactgtgcaagaggaatgatgacttactattacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggaatt ctaggatcc). In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 85 (Mesothelin (P4) scFv amino acid sequence: MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWN WIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPED TAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGS). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 85.

In some alternatives, the scFv is encoded by a nucleic acid sequence set forth in SEQ ID NO: 86 (VAR2CSA (ID1-DBL2Xb) scFv nucleotide sequence: atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaagtctcacgaatgggtataagtgcg acaaatgcaaatcaggcacatcccggtccaaaaaaaaatggatctggaaaaagagcagcgggaatgaggagggcctgcaggaag aatacgctaacacaatcggactgccccctcgaactcaatctctgtaccttgggaacttgcctaaactggaaaacgtctgtgaagacgtc aaggacataaacttcgatacgaaagagaagttcctggctggatgccttatcgtgagctttcacgagggaaagaatctgaagaaaaggt acccacagaataaaaactctggcaataaagagaatttgtgcaaggccctggagtactcttttgcggactatggcgatctcataaagggc acaagcatctgggataatgagtataccaaggatctggagctgaatctgcagaataactttgggaaattgtttgggaagtatattaaaaaa aataataccgccgagcaggacacatcctactcttccctcgacgagctgcgggagtcatggtggaacaccaacaaaaagtatatctgg acggcaatgaaacatggcgcggaaatgaatatcaccacttgtaatgcagacggctctgtcaccgggtccggttcttcttgcgatgatat accaaccatcgatttgattccgcagtatctgagatttctccaagagtgggtggaaaatttttgcgagcagaggcaagctaaggtcaagg acgtgatcaccaattgtaaaagttgcaaagaatcagggaacaagtgcaaaaccgagtgtaagacgaagtgcaaggacgaatgcgag aaatataaaaaattcatcgaggcttgtggaacagccgggggtgggattgggaccgcaggcagcccatggagcaagcggtgggacc aaatctataaacgatacagcaagcacatcgaagatgccaagcggaaccgcaaagctggcacaaaaaactgtgggactagcagcac gactaatgccgcggcaagcactgatgaaaataaatgcgtgcaaagtgacatcgactctttcttcaaacacctgatcgatatcggtcttac tacgccaagcagctacctcagtaatgttctggatgataatatttgtggggcggacaaagcaccttggactacctacaccacctatacca caacagagaagtgtaataaagagagagacaaatcaaagtctcagagctctgatacactggtcgttgtgaatgtgccaagccctttggg gaatacgccctacagatataaatac). In some alternatives, the scFv comprises an amino acid sequence set forth in SEQ ID NO: 87 (VAR2CSA (ID1-DBL2Xb) scFv amino acid sequence: MLLLVTSLLLCELPHPAFLLIPSLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQ EEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLK KRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLF GKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSV TGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTE CKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRK AGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGA DKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKY). In some alternatives, the scFv is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 87.

“Spacer”, can also be referred to as a “linker,” and has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, residues which resides between the light and the heavy chains. The spacer can be 10 to 20 amino acids in length (e.g., at least, equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids or a length within a range defined by any two of the aforementioned lengths). The flexible spacer allows the scFv domain to orient in multiple directions in order to enable an optimized antigen binding specificity. The scFv domain can be preceded by a signal peptide so as to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression, whereby cleavage can occur. A flexible spacer can also allow the scFv domain to orient in different directions to facilitate antigen binding. A good spacer for allowing specific binding of the scFv domain can be determined empirically and is dependent on the scFv domain antigen recognition domain. In some alternatives, the linker comprises optimized spacer lengths so as to improve binding of scFv domain to the target cell, which may increase cytotoxic efficacy. In some alternatives, the linker or spacer between the scFv domain and the transmembrane can be 25 to 55 amino acids in length (e.g., at least, equal to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids or a length within a range defined by any two of the aforementioned lengths. In some alternatives, the spacer is encoded by the nucleic acid sequence set forth in SEQ ID NO: 102 (IgG4 hinge nucleotide sequence: gagagcaagtacggaccgccctgccccccttgccct). In some alternatives, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 103 (IgG4 hinge amino acid sequence: ESKYGPPCPPCP). In some alternatives, the spacer is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 103. In some alternatives, the spacer is encoded by a nucleic acid sequence set forth in SEQ ID NO: 104 (IgG4-CH2(L235D, N297Q) nucleotide sequence; gcccccgagttcgacggcggacccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagccggacccccgagg tgacctgcgtggtggtggacgtgagccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaacgcc aagaccaagcccagagaggaacagttccagagcacctaccgggtggtgtctgtgctgaccgtgctgcaccaggactggctgaacg gcaaagaatacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaag). In some alternatives, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 105 (IgG4-CH2(L235D, N297Q) amino acid sequence; APEFDGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK). In some alternatives, the spacer is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 105. In some alternatives, the spacer is encoded by a nucleic acid sequence set forth in SEQ ID NO: 106 (IgG4-CH3 nucleotide sequence: ggccagcctcgcgagccccaggtgtacaccctgcctccctcccaggaagagatgaccaagaaccaggtgtccctgacctgcctggt gaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcctgagaacaactacaagaccacccctcccgtg ctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagccggtggcaggaaggcaacgtctttagctgcag cgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtccctgggcaag). In some alternatives, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 107 (IgG4 CH3 amino acid sequence: GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK). In some alternatives, the spacer is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 107. In some alternatives, the spacer is encoded by a nucleic acid sequence set forth in SEQ ID NO: 108 (CD8 hinge/Transmembrane domain nucleotide sequence: gaatctaagtacggaccggccaagcctaccaccacccctgcccctagacctccaacacccgccccaacaatcgccagccagcctct gtctctgaggcccgaggcttgtagaccagctgctggcggagccgtgcacaccagaggactggatttcgcctgcgacatctacatctg ggcccctctggccggcacatgtggcgtgctgctgctgagcctcgtgatcacc). In some alternatives, the spacer comprises an amino acid sequence set forth in SEQ ID NO: 109 (CD8 hinge/Transmembrane domain amino acid sequence: ESKYGPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVIT. In some alternatives, the spacer is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 109.

“Transmembrane domain” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a transmembrane region of a protein. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix, or any other structure. In some alternatives herein, the transmembrane domain comprises CD28tm. In some alternatives, the transmembrane domain is encoded by a sequence set forth in SEQ ID NO: 110 (CD28 transmembrane domain nucleotide sequence: atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg). In some alternatives, the transmembrane domain comprises an amino acid sequence set forth in SEQ ID NO: 111 (CD28 transmembrane domain amino acid sequence: MFWVLVVVGGVLACYSLLVTVAFIIFWV). In some alternatives, the transmembrane domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 111. In some alternatives, the transmembrane domain is encoded by a sequence set forth in SEQ ID NO: 112 (CD8 hinge/Transmembrane domain nucleotide sequence: gaatctaagtacggaccggccaagcctaccaccacccctgcccctagacctccaacacccgccccaacaatcgccagccagcctct gtctctgaggcccgaggcttgtagaccagctgctggcggagccgtgcacaccagaggactggatttcgcctgcgacatctacatctg ggcccctctggccggcacatgtggcgtgctgctgctgagcctcgtgatcacc). In some alternatives, the transmembrane domain comprises an amino acid sequence set forth in SEQ ID NO: 113 (CD8 hinge/Transmembrane domain amino acid sequence: ESKYGPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVIT). In some alternatives, the transmembrane domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 113.

A “chimeric cytokine receptor,” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a protein that has an amino acid sequence of a chemokine or portion thereof and is a fusion protein with another protein.

“Intracellular signaling domain,” or “Co-stimulatory domain,” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a signaling moiety that provides to T cells a signal which, in addition to the primary signal provided by for instance the CD3 zeta chain of the TCR/CD3 complex, mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like. A co-stimulatory domain can include all or a portion of, but is not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that specifically binds with CD83. In some alternatives, the co-stimulatory domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and cytokine secretion, and the like. In some alternatives of the signaling domain described herein, the signaling domain comprises CD3ζ, CD28 cytoplasmic domain, and/or 4-1BB. In some alternatives, the intracellular signaling domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 114 (CD3ζ Zeta nucleotide sequence: cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcaga agggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccagg aaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggc aagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccaa gg). In some alternatives, the intracellular signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 115 (CD3 Zeta amino acid sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR). In some alternatives, the intracellular signaling domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 115. In some alternatives, the intracellular signaling domain is encoded by a nucleic acid sequence set forth in SEQ ID NO: 116 (4-1BB nucleotide sequence: aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagct gccgatttccagaagaagaagaaggaggatgtgaactg). In some alternatives, the intracellular signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 117 (4-1BB amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL). In some alternatives, the intracellular signaling domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 117. In some alternatives, the intracellular signaling domain is encoded by a sequence set forth in SEQ ID NO: 118 (CD28 cytoplasmic domain nucleotide sequence: cgcagcaagcggagcagaggcggccacagcgactacatgaacatgacccctagacggcctggccccaccagaaagcactacca gccctacgcccctccccgggactttgccgcctacagaagc). In some alternatives, the intracellular signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 119 (CD28 cytoplasmic domain amino acid sequence: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). In some alternatives, the intracellular signaling domain is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 119.

As used herein, “nucleic acid” or “nucleic acid molecule” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

“DNA spacer” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a spacer comprising nucleotides. A DNA spacer may reside in between a promoter and a gene that is to be expressed that is under control of the promoter, for example. In some alternatives, a DNA spacer is between the inducible promoter and the transgene. In some alternatives, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120 (SEQ ID NO: 120; gctagcgtttaaacttaagcttggtaccgagctcggatccgccacc). In some alternatives, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values.

“Coding for” and “encoding” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A “nucleic acid sequence coding for a polypeptide” includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence.

“Survivin,” is also known as baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, and has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, a protein that is a member of the inhibitor of apoptosis family. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124 (Atgggtgccccgacgttgccccctgcctggcagccctttctcaaggaccaccgcatctctacattcaagaactggcccttcttggag ggctgcgcctgcaccccggagcggatggccgaggctggcttcatccactgccccactgagaacgagccagacttggcccagtgttt cttctgcttcaaggagctggaaggctgggagccagatgacgaccccatagaggaacataaaaagcattcgtccggttgcgctttccttt ctgtcaagaagcagtttgaagaattaacccttggtgaatttttgaaactggacagagaaagagccaagaacaaaattgcaaaggaaac caacaataagaagaaagaatttgaggaaactgtgaagaaagtgcgccgtgccatcgagcagctggctgcaatggat). In some alternatives, Survivin comprises the amino acid sequence set forth in SEQ ID NO: 123 (MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQC FFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAK ETNNKKKEFEETVKKVRRAIEQLAAMD).

“Conditional” or “Inducible” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, nucleic acid construct that includes a promoter that provides for gene expression in the presence of an inducer and does not substantially provide for gene expression in the absence of the inducer.

“Constitutive” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, the nucleic acid construct that includes a promoter that is constitutive providing for expression of a polypeptide that is continuously produced.

“Specific” or “Specificity” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, the characteristic of a ligand for the binding partner or alternatively, the binding partner for the ligand, and can include complementary shape, charge and hydrophobic specificity for binding. Specificity for binding can include stereospecificity, regioselectivity and chemoselectivity. In some alternatives, a method of making a nucleic acid encoding a chimeric antigen receptor is provided such that a nucleic acid encoding a chimeric antigen receptor is generated, wherein the chimeric antigen receptor binds specifically to a desired or selected ligand.

“TamR-tf” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, tamoxifen regulated transcription factors. Without being limiting, tamoxifen regulated transcription factors can include wild-type HEA3, wild-type HEA4, mutant HEA3, mutant HEA4 and/or RelA variants.

“HEA3” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a chimeric transcription factor composed of human subunits including the N-terminal DNA binding domain of Hepatocyte Nuclear Factor 1-alpha (HNF-1α) fused in frame to the mutant tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD) which contains 1 amino acid substitution (G521R), which ablates binding of estradiol but confers nanomolar estrogen analogues, that is in turn fused to the p65 activation domain of NF-κB (p65).

HEA-3 has been described by Roscilli et al. (Molecular Therapy, Vol. 6, Issue 5, pp. 653-663 (2002)) as a novel 4-hydroxytamoxifen (4-OHT) dependent regulator which was shown to have long term control of gene expression in mouse skeletal muscle (Roscilli et al.; included by reference in its entirety herein.)

“HEA4” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a chimeric transcription factor composed of human subunits including the N-terminal DNA binding domain of Hepatocyte Nuclear Factor 1-alpha (HNF-1α) fused in frame to the tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD) which contains 3 amino acid substitutions (G400V, M543A, L544A) that ablate binding of estradiol but confers nanomolar sensitivity to estrogen analogues, that is in turn fused to the p65 activation domain of NF-κB (p65).

“Regulate” or “modulate” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, the act of controlling a biological process, or to exert a modifying or controlling influence on a biological or cellular process or pathway.

“Cytotoxic T lymphocyte” (CTL) has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a T lymphocyte that expresses CD8 on the surface thereof (e.g., a CD8 + T cell). In some alternatives, such cells are preferably “memory” T cells (TM cells) that are antigen-experienced. In some alternatives, the host cells comprising the alternative systems provided herein are CTL cells.

“Central memory” T cell (or “TCM”) has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, an antigen experienced CTL that expresses CD62L, CCR-7 and/or CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA, as compared to naïve cells. In some alternatives, central memory cells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO, and/or CD95, and may have decreased expression of CD54RA, as compared to naïve cells. “Effector memory” T cell (or “TEM”) as used herein refers to an antigen experienced T cell that does not express or has decreased expression of CD62L on the surface thereof, as compared to central memory cells, and does not express or has a decreased expression of CD45RA, as compared to naïve cell. In some alternatives, effector memory cells are negative for expression of CD62L and/or CCR7, as compared to naïve cells or central memory cells, and may have variable expression of CD28 and/or CD45RA. In some alternatives, the host cells comprising the alternative systems provided herein are TCM cells. In some alternatives of the host cells provided herein, the host cell is a central memory T cell.

“Naïve” T cells has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a non-antigen experienced T lymphocyte that expresses CD62L and/or CD45RA, and does not express CD45RO−, as compared to central or effector memory cells. In some alternatives, naïve CD8+T lymphocytes are characterized by the expression of phenotypic markers of naïve T cells including CD62L, CCR7, CD28, CD127, and/or CD45RA. In some alternatives, the host cells comprising the alternative systems provided herein are naïve T cells.

“Effector” “T E ” T cells has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, antigen experienced cytotoxic T lymphocyte cells that do not express or have decreased expression of CD62L, CCR7, and/or CD28, and are positive for granzyme B and/or perforin, as compared to central memory or naïve T cells. In some alternatives, the host cells comprising the alternative systems provided herein are effector T cells.

“Enriched” and “depleted” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, amounts of cell types in a mixture and subjecting of the mixture of the cells to a process or step, which results in an increase in the number of the “enriched” type and a decrease in the number of the “depleted” cells. Thus, depending upon the source of the original population of cells subjected to the enriching process, a mixture or composition may contain 60, 70, 80, 90, 95, or 99 percent or more (in number or count) of the “enriched” cells and/or 40, 30, 20, 10, 5 or 1 percent or less (in number or count) of the “depleted” cells. In some alternatives, an in vitro method for preparing a host cell of any one of any one of the alternatives is provided herein. The in vitro method comprises a) providing a system of any one of the alternatives described herein, and b) introducing the system into an isolated T lymphocyte population and expanding each T lymphocyte population in vitro. The process of expanding each T lymphocyte population is a process of enriching the T lymphocyte population.

“Epitope” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a part of an antigen or molecule that is recognized by the immune system including antibodies, T cells, and/or B cells. Epitopes usually have at least 7 amino acids and can be linear or conformational. In some alternatives herein, an antibody or ligand binding domain can be optimized to bind a specific epitope. Techniques for optimizing binding can be appreciated by those skilled in the art. For example, a ligand binding motif can be optimized by point mutation in order to increase binding affinity and/or epitope recognition.

“Isolated,” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, various polypeptides or nucleic acids that have been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide or nucleic acid is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide or nucleic acid, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated can also be used to describe isolation of a subpopulation of cells, such as, for example isolation of subpopulations of CD4+ and/or CD8+T lymphocytes during methods of manufacturing cells for use.

“Intracellular signaling domain” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, all or a portion of one or more domains of a molecule (here the chimeric receptor molecule) that provides for activation of a lymphocyte. Intracellular domains of such molecules mediate a signal by interacting with cellular mediators to result in proliferation, differentiation, activation and other effector functions. In some alternatives, such molecules include all or portions of CD28, CD3, or 4-1BB, or combinations thereof.

“Ligand” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a substance that binds specifically to another substance to form a complex. Examples of ligands include epitopes on antigens, molecules that bind to receptors, substrates, inhibitors, hormones, and/or activators. “Ligand binding domain” as used herein refers to substance or portion of a substance that binds to a ligand. Examples of ligand binding domains include antigen binding portions of antibodies, extracellular domains of receptors, and/or active sites of enzymes. “Operably linked” as used herein refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two proteins coding regions, in the same reading frame.

“Tumor specific molecule,” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, tumor-typical antigens, proteins or molecules expressed by a tumor. In some of the alternatives described herein, the tumor specific molecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof.

A “viral molecule” or “viral specific molecule” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a protein encoded by a viral RNA or DNA, or a molecule that is expressed by a cell in reaction to a viral infection. In some alternatives described herein the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein.

“Percent (%) amino acid sequence identity” with respect to the chimeric receptor polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence for each of the ligand binding domain, spacer, transmembrane domain, and/or the lymphocyte activating domain, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For example, % amino acid sequence identity values generated using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology, 266:460-480 (1996); herein incorporated by reference] uses several search parameters, most of which are set to the default values. Those that are not set to default values (i.e., the adjustable parameters) are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11 and scoring matrix=BLOSUM62. A % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between each or all of the polypeptide amino acid sequence of the reference chimeric receptor sequence and the comparison amino acid sequence of interest as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the polypeptide of interest. In some alternatives, the percent sequence identity of amino acids or nucleic acids are determined by computer software.

“Substantially purified” refers to a molecule that has 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less other molecule types or other cell types. A substantially purified cell also refers to a cell, which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells.

“Not substantially found” when used in reference the presence of a tumor antigen or other molecules on normal cells refers to the percentage of a normal cell type that has the antigen or molecule, and/or the density of the antigen on the cells. In some alternatives, not substantially found means that the antigen or molecule is found on less than 50% of normal cell type and/or at a 50% less density as compared to the amount of cells or antigen found on a tumor cell or other diseased cell.

“T cells” or “T lymphocytes” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, can be T-cells from any mammal, preferably a primate, species, including monkeys, dogs, and/or humans. In some alternatives of the host cells provided herein, the host cells are T cells, wherein the T cells are allogeneic (from the same species but different donor) as the recipient subject; in some alternatives the T cells are autologous (the donor and the recipient are the same); in some alternatives the T cells are syngeneic (the donor and the recipients are different but are identical twins).

“Vector” or “construct” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, nucleic acid used to introduce heterologous nucleic acids into a cell that has regulatory elements to provide expression of the heterologous nucleic acids in the cell. Vectors include but are not limited to plasmid, minicircles, yeast, transposons, and viral genomes. In some alternatives, the vectors are plasmid, minicircles, yeast, transposons, or viral genomes. In some alternatives, the transposons are piggy bac transposons. In some alternatives of the systems provided herein, the first and/or second nucleic acid reside on a vector. In some alternatives, the vector is a viral vector.

“Transgene” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, a gene or genetic material that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. In some alternatives described herein, a method is provided for inducible expression of a transgene in cells, the system comprising: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes the transgene; and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by a drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the transgene encodes a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity. In some alternatives, the transgene encodes a protein that modulates T cells.

A viral vector can be used to deliver genetic material into a cell. Delivery of the vector can be performed by transduction and the technique is known to those skilled in the art. In some alternatives herein, a vector for introducing the nucleic acid into a cell is an adenovirus viral vector or a lentivirus viral vector.

“Minicircles,” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, small circular plasmid derivatives that have been freed or liberated from prokaryotic vector components. Minicircles can serve as an expression vector, where they have been applied as transgene carriers for the genetic modification of mammalian cells, with the advantage that, since they contain no bacterial DNA sequences, they are less likely to be perceived as foreign and destroyed. As such, typical transgene delivery methods involve plasmids, which contain foreign DNA. The smaller size of minicircles also extends their cloning capacity and facilitates their delivery into cells. Without being limiting, the preparation of minicircles can follow a two-step procedure, which can involve production of a parental plasmid (bacterial plasmid with eukaryotic inserts) in E. coli and induction of a site-specific recombinase at the end of this process but still in bacteria. These steps can be followed by the excision of prokaryotic vector parts via two recombinase-target sequences at both ends of the insert and recovery of the resulting minicircle (vehicle for the highly efficient modification of the recipient cell) and the miniplasmid by capillary gel electrophoresis (CGE).

The purified minicircle can be transferred into the recipient cell by transfection, by electroporation, or by other methods known to those skilled in the art. Conventional minicircles can lack an origin of replication, so they cannot replicate within the target cells and the encoded genes will disappear as the cell divides (which can be either an advantage or disadvantage depending on whether the application demands persistent or transient expression). Some alternatives utilize a gene delivery polynucleotide for stable insertion of a nucleic acid into a gene, wherein the nucleic acid for insertion is flanked by inverted terminal repeat gene sequences in the gene delivery polynucleotide, and wherein the gene delivery polynucleotide is selectable. In some alternatives, the gene delivery polynucleotide is a minicircle.

Plasmids containing transposon systems such as Sleeping Beauty (SB) or piggyBac offer a non-viral approach for stably introducing genes into T-cells. Recently, the piggyBac system was used to produce stably-transfected mammalian cells expressing multiple transgenes of interest by delivery of multiple transposons. The SB system, first reactivated for mammalian cell use by Ivics and coworkers, has been used as the gene delivery modality in clinical trials of T-cell immunotherapy. Gene integration by SB has weaker preference for transcriptional units and their regulatory sequences compared to the γ-retroviral and lentiviral vectors and is therefore considered to be safer. In some alternatives described herein, genetic modification by minicircles comprising the Sleeping Beauty system are contemplated. In some alternatives described herein, genetic modification by minicircles comprising the piggyBac system are contemplated. In some alternatives described herein, genetic modification by minicircles comprising the Sleeping Beauty system are contemplated.

“Apoptosis” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, the process of programmed cell death (PCD) that can occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. In apoptosis, a cell initiates intracellular apoptotic signaling in response to stress, which can bring about cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia and increased intracellular calcium concentration, for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, can also help regulate apoptosis.

Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows apoptotic signals to cause cell death, or the process to be stopped, should the cell no longer need to die. Several proteins are involved, but two main methods of regulation have been identified: targeting mitochondria functionality, or directly transducing the signal via adaptor proteins to the apoptotic mechanisms. Another extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain.

Apoptosis can be regulated by many factors. These factors can include but are not limited to genes that can express IL-2, IL-7, IL-15, Chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. IL-15 regulates T and natural killer cell activation and proliferation. In rodent lymphocytes, IL-15 was shown to prevent apoptosis by inducing an apoptosis inhibitor, BCL2L1/BCL-X(L). In humans with celiac disease, IL-15 similarly suppresses apoptosis in T-lymphocytes by inducing Bcl-2 and/or BCL-xL. Bcl-2 (B-cell lymphoma 2), encoded in humans by the BCL2 gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inducing (pro-apoptotic) it or inhibiting it (anti-apoptotic). Bcl-2 is specifically considered as an important anti-apoptotic protein and is, thus classified as an oncogene. Protein kinase B (PKB), also known as Akt, is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. In some alternatives, a system for inducible expression of a gene that regulates a T cell function is provided, wherein the system comprises: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the polypeptide that regulates apoptosis or modulates checkpoint signaling comprises IL-2, IL-7, IL-15, chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2 or PD-1CD28 chimeras.

“Checkpoint signaling” has its plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, blocking the cell cycle at specific transition points, and/or checkpoints to ensure that the events of the cell cycle take place in the correct order. Checkpoint signaling can also be activated. By way of example and not of limitation, checkpoint signaling can occur by damage to the DNA so that the cell cycle does not have to proceed until the damage is repaired. “Cell cycle checkpoints” are control mechanisms in eukaryotic cells which ensure proper division of the cell. Each checkpoint serves as a potential halting point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring when favorable conditions are met. Currently, there are three known checkpoints: the G1 checkpoint, also known as the restriction or start checkpoint; the G2/M checkpoint; and the metaphase checkpoint, also known as the spindle checkpoint. The biochemical pathways that restrain cell cycle transition and/or induce cell death after stress are known as cell cycle checkpoints. These checkpoints maintain the fidelity of DNA replication, repair, and division. Polypeptides that can regulate checkpoint signaling can include but are not limited to p53, p107, p130, and transcriptional repressor Rb. In some alternatives of the systems provided herein, the polypeptide that modulates checkpoint signaling comprises p53, p107, p130 or transcriptional repressor Rb.

“Negative checkpoint regulators” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, factors that can restrict the ability of T-cell responses to effectively attack tumors. They are also referred to as negative checkpoint signaling. In some alternatives, a system for inducible expression of a transgene is provided. In some alternatives, the transgene encodes a chimeric antigen receptor. In some alternatives, a system for inducible expression of chimeric antigen receptor in cells is provided. In some alternatives of the systems for inducible expression of a gene that regulates a T cell function, the polypeptide that modulates checkpoint signaling is a negative checkpoint regulator comprising PD-1, VISTA, LAG-3 or TIM3. In some alternatives, the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3.

In another example, cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins can also play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression. In some alternatives, a system for inducible expression of a transgene is provided. In some alternatives, the transgene encodes a chimeric antigen receptor. In some alternatives, a system for inducible expression of a transgene is provided. In some alternatives, the transgene encodes a chimeric antigen receptor. In some alternatives, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, is provided. In some alternatives of the system for inducible expression of a gene that regulates a T cell function, the polypeptide modulates checkpoint signaling. In some alternatives, the polypeptide that modulates checkpoint signaling inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3.

“T cell precursors” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, lymphoid precursor cells that can migrate to the thymus and become T cell precursors, which do not express a T cell receptor. All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors (lymphoid progenitor cells) from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classed as double-negative (CD4 − CD8 − ) cells. As they progress through their development, they become double-positive thymocytes (CD4 + CD8 + ), and finally mature to single-positive (CD4 + CD8 − or CD4 − CD8 + ) thymocytes that are then released from the thymus to peripheral tissues.

About 98% of thymocytes die during the development processes in the thymus by failing either positive selection or negative selection, whereas the other 2% survive and leave the thymus to become mature immunocompetent T cells.

The double negative (DN) stage of the precursor T cell is focused on producing a functional β-chain whereas the double positive (DP) stage is focused on producing a functional α-chain, ultimately producing a functional αβ T cell receptor. As the developing thymocyte progresses through the four DN stages (DN1, DN2, DN3, and DN4), the T cell expresses an invariant α-chain but rearranges the β-chain locus. If the rearranged 3-chain successfully pairs with the invariant α-chain, signals are produced which cease rearrangement of the β-chain (and silence the alternate allele) and result in proliferation of the cell. Although these signals require this pre-TCR at the cell surface, they are dependent on ligand binding to the pre-TCR. These thymocytes will then express both CD4 and CD8 and progresses to the double positive (DP) stage where selection of the α-chain takes place. If a rearranged β-chain does not lead to any signaling (e.g. as a result of an inability to pair with the invariant α-chain), the cell may die by neglect (lack of signaling).

“Hematopoietic stem cells” or “HSC” have their plain and ordinary meaning when read in light of the specification, and may include but is not limited to, for example, precursor cells that can give rise to myeloid cells such as, for example, macrophages, monocytes, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells and lymphoid lineages (such as, for example, T-cells, B-cells, NK-cells). HSCs have a heterogeneous population in which three classes of stem cells exist, which are distinguished by their ratio of lymphoid to myeloid progeny in the blood (L/M). In some alternatives described herein a host cell comprising a system of any one of the alternatives described herein is provided. In some alternatives of the host cells provided herein, the host cells are hematopoietic stem cells.

This disclosure provides for a system that has an inducible component for expression of transgenes and a constitutive component for expression of transgenes. The system can be tailored to provide for regulated expression of one or more transgenes to provide for functional characteristics in the transduced cells.

Although, T cell (CAR-T) adoptive therapy clinical trials (chimeric antigen receptor expressing T cells) can demonstrate potent anti-tumor activity, significant toxicities can arise, for example, by engraftment-induced cytokine storm (cytokine release syndrome), tumor lysis syndromes (TLS) and ongoing B cell cytopenias, each of which are attributable to unregulated functional outputs of constitutively expressed CARs. Such toxicities can threaten to limit the applicability of CAR-T cell adoptive therapy, in some patients. Clinical trials using transgene-modified adoptive T cell immunotherapies have tested T cells that can constitutively express the transgene, or are always in the “ON” state, contributing in large part to transgene associated side-effects. Methods to eliminate CAR-T cells such as suicide gene-mediated elimination of CAR-T cells, for example, can ameliorate such toxicities; however, this approach risks premature attenuation of anti-tumor activity and can significantly impact curative potential.

Current small molecule-regulated transgene expression technologies rely on a variety of drug inputs, which include, for example, macrolides, ecdysones and rapamycin analogs. However, use of these drugs may be limited due to the toxic off target effects, unfavorable biodistribution and pharmacodynamics profiles, limited output dynamic range, and/or limited availability as FDA-approved commercially available pharmaceuticals. Furthermore, many of these systems use chimeric transcriptional regulators built from xenogeneic components, thus introducing the complication of immunogenicity when applying these systems to human therapeutics.

Small molecule regulated transgene expression is a highly sought technology in biomedicine, particularly in CAR T cell immunotherapy. Competing systems typically use chimeric transcription factors composed of xenogeneic domains and drug inducers that are not currently FDA approved.

As such, there is a need to identify methods for controlling chimeric receptor expression that are important for therapeutic activity and cell populations that will provide enhanced survival and efficacy in vivo while minimizing adverse side effects of the drugs that induce chimeric antigen receptor expression. There is also a need for expression systems and methods for modulating cells for use in cell therapy, such as for modulating expression of recombinant antigen receptors such as CARs and/or other molecules expressed by such cells, such as to improve therapeutic activity, enhance survival and/or efficacy in vivo and/or minimize adverse side effects. Described herein, are methods that surprisingly led to enhanced survival and efficacy of the treatments in vivo, with minimized adverse side effects.

In some alternatives, a transgene under the control of the inducible promoter, is provided. In some alternatives, the transgene encodes a chimeric antigen receptor (CAR). The inducible promoter provides for the capacity to terminate CAR expression in cells while providing for reactivation of the cells at a later date (e.g. in the case of relapse). In addition, the cycling of CAR T cells through on and off periods can minimize exhaustion and/or energy due to chronic stimulation of the T cell receptors.

In some alternatives, a gene is under the control of the inducible promoter. In some alternatives, the gene encodes a chimeric cytokine receptor.

The design of the vectors also provides for additional transgenes that can enhance one or more functional characteristics of transduced cells, such as enhanced tumor potency, survival and proliferation of transduced cells. In some alternatives, these transgenes are under the control of an inducible promoter. Such transgenes include, without limitation, genes that promote survival and proliferation, genes that prevent apoptosis, and genes that regulate checkpoint signaling. Such genes include genes encoding IL-2, IL-15, chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. In some alternatives, the transgenes are genes encoding IL-2, IL-7, IL-15, chemokine receptors, Bcl2, chimeric cytokine receptors, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. In some alternatives, the gene that modulates checkpoint signaling, encodes a polypeptide that inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3. In some alternatives, the gene is under a control of an inducible promoter. In some alternatives, the gene under control of an inducible promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 88 (CCR(CD122) nucleotide sequence: atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagattgtgatattgaaggtaaagatg gcaaacaatatgagagtgttctaatggtcagcatcgatcaattattggacagcatgaaagaaattggtagcaattgcctgaataatgaatt taacttttttaaaagacatatctgtgatgctaataaggaaggtatgtttttattccgtgctgctcgcaagttgaggcaatttcttaaaatgaata gcactggtgattttgatctccacttattaaaagtttcagaaggcacaacaatactgttgaactgcactggccaggttaaaggaagaaaac cagctgccctgggtgaagcccaaccaacaaagagtttggaagaaaataaatctttaaaggaacagaaaaaactgaatgacttgtgttt cctaaagagactattacaagagataaaaacttgttggaataaaattttgatgggcactaaagaacacggaggcggtgggagcggagg cggtgggagcatgacaattctaggtacaacttttggcatggttttttctttacttcaagtcgtttctggagaaagtggctatgctcaaaatgg agacttggaagatgcagaactggatgactactcattctcatgctatagccagttggaagtgaatggatcgcagcactcactgacctgtg cttttgaggacccagatgtcaacatcaccaatctggaatttgaaatatgtggggccctcgtggaggtaaagtgcctgaatttcaggaaa ctacaagagatatatttcatcgagacaaagaaattcttactgattggaaagagcaatatatgtgtgaaggttggagaaaagagtctaacc tgcaaaaaaatagacctaaccactatagttaaacctgaggctccttttgacctgagtgtcatctatcgggaaggagccaatgactttgtg gtgacatttaatacatcacacttgcaaaagaagtatgtaaaagttttaatgcacgatgtagcttaccgccaggaaaaggatgaaaacaa atggacgcatgtgaatttatccagcacaaaactgacactcctgcagagaaagctccaaccggcagcaatgtatgagattaaagttcga tccatccctgatcactattttaaaggcttctggagtgaatggagtccaagttattacttcagaactccagagatcaataatagctcagggg agatggatcctatcttactaaccatcagcattttgagttttttctctgtcgctctgttggtcatcttggcctgtgtgttatggaactgcaggaac accgggccatggctgaagaaggtcctgaagtgtaacaccccagacccctcgaagttcttttcccagctgagctcagagcatggagga gacgtccagaagtggctctcttcgcccttcccctcatcgtccttcagccctggcggcctggcacctgagatctcgccactagaagtgct ggagagggacaaggtgacgcagctgctcctgcagcaggacaaggtgcctgagcccgcatccttaagcagcaaccactcgctgacc agctgcttcaccaaccagggttacttcttcttccacctcccggatgccttggagatagaggcctgccaggtgtactttacttacgacccct actcagaggaagaccctgatgagggtgtggccggggcacccacagggtcttccccccaacccctgcagcctctgtcaggggagga cgacgcctactgcaccttcccctccagggatgacctgctgctcttctcccccagtctcctcggtggccccagccccccaagcactgcc cctgggggcagtggggccggtgaagagaggatgcccccttctttgcaagaaagagtccccagagactgggacccccagcccctgg ggcctcccaccccaggagtcccagacctggtggattttcagccaccccctgagctggtgctgcgagaggctggggaggaggtccct gacgctggccccagggagggagtcagtttcccctggtccaggcctcctgggcagggggagttcagggcccttaatgctcgcctgcc cctgaacactgatgcctacttgtccctccaagaactccagggtcaggacccaactcacttggtg). In some alternatives, the sequence under control of an inducible promoter encodes for an amino acid sequence set forth in SEQ ID NO: 89 (CCR(CD122) amino acid sequence: MLLLVTSLLLCELPHPAFLLIPDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLN NEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCT GQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGT KEHGGGGSGGGGSMTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCY SQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLI GKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVIYREGANDFVVTFNTSHLQKKYV KVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGF WSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWNCRNTGPWLKK VLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQ LLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPD EGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGE ERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGV SFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 89.

In some alternatives, the gene under control of an inducible promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 90 (PD1ECD-IFNalpha nucleotide sequence nucleotide sequence: atgcagatccctcaggccccttggcctgtcgtgtgggctgtgctgcagctgggatggcggcctggctggtttctggacagccccgac agaccctggaacccccctacattttcccctgccctgctggtcgtgaccgagggcgacaatgccaccttcacctgtagcttcagcaaca ccagcgagagcttcgtgctgaactggtacagaatgagccccagcaaccagaccgacaagctggccgccttccccgaggatagatct cagcccggccaggactgccggttcagagtgacccagctgcccaacggccgggacttccacatgtctgtcgtgcgggccagacgga acgacagcggcacatatctgtgcggcgccatcagcctggcccccaaggcccagatcaaagagagcctgagagccgagctgagag tgaccgagagaagggccgaagtgcctaccgcccaccctagcccatctccaagacctgccggccagttccagacactcgtgggcgg aggatgcgacctgcctcagacacacagcctgggcagcagacggaccctgatgctgctggcccagatgcggaagatcagcctgttc agctgcctgaaggaccggcacgacttcggcttccctcaggaagagttcggcaaccagtttcagaaggccgagacaatccccgtgct gcacgagatgatccagcagatcttcaacctgttctccaccaaggacagcagcgccgcctgggacgagacactgctggacaagttcta caccgagctgtaccagcagctgaatgacctggaagcctgcgtgatccagggcgtgggcgtgacagagacacccctgatgaaggaa gatagcatcctggccgtgcgcaagtacttccagcggatcaccctgtacctgaaagagaagaagtacagcccctgcgcctgggaggt cgtgcgcgccgagatcatgagaagcttcagcctgagcaccaacctgcaggaaagcctgcggagcaaagaa). In some alternatives, the sequence under control of an inducible promoter encodes the amino acid sequence set forth in SEQ ID NO: 91 (PD1ECD-IFNalpha amino acid sequence: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVR ARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV GGGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIP VLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLM KEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 91.

In some alternatives, the gene under control of an inducible promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 92 (STAT5a nucleotide sequence:

atggcgggctggatccaggcccagcagctgcagggagacgcgctgcgccag

atgcaggtgctgtacggccagcacttccccatcgaggtccggcactacttg

gcccagtggattgagagccagccatgggatgccattgacttggacaatccc

caggacagagcccaagccacccagctcctggagggcctggtgcaggagctg

cagaagaaggcggagcaccaggtgggggaagatgggtttttactgaagatc

aagctggggcactacgccacgcagctccagaaaacatatgaccgctgcccc

ctggagctggtccgctgcatccggcacattctgtacaatgaacagaggctg

gtccgagaagccaacaattgcagctctccggctgggatcctggttgacgcc

atgtcccagaagcaccttcagatcaaccagacatttgaggagctgcgactg

gtcacgcaggacacagagaatgagctgaagaaactgcagcagactcaggag

tacttcatcatccagtaccaggagagcctgaggatccaagctcagtttgcc

cagctggcccagctgagcccccaggagcgtctgagccgggagacggccctc

cagcagaagcaggtgtctctggaggcctggttgcagcgtgaggcacagaca

ctgcagcagtaccgcgtggagctggccgagaagcaccagaagaccctgcag

ctgctgcggaagcagcagaccatcatcctggatgacgagctgatccagtgg

aagcggcggcagcagctggccgggaacggcgggccccccgagggcagcctg

gacgtgctacagtcctggtgtgagaagttggccgagatcatctggcagaac

cggcagcagatccgcagggctgagcacctctgccagcagctgcccatcccc

ggcccagtggaggagatgctggccgaggtcaacgccaccatcacggacatt

atctcagccctggtgaccagcacattcatcattgagaagcagcctcctcag

gtcctgaagacccagaccaagtttgcagccaccgtacgcctgctggtgggc

gggaagctgaacgtgcacatgaatcccccccaggtgaaggccaccatcatc

agtgagcagcaggccaagtctctgcttaaaaatgagaacacccgcaacgag

tgcagtggtgagatcctgaacaactgctgcgtgatggagtaccaccaagcc

acgggcaccctcagtgcccacttcaggaacatgtcactgaagaggatcaag

cgtgctgaccggcggggtgcagagtccgtgacagaggagaagttcacagtc

ctgtttgagtctcagttcagtgttggcagcaatgagcttgtgttccaggtg

aagactctgtccctacctgtggttgtcatcgtccacggcagccaggaccac

aatgccacggctactgtgctgtgggacaatgcctttgctgagccgggcagg

gtgccatttgccgtgcctgacaaagtgctgtggccgcagctgtgtgaggcg

ctcaacatgaaattcaaggccgaagtgcagagcaaccggggcctgaccaag

gagaacctcgtgttcctggcgcagaaactgttcaacaacagcagcagccac

ctggaggactacagtggcctgtccgtgtcctggtcccagttcaacagggag

aacttgccgggctggaactacaccttctggcagtggtttgacggggtgatg

gaggtgttgaagaagcaccacaagccccactggaatgatggggccatccta

ggttttgtgaataagcaacaggcccacgacctgctcatcaacaagcccgac

gggaccttcttgttgcgctttagtgactcagaaatcgggggcatcaccatc

gcctggaagtttgattccccggaacgcaacctgtggaacctgaaaccattc

accacgcgggatttctccatcaggtccctggctgaccggctgggggacctg

agctatctcatctatgtgtttcctgaccgccccaaggatgaggtcttctcc

aagtactacactcctgtgctggctaaagctgttgatggatatgtgaaacca

cagatcaagcaagtggtccctgagtttgtgaatgcatctgcagatgctggg

ggcagcagcgccacgtacatggaccaggccccctccccagctgtgtgcccc

caggctccctataacatgtacccacagaaccctgaccatgtactcgatcag

gatggagaattcgacctggatgagaccatggatgtggccaggcacgtggag

gaactcttacgccgaccaatggacagtcttgactcccgcctctcgccccct

gccggtcttttcacctctgccagaggctccctctca).

In some alternatives, the sequence under control of an inducible promoter encodes for the amino acid sequence set forth in SEQ ID NO: 93 (STAT5a amino acid sequence: MAGWIQAQQLQGDALRQMQVLYGQHFPIEVRHYLAQWIESQPWDAIDLDNPQDRA QATQLLEGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQKTYDRCPLELVRCIRHI LYNEQRLVREANNCSSPAGILVDAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQ EYFIIQYQESLRIQAQFAQLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYR VELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKL AEIIWQNRQQIRRAEHLCQQLPIPGPVEEMLAEVNATITDIISALVTSTFIIEKQPPQVL KTQTKFAATVRLLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNECSGEILNN CCVMEYHQATGTLSAHFRNMSLKRIKRADRRGAESVTEEKFTVLFESQFSVGSNELV FQVKTLSLPVVVIVHGSQDHNATATVLWDNAFAEPGRVPFAVPDKVLWPQLCEALN MKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGWNY TFWQWFDGVMEVLKKHHKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSEI GGITIAWKFDSPERNLWNLKPFTTRDFSIRSLADRLGDLSYLIYVFPDRPKDEVFSKY YTPVLAKAVDGYVKPQIKQVVPEFVNASADAGGSSATYMDQAPSPAVCPQAPYNM YPQNPDHVLDQDGEFDLDETMDVARHVEELLRRPMDSLDSRLSPPAGLFTSARGSLS). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 93.

In some alternatives, the gene under control of an inducible promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 94 (Constitutively active-STAT5a nucleotide sequence:

atggcgggctggatccaggcccagcagctgcagggagacgcgctgcgccag

atgcaggtgctgtacggccagcacttccccatcgaggtccggcactacttg

gcccagtggattgagagccagccatgggatgccattgacttggacaatccc

caggacagagcccaagccacccagctcctggagggcctggtgcaggagctg

cagaagaaggcggagcaccaggtgggggaagatgggtttttactgaagatc

aagctggggcactacgccacgcagctccagaaaacatatgaccgctgcccc

ctggagctggtccgctgcatccggcacattctgtacaatgaacagaggctg

gtccgagaagccaacaattgcagctctccggctgggatcctggttgacgcc

atgtcccagaagcaccttcagatcaaccagacatttgaggagctgcgactg

gtcacgcaggacacagagaatgagctgaagaaactgcagcagactcaggag

tacttcatcatccagtaccaggagagcctgaggatccaagctcagtttgcc

cagctggcccagctgagcccccaggagcgtctgagccgggagacggccctc

cagcagaagcaggtgtctctggaggcctggttgcagcgtgaggcacagaca

ctgcagcagtaccgcgtggagctggccgagaagcaccagaagaccctgcag

ctgctgcggaagcagcagaccatcatcctggatgacgagctgatccagtgg

aagcggcggcagcagctggccgggaacggcgggccccccgagggcagcctg

gacgtgctacagtcctggtgtgagaagttggccgagatcatctggcagaac

cggcagcagatccgcagggctgagcgcctctgccagcagctgcccatcccc

ggcccagtggaggagatgctggccgaggtcaacgccaccatcacggacatt

atctcagccctggtgaccagcacattcatcattgagaagcagcctcctcag

gtcctgaagacccagaccaagtttgcagccaccgtacgcctgctggtgggc

gggaagctgaacgtgcacatgaatcccccccaggtgaaggccaccatcatc

agtgagcagcaggccaagtctctgcttaaaaatgagaacacccgcaacgag

tgcagtggtgagatcctgaacaactgctgcgtgatggagtaccaccaagcc

acgggcaccctcagtgcccacttcaggaacatgtcactgaagaggatcaag

cgtgctgaccggcggggtgcagagtccgtgacagaggagaagttcacagtc

ctgtttgagtctcagttcagtgttggcagcaatgagcttgtgttccaggtg

aagactctgtccctacctgtggttgtcatcgtccacggcagccaggaccac

aatgccacggctactgtgctgtgggacaatgcctttgctgagccgggcagg

gtgccatttgccgtgcctgacaaagtgctgtggccgcagctgtgtgaggcg

ctcaacatgaaattcaaggccgaagtgcagagcaaccggggcctgaccaag

gagaacctcgtgttcctggcgcagaaactgttcaacaacagcagcagccac

ctggaggactacagtggcctgtccgtgtcctggtcccagttcaacagggag

aacttgccgggctggaactacaccttctggcagtggtttgacggggtgatg

gaggtgttgaagaagcaccacaagccccactggaatgatggggccatccta

ggttttgtgaataagcaacaggcccacgacctgctcatcaacaagcccgac

gggaccttcttgttgcgctttagtgactcagaaatcgggggcatcaccatc

gcctggaagtttgattccccggaacgcaacctgtggaacctgaaaccattc

accacgcgggatttctccatcaggtccctggctgaccggctgggggacctg

agctatctcatctatgtgrncctgaccgccccaaggatgaggtcttctcca

agtactacactcctgtgctggctaaagctgttgatggatatgtgaaaccac

agatcaagcaagtggtccctgagrngtgaatgcatttgcagatgctggggg

cagcagcgccacgtacatggaccaggccccctccccagctgtgtgccccca

ggctccctataacatgtacccacagaaccctgaccatgtactcgatcagga

tggagaattcgacctggatgagaccatggatgtggccaggcacgtggagga

actcttacgccgaccaatggacagtcttgactcccgcctctcgccccctgc

cggtcttttcacctctgccagaggctccctctca).

In some alternatives, the sequence under control of an inducible promoter encodes for the amino acid sequence set forth in SEQ ID NO: 95 (Constitutively active-STAT5a amino acid sequence: MAGWIQAQQLQGDALRQMQVLYGQHFPIEVRHYLAQWIESQPWDAIDLDNPQDRA QATQLLEGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQKTYDRCPLELVRCIRHI LYNEQRLVREANNCSSPAGILVDAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQ EYFIIQYQESLRIQAQFAQLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYR VELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKL AEIIWQNRQQIRRAERLCQQLPIPGPVEEMLAEVNATITDIISALVTSTFIIEKQPPQVL KTQTKFAATVRLLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNECSGEILNN CCVMEYHQATGTLSAHFRNMSLKRIKRADRRGAESVTEEKFTVLFESQFSVGSNELV FQVKTLSLPVVVIVHGSQDHNATATVLWDNAFAEPGRVPFAVPDKVLWPQLCEALN MKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGWNY TFWQWFDGVMEVLKKHHKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSEI GGITIAWKFDSPERNLWNLKPFTTRDFSIRSLADRLGDLSYLIYVFPDRPKDEVFSKY YTPVLAKAVDGYVKPQIKQVVPEFVNAFADAGGSSATYMDQAPSPAVCPQAPYNM YPQNPDHVLDQDGEFDLDETMDVARHVEELLRRPMDSLDSRLSPPAGLFTSARGSLS). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 95.

In some alternatives, the gene under control of an inducible promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 96 (Dominant negative TGFbRII nucleotide sequence:

atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgtgg

acgcgtatcgccagcacgatcccaccgcacgttcagaagtcggttaataac

gacatgatagtcactgacaacaacggtgcagtcaagtttccacaactgtgt

aaattttgtgatgtgagattttccacctgtgacaaccagaaatcctgcatg

agcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtg

gctgtatggagaaagaatgacgagaacataacactagagacagtttgccat

gaccccaagctcccctaccatgactttattctggaagatgctgcttctcca

aagtgcattatgaaggaaaaaaaaaagcctggtgagactttcttcatgtgt

tcctgtagctctgatgagtgcaatgacaacatcatcttctcagaagaatat

aacaccagcaatcctgacttgttgctagtcatatttcaagtgacaggcatc

agcctcctgccaccactgggagttgccatatctgtcatcatcatcttctac

tgctaccgcgttaaccggcagcagaagctgagttcaacctgggaaaccggc

aagacgcggaagctcatggagttcagcgagcactgtgccatcatcctggaa

gatgaccgctctgacatcagctccacgtgtgccaacaacatcaaccacaac

acagagctgctgcccattgagctggacaccctggtggggaaaggtcgcttt

gctgaggtctataaggccaagctgaagcagaacacttcagagcagtttgag

acagtggcagtcaagatcttt).

In some alternatives, the sequence under control of an inducible promoter encodes for the amino acid sequence set forth in SEQ ID NO: 97 (Dominant negative TGFbRII amino acid sequence: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV RFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLP PLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCAN NINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIF). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 97.

In some alternatives, the gene under control of an inducible promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 98 (Dominant negative SHP1 nucleotide sequence:

atggtgaggtggtttcaccgagacctcagtgggctggatgcagagaccctg

ctcaagggccgaggtgtccacggtagcttcctggctcggcccagtcgcaag

aaccagggtgacttctcgctctccgtcagggtgggggatcaggtgacccat

attcggatccagaactcaggggatttctatgacctgtatggaggggagaag

tttgcgactctgacagagctggtggagtactacactcagcagcagggtgtc

ctgcaggaccgcgacggcaccatcatccacctcaagtacccgctgaactgc

tccgatcccactagtgagaggtggtaccatggccacatgtctggcgggcag

gcagagacgctgctgcaggccaagggcgagccctggacgtttcttgtgcgt

gagagcctcagccagcctggagacttcgtgctttctgtgctcagtgaccag

cccaaggctggcccaggctccccgctcagggtcacccacatcaaggtcatg

tgcgagggtggacgctacacagtgggtggtttggagaccttcgacagcctc

acggacctggtggagcatttcaagaagacggggattgaggaggcctcaggc

gcctttgtctacctgcggcagccgtactatgccacgagggtgaatgcggct

gacattgagaaccgagtgttggaactgaacaagaagcaggagtccgaggat

acagccaaggctggcttctgggaggagtttgagagtttgcagaagcaggag

gtgaagaacttgcaccagcgtctggaagggcagcggccagagaacaagggc

aagaaccgctacaagaacattctcccctttgaccacagccgagtgatcctg

cagggacgggacagtaacatccccgggtccgactacatcaatgccaactac

atcaagaaccagctgctaggccctgatgagaacgctaagacctacatcgcc

agccagggctgtctggaggccacggtcaatgacttctggcagatggcgtgg

caggagaacagccgtgtcatcgtcatgaccacccgagaggtggagaaaggc

cggaacaaatgcgtcccatactggcccgaggtgggcatgcagcgtgcttat

gggccctactctgtgaccaactgcggggagcatgacacaaccgaatacaaa

ctccgtaccttacaggtctccccgctggacaatggagacctgattcgggag

atctggcattaccagtacctgagctggcccgaccatggggtccccagtgag

cctgggggtgtcctcagcttcctggaccagatcaaccagcggcaggaaagt

ctgcctcacgcagggcccatcatcgtgcactccagcgccggcatcggccgc

acaggcaccatcattgtcatcgacatgctcatggagaacatctccaccaag

ggcctggactgtgacattgacatccagaagaccatccagatggtgcgggcg

cagcgctcgggcatggtgcagacggaggcgcagtacaagttcatctacgtg

gccatcgcccagttcattgaaaccactaagaagaagctggaggtcctgcag

tcgcagaagggccaggagtcggagtacgggaacatcacctatcccccagcc

atgaagaatgcccatgccaaggcctcccgcacctcgtccaaacacaaggag

gatgtgtatgagaacctgcacactaagaacaagagggaggagaaagtgaag

aagcagcggtcagcagacaaggagaagagcaagggttccctcaagaggaag

tga).

In some alternatives, the sequence under control of an inducible promoter encodes for the amino acid sequence set forth in SEQ ID NO: 99: Dominant negative SHP1 amino acid sequence: MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQN SGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYH GHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKV MCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIEN RVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILP FDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQM AWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLR TLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIV HSSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIY VAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYEN LHTKNKREEKVKKQRSADKEKSKGSLKRK). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 99.

In some alternatives, the gene under control of an inducible promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 100 (PD1TM-MyD88 nucleotide sequence:

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagca ttcctcctgatcccaggcggaggagtgggagtcgtgggcggactgctggga tctctggtgctgctcgtgtgggtgctggccgtgattggcggaggaatggct gctggcggacctggcgctggatctgctgctcctgtgtctagcaccagcagc ctgcctctggccgccctgaatatgagagtgcggcggagactgagcctgttc ctgaacgtgcggacacaggtggccgccgattggacagctctggccgaggaa atggacttcgagtacctggaaatccggcagctggaaacccaggccgaccct acaggacgcctgctggatgcttggcagggcagaccaggcgcttctgtgggg agactgctggaactgctgaccaagctgggccgggacgacgtgctgctggaa ctgggccctagcatcgaagaggactgccagaagtacatcctgaagcagcag caggaagaggccgagaagcctctgcaggtggcagccgtggatagcagcgtg ccaagaacagctgagctggccggaatcaccaccctggacgatcctctgggc cacatgcccgagagattcgacgccttcatctgctactgccccagcgacatc cagttcgtgcaggaaatgatcagacagctggaacagaccaactaccggctg aagctgtgcgtgtccgaccgggatgtgctgcctggcacctgtgtgtggtct atcgccagcgagctgatcgagaagcggtgcagacggatggtcgtggtggtg tccgacgactacctgcagtccaaagagtgcgacttccagaccaagttcgcc ctgagcctgagccctggcgcccaccagaagagactgatccccatcaagtac aaggccatgaagaaagagttccccagcatcctgcggttcatcaccgtgtgc gactacaccaacccctgcaccaagtcctggttctggaccagactggccaag gccctgtctctgcc).

In some alternatives, the gene under control of an inducible promoter encodes for an amino acid sequence set forth in SEQ ID NO: 101 (PD1TM-MyD88 amino acid sequence: MLLLVTSLLLCELPHPAFLLIPGGGVGVVGGLLGSLVLLVWVLAVIGGGMAAGGPG AGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQL ETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQ QEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMI RQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDF QTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKAL SL). In some alternatives, the sequence under control of an inducible promoter is encoded by a nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 101.

The disclosure provides for a system comprising first and second nucleic acids, and vectors and host cells including such nucleic acids. Each of the first and second nucleic acids comprise a number of modular components that can be excised and replaced with other components in order to customize the system for a specific target cell. In some alternatives, the first nucleic acid includes an inducible promoter for control of the expression of the genes (e.g., polynucleotide coding for a chimeric antigen receptor) in an on and off manner as needed, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In other alternatives, the second nucleic acid comprises a constitutive promoter that provides for expression of a transcriptional activator. In some alternatives, the gene encodes for a chimeric antigen receptor. In some alternatives, the gene encodes for a chimeric antigen receptor AND a transcriptional activator (encoded by one plasmid).

Inducible System.

The disclosure provides a system useful for providing regulated expression of transgenes in cells, such as mammalian cells. Such transgenes include, without limitation, T cell receptors, affinity matured T cell receptors, chimeric antigen receptors, chemokine receptors, chimeric chemokine receptors, cytokines, genes that inhibit apoptosis, and/or genes that modulate checkpoint signaling. In some alternatives, the polypeptide that modulates checkpoint signaling inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3. In some alternatives, the system contains a number of modular components that provide for easy substitution of elements of the nucleic acid. In some alternatives of the system, the system provides regulation of expression of transgenes in cells, such as mammalian cells, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the transgenes code for T cell receptors, affinity matured T cell receptors, chimeric antigen receptors, chemokine receptors, chimeric chemokine receptors, cytokines, genes that regulate apoptosis, and/or genes that modulate checkpoint signaling. In some alternatives, the gene that modulates checkpoint signaling encodes a polypeptide inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3.

In some alternatives, a system for inducible expression of a transgene is provided. In some alternatives, the transgene encodes a chimeric antigen receptor. In some alternatives, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, is provided. In some alternatives, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, comprises: a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, viral molecule, or any other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain, and a fourth polynucleotide, which encodes an intracellular signaling domain, and a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the second promoter is constitutive or inducible.

In some alternatives, a polynucleotide coding for a chimeric antigen receptor comprises a polynucleotide coding for a ligand binding domain, wherein the target molecule is a tumor specific antigen, a polynucleotide coding for a polypeptide spacer wherein the spacer is optionally optimized; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for an intracellular signaling domain. In some alternatives, an expression vector comprises a first and/or second nucleic acid, as described herein. Polypeptides encoded by all of or a portion of the chimeric receptor nucleic acids are also included herein.

In other alternatives, a first nucleic acid comprises a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a polynucleotide coding for a gene that promotes cell survival and proliferation, a gene that regulates apoptosis, and/or a gene that modulates checkpoint signaling. Such genes include genes encoding IL-2, IL-7, IL-15, chemokine receptors, chimeric chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. In some alternatives, the gene that modulates checkpoint signaling encodes a polypeptide that inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3.

In some alternatives, the system is for inducible expression of a gene that regulates a T cell function.

Inducible Promoters.

A system comprises a first nucleic acid comprising a first promoter inducible by a drug. By utilizing an inducible promoter, transgene expression can be turned on and off in order to avoid toxic side effects and/or to allow the cells to rest during remission. Another purpose of the inducible promoter system is to improve anti-tumor efficacy by turning on genes that enhance T cell function. Although several inducible promoter systems are known, clinical applicability of these systems is limited due to toxic off target effects, unfavorable biodistribution and pharmacodynamics profiles, limited output dynamic range, and/or limited availability as FDA-approved commercially available pharmaceuticals. Furthermore, many of these systems use chimeric transcriptional regulators built from xenogeneic components, thus introducing the complication of immunogenicity when applying these systems to human therapeutics. In some alternatives, the first nucleic acid comprises a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from the group consisting of a tumor specific molecule, a viral specific molecule, and other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain.

In some alternatives, a first promoter is inducible by a drug. The drug is selected based on safety record, favorable pharmacokinetic profile, tissue distribution, a low partition coefficient between the extracellular space and cytosol, low immunogenicity, low toxicities, and/or high expression in lymphocytes. In a specific alternative, a drug is selected that is FDA approved, provides for transgene expression in lymphocytes, does not activate other undesirable gene expression, and induces a promoter that does not contain any xenogeneic components. In some alternatives, the inducible promoter is activated by a transcriptional activator that interacts with a drug. The transcriptional activator is activated or able to bind to and activate the inducible promoter in the presence of the drug.

A specific alternative of a drug is a drug that binds to an estrogen receptor ligand binding domain of a transcriptional activator. In some alternatives, the drug includes tamoxifen, its metabolites, analogs, and pharmaceutically acceptable salts and/or hydrates or solvates thereof. In some alternatives, the drug includes tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8.

Tamoxifen, CAS RN: 10540-29-1, useful in some approaches described herein, is also known as 2-(4-((1Z)-1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-ethanamine, or (Z)-2-(para-(1,2-Diphenyl-1-butenyl)phenoxy)-N,N-dimethylamine (IUPAC), and has a molecular formula of C26H29NO and a molecular weight (M.W.) of 371.52 g/mol. Tamoxifen is a Selective Estrogen Receptor Modulator with tissue-specific activities. Tamoxifen acts as an anti-estrogen (inhibiting agent) agent in the mammary tissue, but as an estrogen (stimulating agent) in cholesterol metabolism, bone density, and cell proliferation in the endometrium. Tamoxifen is frequently administered orally as a pharmaceutically acceptable salt. For example, Tamoxifen citrate (RN 54965-24-1, M.W. 563.643) is indicated for treatment of metastatic breast cancer, and as an adjuvant for the treatment of breast cancer in women following mastectomy axillary dissection, and breast irradiation. Tamoxifen citrate is also indicated to reduce incidence of breast cancer in women at high risk for breast cancer.

Metabolites of tamoxifen in rat, mouse and human breast cancer patients, useful in some approaches described herein, include major metabolites N-desmethyltamoxifen (RN 31750-48-8, M.W. 357.494) and 4-hydroxytamoxifen (4-OHT) (RN 68392-35-8, M.W. 387.52, Afimoxifene), and are disclosed in Robinson et al. (Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient. Drug Metab Dispos January 1991 19:36-43; incorporated by reference in its entirety herein). Additional cytochrome P-450 metabolites, useful in some approaches described herein, include cis-4-hydroxytamoxifen (RN 174592, M.W. 387.52; Afimoxifene, E-isomer), and 4′-hydroxytamoxifen ((Z)-4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-phenylbut-1-en-2-yl)phenol) as disclosed in Crewe et al. (Metabolism of Tamoxifen by recombinant human cytochrome P-450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen, Drug Metab Dispos, 30(8): 869-874, 2002, FIG. 1 ; incorporated by reference in its entirety herein).

Compounds with structural similarity to tamoxifen, useful in some approaches described herein, include, but are not limited to, cis-tamoxifen (RN 13002-65-8, M.W. 371.521), 4-methyltamoxifen (RN 73717-95-5, M.W. 385.548), N-desmethyltamoxifen (RN 31750-48-8, M.W. 357.494), (Z)-desethyl methyl tamoxifen (RN 15917-50-7, M.W. 357.494), (E)-desethyl methyl tamoxifen (RN 31750-45-5, M.W. 357.494), trans-4-hydoxytamoxifen (RN 68047-06-3, M.W. 387.52), Afimoxifene (RN 68392-35-8, M.W. 387.52, 4-hydroxytamoxifen), Afimoxifene, E-isomer (RN 174592-47-3, M.W. 387.52), 4-chlorotamoxifen (RN 77588-46-6, M.W. 405.966), 4-fluorotamoxifen (RN 73617-96-6, M.W. 389.511), Toremifene (RN 89778-26-7, M.W. 405.966), desethyl tamoxifen (RN 19957-51-8, M.W. 343.47), (E)-desethyl tamoxifen (RN 97151-10-5, M.W. 343.47), (Z)-desethyl tamoxifen (RN 97151-11-6, M.W. 343.47), Miproxifene (RN 129612-87-9, M.W. 429.6), 2-(p-(beta-ethyl-alpha-phenyl styryl)phenoxy)triethylamine (RN 749-86-0, M.W. 399.575), Droloxifene (RN 82413-20-5, M.W. 387.52), 4-iodo-tamoxifen (RN 116057-68-2, M.W. 497.413), dihydrotamoxifen (RN 109640-20-2, M.W. 373.537), (E)-N,N-dimethyl-2-(4-(1-(2-methylphenyl)-2-phenyl-1-butenyl)phenoxy)ethanamine (RN 97150-96-4, M.W. 385.548), or 4-hydroxytoremifene (RN 110503-62-3, M.W. 421.965); and/or pharmaceutically acceptable salts and/or hydrates or solvates thereof.

For example, citrate salts of tamoxifen, or citrate salts of compounds with structural similarity to tamoxifen, useful in some approaches described herein, include, but are not limited to tamoxifen citrate (RN 54965-24-1, M.W. 563.64), 2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylethylamine citrate (RN 7244-97-5, 563.64), (E)-tamoxifen citrate (RN 76487-65-5, M.W. 563.64), Toremifene citrate (RN 89778-27-8, M.W. 598.088), Droloxifene citrate (RN 97752-20-0, M.W. 579.64), 2-(p-(1,2-bis(p-methoxyphenyl)-1-butenyl)phenoxy)triethylamine citrate (RN 42920-39-8, M.W. 651.748), 2-(4-(1,2-diphenylethenyl)phenoxy)-N,N-diethyl-ethanamine 2-hydroxy-1,2,3-propanetricarboxylate (RN 40297-42-5, M.W. 563.643), 2-(p-(alpha-phenyl styryl)phenoxy)triethylamine citrate (RN 102433-95-4, M.W. 563.64), 2-(p-(2-(p-methoxyphenyl)-1-phenyl-1-butenyl)phenoxy)triethylamine citrate (1:1) (RN 42824-34-0, M.W. 637.72), 2-(p-(1-(p-methoxyphenyl)-2-phenylpropenyl)phenoxy)triethylamine citrate (RN 13554-24-0, M.W. 607.696), 2-(p-(alpha-(p-methoxyphenyl)styryl)phenoxy)triethylamine citrate monohydrate (RN 13542-71-7, M.W. 593.669), 2-(p-(p-methoxy-alpha-phenylphenethyl) phenoxy)triethylamine citrate (RN 16421-72-0, M.W. 595.685), alpha-(p-(2-(diethylamino)ethoxy)phenyl)-beta-ethyl-p-methoxy-alpha-phenylphenethyl alcohol citrate (1:1) (RN 35263-93-5, M.W. 639.737), 1-(p-(2-(diethylamino)ethoxy)phenyl)-2-(p-methoxyphenyl)-1-phenylethanol citrate (M.W. 611.68), alpha-p-(2-(diethyl amino)ethoxy)phenyl)-beta-ethyl-alpha-(p-hydroxyphenyl)-p-methoxyphenethyl alcohol citrate (RN 35263-96-8, M.W. 655.737), and/or 2-(p-(p-methoxy-alpha-methylphenethyl)phenoxy)-triethylamine citrate (RN 15624-34-7, M.W. 533.614).

In some alternatives, an effective amount of the drug for inducing expression is an amount that provides for an increase in transgene expression over uninduced and/or basal level of expression. In some alternatives, this amount can be readily determined using known dosages and pharmacokinetic profile of the drug. In the systems provided herein, wherein the systems are inducible by the drug, the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor.

In some alternatives, the inducible promoter has a low level of basal activity. When a lentiviral vector is used, the level of basal activity in uninduced cells is 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less, or any level of basal activity in between a range defined by any two aforementioned values, as compared to when cells are induced to express the gene. The level of basal activity can be determined by measuring the amount of the expression of the transgene (e.g. marker gene) in the absence of the inducer (e.g. drug) using flow cytometry.

In some alternatives, the inducible promoter provides for a high level of induced activity, as compared to uninduced or basal activity. In some alternatives, the level of activity in the induced state is 2, 4, 6, 8, or 10 fold or greater than the activity level in the uninduced state. In some alternatives, transgene expression under control of the inducible promoter is turned off in the absence of a transactivator in less than 10, 8, 6, 4, 2, or 1 days excluding 0 days.

In some alternatives, an inducible promoter can be designed and/or modified to provide for a low level of basal activity, a high level of inducibility, and/or a short time for reversibility. In some alternatives, the inducible promoter is the 7×HBD/mE1b promoter. An exemplary sequence for the promoter is set forth in SEQ ID NO: 23 (SEQ ID NO: 23; tagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataat ctacaatagttaataatctacaagagctcagggtatataatg). For example, in the 7×HBD/mE1b promoter, mutations can be made to enhance the binding of the transcriptional activator. In some alternatives, a DNA spacer is between the inducible promoter and the transgene. In some alternatives, the DNA spacer comprises a sequence set forth in SEQ ID NO: 120 (SEQ ID NO: 120; gctagcgtttaaacttaagcttggtaccgagctcggatccgccacc). In some alternatives, the DNA spacer comprises a sequence that has 95%, 90%, 85% or 80% sequence identity to SEQ ID NO: 120 or any percent sequence identity in between a range defined by any two aforementioned values.

In some alternatives, the system employs a synthetic transcriptional activator which, in the presence of the drug (e.g., tamoxifen), binds a synthetic promoter upstream of a transgene to induce expression. In some alternatives, the transcriptional activator is HEA3. An exemplary amino acid sequence is set forth in SEQ ID NO: 1 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 1. The mutant tamoxifen-specific ligand binding domain of the estrogen receptor ligand binding domain (ER-LBD) is found at amino acids 282-595 of the TamR-tf and has a mutation at position 521. The p65 activation domain of NF-κB (p65 or TAD) is found at amino acids 596 to 862. SEQ ID NO: 1 comprises one point mutation in the ER-LBD that ablates binding to endogenous estrogen, but confers nanomolar specificity to 4-OHT, fulvestrant and other estrogen analogs.

In some alternatives, the HEA3 transcription factor comprises at least one point mutation that ablates binding to the endogenous estrogen but confers nanomolar specificity tamoxifen metabolite, 4-hydroxytamoxifen, fulvestrant, and other estrogen analogs. In some alternatives, the one point mutation is at position 521 of the sequence of the wild-type HEA3. In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 1 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). SEQ ID NO: 1 comprises one point mutation in the ER-LBD that ablates binding to endogenous estrogen, but confers nanomolar specificity to 4-OHT, fulvestrant and other estrogen analogs.

In some alternatives, the HEA3 transcription factor comprises at least one point mutation in the p65 domain that enhances transcriptional activity. In some alternatives, the point mutation is at position 846 of the sequence of the wild-type HEA3 (position 536 in the RelA protein, which correspond to position 846 in HEA3). In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 2 (SEQ ID NO: 2; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 2. SEQ ID NO: 2 comprises a point mutation in HEA3, in the p65 domain which enhances transcriptional activity and is referred to as HEA3 (p65/S536E). The p65/S536E position in the RelA protein corresponds to position 846 in wild type HEA3 and wild type HEA4.

In some alternatives, the HEA3 transcription factor comprises at least one point mutation in the p65 domain that enhances transcriptional activity. In some alternatives, the point mutation is at position 621 of the sequence of the wild-type HEA3 (This is position 310 of the WT RelA protein). In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 3 (SEQ ID NO: 3; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FQSSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 3. SEQ ID NO: 3 comprises a sequence of HEA3 containing one point mutation (K310Q positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as HEA3 (p65/K310Q). K310Q refers to position 310 of RelA. This position thus corresponds to position 621 of WT HEA3 AND WT HEA4.

In some alternatives, the HEA3 transcription factor comprises at least three point mutations in the estrogen receptor ligand binding domain that ablates binding to endogenous estrogen, and confer nanomolar sensitivity to CMP8 and 4-hydroxytamoxifen. In some alternatives, the point mutations are at positions 384, 421 and 521 of the sequence of the wild-type HEA3. In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 8 (SEQ ID NO: 8; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF KSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDF SALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 8. SEQ ID No: 8 comprises the sequence of HEA3 containing three point mutations (L384M, M421G, G521R) in the ER-LBD that ablate binding to endogenous estrogen, and confer nanomolar sensitivity to CMP8 and 4-hydroxytamoxifen. This is referred to as CMP8-HEA3.

In some alternatives, the HEA3 transcription factor is a CMP8-responsive HEA3 transcription factor. In some alternatives, the CMP8-responsive HEA3 transcription factor comprises at least one point mutation in the p65 domain that enhances transcriptional activity. In some alternatives, the point mutation is at position 846 of the sequence of the CMP8-responsive HEA3 (position 536 in the RelA protein, which correspond to position 846 in HEA3). In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 9 (SEQ ID NO: 9; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF KSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADMDF SALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 9. SEQ ID NO: 9 is the sequence of CMP8-HEA3 containing one point mutation (S536E) in the p65 domain that enhances transcriptional activity. This is referred to as CMP8-HEA3 (p65/S536E positions in the RelA protein, which correspond to position 846 in HEA3).

In some alternatives, the HEA3 transcription factor is a CMP8-responsive HEA3 transcription factor. In some alternatives, the CMP8-responsive HEA3 transcription factor comprises at least one point mutation in the p65 domain that enhances transcriptional activity. In some alternatives, the point mutation is at position 310 of the sequence of the RelA protein which corresponds to position 621 of the wild-type HEA3 protein, and the wild-type HEA4 protein. In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 10 (SEQ ID NO: 10; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF QSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDF SALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 10. SEQ ID NO: 10 is the sequence of CMP8-HEA3 containing one point mutation (K310Q positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as CMP8-HEA3 (p65/K310Q). Position 310 of the RelA protein is position 621 of wild-type HEA3 and wild-type HEA4.

In some alternatives, the HEA3 transcription factor is a CMP8-responsive HEA3 transcription factor. In some alternatives, the CMP8-responsive HEA3 transcription factor comprises at least two point mutations in the p65 domain that enhances transcriptional activity. In some alternatives, the point mutations are at positions 621 of the sequence of the wild-type HEA3 (The 310 position in the RelA protein corresponds to position 621 in wild type HEA3). In some alternatives, the amino acid sequence comprises a sequence set forth in SEQ ID NO: 11 (SEQ ID NO: 11; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF QSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADMDF SALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 11. SEQ ID NO: 11 comprises the sequence of CMP8-HEA3 containing two point mutations (K310Q and S536E are positions in the RelA protein, which correspond to positions 621 (K310A of the RelA protein) and 846 (S536E of the RelA protein) in both HEA3 and HEA4) in the p65 domain that enhance transcriptional activity. This is referred to as CMP8-HEA3 (p65/S536E/K310Q positions in the RelA protein, which correspond to position 621 and 846 in both HEA3 and HEA4.

In some alternatives, the system employs a synthetic transcriptional activator which, in the presence of the drug, binds a synthetic promoter upstream of a transgene to induce expression. In some alternatives, the transcriptional activator is HEA4. HEA4 comprises an estrogen receptor ligand binding domain ((ER-LBD) and a p65 domain. In some alternatives, the HEA4 further comprises an HNF1alpha DNA binding domain. In some alternatives, the HEA4 comprises at last three point mutations in the ER-LBD that ablate binding to endogenous estrogen, and confer higher sensitivity to 4-hydroxytamoxifen, fulvestrant, and estrogen analogs compared to wild-type HEA3. In some alternatives, the positions of the three point mutations are at positions 400, 543 and 544 in HEA4 transcription factor. In some alternatives, the amino acid sequence of the HEA4 comprising the at least three mutations comprise an amino acid sequence set forth in SEQ ID NO: 4 (SEQ ID NO: 4; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 4. SEQ ID NO: 4 comprises the sequence of wild-type HEA4 containing three point mutations (G400V, M543A, L544A) in the ER-LBD that ablate binding to endogenous estrogen, and confer higher sensitivity to 4-hydroxytamoxifen, fulvestrant, and estrogen analogs compared to wild-type HEA3.

In some alternatives, the HEA4 transcription factor comprises at least one point mutation. In some alternatives, the at least one mutation is in the p65 domain and enhances transcriptional activity. In some alternatives, the at least one mutation is at position 846 of the wild type HEA4 transcription factor. In some alternatives, the amino acid sequence of the HEA4 comprising the at least one mutation comprises an amino acid sequence set forth in SEQ ID NO: 5 (SEQ ID NO: 5; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 5. SEQ ID NO: 5 comprises the sequence of HEA4 containing one point mutation (S536E positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as HEA4 (p65/S536E). The mutation S536E refers to the mutation at position 536 of WT RelA. This position translates to position 846 of HEA3. The mutation K310Q refers to the mutation at position 310 of WT RelA. This position translates to position 621 of HEA3.

In some alternatives, the HEA4 transcription factor comprises at least one point mutation. In some alternatives, the at least one mutation is in the p65 domain and enhances transcriptional activity. In some alternatives, the at least one mutation is at position 621 of the HEA4 transcription factor. In some alternatives, the amino acid sequence of the HEA4 comprising the at least one mutation comprises an amino acid sequence set forth in SEQ ID NO: 6 (SEQ ID NO: 6; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FQSSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 6. SEQ ID NO: 6 comprises the sequence of HEA4 containing one point mutation (K310Q positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as HEA4 (p65/K310Q). K310Q refers to the position in RelA, which corresponds to position 621 in WT HEA4.

In some alternatives, the HEA4 transcription factor comprises at least two point mutations. In some alternatives, the at least two mutations are in the p65 domain and enhances transcriptional activity. In some alternatives, at least two mutations are at positions 846 and 621 of the HEA4 transcription factor. In some alternatives, the amino acid sequence of the HEA4 comprising the at least two mutations comprise an amino acid sequence set forth in SEQ ID NO: 7 (SEQ ID NO: 7; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FQSSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADM DFSALLSQISS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 7. SEQ ID NO: 7 comprises the sequence of HEA4 containing two point mutations (K310Q and S536E, positions in the RelA protein) in the p65 domain that enhance transcriptional activity. This is referred to as HEA4 (p65/S536E/K310Q positions in the RelA protein). K310Q in RelA corresponds to position 621 in HEA4; S536E corresponds to position 846 in HEA4.

Additional chimeric transcription factors are also contemplated herein. In some alternatives, the HEA4 transcription factor comprises HNF1alpha, an ER-LBD that is the same as HEA4, and a VP64 activation domain instead of the p65 activation domain nucleotide sequence (referred herein as HEA4(VP64), and is encoded by SEQ ID NO: 24 (atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcagg cactcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagct ggccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcacccccccc atcctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccc tggcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaac cagagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcgg aagcagagagaggtggcccagcagtttacacacgccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccac caagaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaacccc agcaaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcag ggcctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagct gtctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagc ctgaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagc gaggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggc ttcgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagc atggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggaga tcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcct gctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaaga tcaccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatc ctgagccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacct gctgctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctca cctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgt ggacgctctggacgacttcgaccttgacatgttgggttccgacgcgctggatgattttgatttggacatgctgggaagcgacgcactgg atgactttgatctcgatatgctcggctctgacgcattggacgacttcgacttggatatgctgggttct; HEA4 (VP64)). In some alternatives, HEA4(VP64) comprises an amino acid sequence set forth in SEQ ID NO: (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVDALDDFDLDMLGSDALDDFDLD MLGSDALDDFDLDMLGSDALDDFDLDMLGS). Some alternatives include a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 25. In some alternatives herein, the chimeric transcription factor is HEA4(VP64), and is encoded by SEQ ID NO: 24. In some alternatives, HEA4(VP64) comprises an amino acid sequence set forth in SEQ ID NO: 25.

In some alternatives, HEA4 comprises HNF1alpha, an ER-LBD that is the same as HEA4, and an HSF1 activation domain instead of a p65 activation domain. HEA4 (HSF-1) comprises HNF1alpha, ER-LBD that is the same as HEA4, and HSF1 activation domain instead of a p65 activation domain, and is encoded by SEQ ID NO: 26 (atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcagg cactcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagct ggccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcacccccccc atcctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccc tggcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaac cagagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcgg aagcagagagaggtggcccagcagtttacacacgccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccac caagaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaacccc agcaaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcag ggcctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagct gtctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagc ctgaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagc gaggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggc ttcgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagc atggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggaga tcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcct gctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaaga tcaccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatc ctgagccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacct gctgctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctca cctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgt ggaaaagtgcctcagcgtagcctgcctggacaagaatgagctcagtgaccacttggatgctatggactccaacctggataacctgca gaccatgctgagcagccacggcttcagcgtggacaccagtgccctgctggacctgttcagcccctcggtgaccgtgcccgacatga gcctgcctgaccttgacagcagcctggccagtatccaagagctcctgtctccccaggagccccccaggcctcccgaggcagagaac agcagcccggattcagggaagcagctggtgcactacacagcgcagccgctgttcctgctggaccccggctccgtggacaccggga gcaacgacctgccggtgctgtttgagctgggagagggctcctacttctccgaaggggacggcttcgccgaggaccccaccatctcc ctgctgacaggctcggagcctcccaaagccaaggaccccactgtctcc). In some alternatives, herein the chimeric transcription factor is HEA4(HSF-1) and is encoded by SEQ ID NO: 26. In some alternatives, herein the chimeric transcription factor is HEA4(HSF-1) and comprises an amino acid sequence set forth in SEQ ID NO: 27 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEKCLSVACLDKNELSDHLDAMDS NLDNLQTMLSSHGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPP EAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDP TISLLTGSEPPKAKDPTVS). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 27.

In some alternatives herein the chimeric transcription factor is HEA4(Arnt). HEA4(Arnt) comprises HNF1alpha, ER-LBD that is the same as HEA4, and an Arnt activation domain instead of an p65 activation domain amino acid sequence. In some alternatives, HEA4(Arnt) is encoded by a sequence set forth in SEQ ID NO: 28 (atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcagg cactcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagct ggccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcacccccccc atcctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccc tggcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaac cagagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcgg aagcagagagaggtggcccagcagtttacacacgccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccac caagaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaacccc agcaaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcag ggcctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagct gtctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagc ctgaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagc gaggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggc ttcgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagc atggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggaga tcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcct gctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaaga tcaccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatc ctgagccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacct gctgctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctca cctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgt gcctgaggtcttccaggagatgctgtccatgctgggagatcagagcaacagctacaacaatgaagaattccctgatctaactatgtttc cccccttttcagaa). In some alternatives, HEA4(Arnt) comprises an amino acid sequence set forth in SEQ ID NO: 29 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVPEVFQEMLSMLGDQSNSYNNEEF PDLTMFPPFSE). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 29.

“Selectable marker cassette,” has its plain and ordinary meaning when read in light of the specification, and may be used, for example, to describe a gene that encodes a “selectable marker” and is introduced into a vector or a cell that confers a trait for artificial selection. A selectable marker cassette can be a screenable marker to allow a researcher to distinguish between wanted and unwanted cells, or to enrich for a specific cell type. In some alternatives, a gene delivery polynucleotide is provided. In some alternatives, the gene delivery polynucleotide comprises a selectable marker cassette. The selectable marker cassette as described herein, encodes a protein (the selectable marker) that is used for selecting or tracking a protein or cell that has a protein of interest. In the alternatives described herein, the fusion protein provided herein can comprise a marker sequence that can be selected in experiments, such as flow cytometry. In some alternatives, the marker is the protein Her2tG.

In some alternatives, the selectable marker is encoded by an EGFRt nucleotide sequence, wherein the selectable maker is encoded by a sequence set forth in SEQ ID NO: 30 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcct gccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatca cagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaa gcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgt gataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattat aagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccgga gcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagccaagg gagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggacc agacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaaca ccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttg aaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctgg ggatcggcctcttcatgtga). In some alternatives, the selectable marker comprises an EGFRt amino acid sequence set forth in SEQ ID NO: 31 (MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGE PREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGE NNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLL VVALGIGLFM). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 31.

In some alternatives, the selectable marker is encoded by a Her2t nucleotide sequence, set forth in SEQ ID NO: 32 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccatgccaccctgagtgtcagcccca gaatggctcagtgacctgttttggaccggaggctgaccagtgtgtggcctgtgcccactataaggaccctcccttctgcgtggcccgct gccccagcggtgtgaaacctgacctctcctacatgcccatctggaagtttccagatgaggagggcgcatgccagccttgccccatca actgcacccactcctgtgtggacctggatgacaagggctgccccgccgagcagagagccagccctctgacgtccatcatctctgcg gtggttggcattctgctggtcgtggtcttgggggtggtctttgggatcctcatctga). In some alternatives, the selectable marker comprises the Her2t amino acid sequence set forth in SEQ ID NO: 33 (MLLLVTSLLLCELPHPAFLLIPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCV ARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSII SAVVGILLVVVLGVVFGILI). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 33.

In some alternatives, the selectable marker is encoded by the Her2tG nucleotide sequence set forth in SEQ ID NO: 34 (atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccatgccaccctgagtgtcagccccag aatggctcagtgacctgttttggaccggaggctgaccagtgtgtggcctgtgcccactataaggaccctcccttctgcgtggcccgctg ccccagcggtgtgaaacctgacctctcctacatgcccatctggaagtttccagatgaggagggcgcatgccagccttgccccatcaa ctgcacccactcctgtgtggacctggatgacaagggctgccccgccgagcagagagccagcccgttaacgggtggaggcagcgg aggtggctccatcatctctgcggtggttggcattctgctggtcgtggtcttgggggtggtctttgggatcctcatctga). In some alternatives, the selectable marker comprises the Her2tG amino acid sequence, set forth in SEQ ID NO: 35 (MLLLVTSLLLCELPHPAFLLIPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCV ARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTGG GSGGGSIISAVVGILLVVVLGVVFGILI). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 35.

In some alternatives, the selectable marker is encoded by the CD19t nucleotide sequence, set forth in SEQ ID NO: 36 (atgccacctcctcgcctcctcttcttcctcctcttcctcacccccatggaagtcaggcccgaggaacctctagtggtgaaggtggaag agggagataacgctgtgctgcagtgcctcaaggggacctcagatggccccactcagcagctgacctggtctcgggagtccccgctt aaacccttcttaaaactcagcctggggctgccaggcctgggaatccacatgaggcccctggccatctggcttttcatcttcaacgtctct caacagatggggggcttctacctgtgccagccggggcccccctctgagaaggcctggcagcctggctggacagtcaatgtggagg gcagcggggagctgttccggtggaatgtttcggacctaggtggcctgggctgtggcctgaagaacaggtcctcagagggccccagc tccccttccgggaagctcatgagccccaagctgtatgtgtgggccaaagaccgccctgagatctgggagggagagcctccgtgtgtc ccaccgagggacagcctgaaccagagcctcagccaggacctcaccatggcccctggctccacactctggctgtcctgtggggtacc ccctgactctgtgtccaggggccccctctcctggacccatgtgcaccccaaggggcctaagtcattgctgagcctagagctgaagga cgatcgcccggccagagatatgtgggtaatggagacgggtctgttgttgccccgggccacagctcaagacgctggaaagtattattgt caccgtggcaacctgaccatgtcattccacctggagatcactgctcggccagtactatggcactggctgctgaggactggtggctgga aggtctcagctgtgactttggcttatctgatcttctgcctgtgttcccttgtgggcattcttcatcttcaaagagccctggtcctgaggagga aaagataa). In some alternatives, the selectable marker comprises the CD19t amino acid sequence, set forth in SEQ ID NO: 37 (MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESP LKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVN VEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGE PPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLS LELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWH WLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 37.

In some alternatives, the selectable marker is encoded by the DHFRdm nucleotide sequence set forth in SEQ ID NO: 38 (atggttggttcgctaaactgcatcgtcgctgtgtcccagaacatgggcatcggcaagaacggggacttcccctggccaccgctcag gaatgaatccagatatttccagagaatgaccacaacctcttcagtagaaggtaaacagaatctggtgattatgggtaagaagacctggt tctccattcctgagaagaatcgacctttaaagggtagaattaatttagttctcagcagagaactcaaggaacctccacaaggagctcattt tctttccagaagtctagatgatgccttaaaacttactgaacaaccagaattagcaaataaagtagacatggtctggatagttggtggcagt tctgtttataaggaagccatgaatcacccaggccatcttaaactatttgtgacaaggatcatgcaagactttgaaagtgacacgttttttcc agaaattgatttggagaaatataaacttctgccagaatacccaggtgttctctctgatgtccaggaggagaaaggcattaagtacaaattt gaagtatatgagaagaatgat). In some alternatives, the selectable marker comprises the DHFRdm amino acid sequence, set forth in SEQ ID NO: 39 (VGSLNCIVAVSQNMGIGKNGDFPWPPLRNESRYFQRMTTTSSVEGKQNLVIMGKKT WFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQE EKGIKYKFEVYEKND). Some alternatives include a nucleic acid comprising a nucleic acid sequence that encodes SEQ ID NO: 39.

In some alternatives, a system for inducible expression of a chimeric antigen receptor is provided, wherein the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor comprising a ligand binding domain specific for a ligand selected from a tumor specific molecule, viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand can elicit recognition, modulation, inhibition, and/or elimination by a lymphocyte; a polynucleotide coding for a polypeptide spacer, wherein the spacer is preferably optimized; a polynucleotide coding for a transmembrane domain; and a polynucleotide coding for an intracellular signaling domain; and b) a second nucleic acid comprising a second promoter operably linked to nucleic acid coding for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the drug is tamoxifen and/or its metabolites. In some alternatives, the second promoter is constitutive or inducible. In some alternatives, the first promoter comprises a nucleic acid sequence of SEQ ID NO: 23 (SEQ ID NO: 23; tagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataat ctacaatagttaataatctacaagagctcagggtatataatg). In some alternatives, the second promoter is a constitutive promoter. In some alternatives, the second promoter is the EF1αp. In some alternatives, the transcriptional activator comprises an amino acid sequence set forth in SEQ ID NO's: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. In some alternatives, the first nucleic acid further comprises a first vector and the second nucleic acid further comprises a second vector. In some alternatives, both vectors are packaged in a viral vector. In some alternatives, the viral vector is a lentivirus. In some alternatives, the first and second nucleic acid comprise a vector. In some alternatives, the first nucleic acid further comprises a nucleic acid sequence coding for a selectable marker. In some alternatives, the second nucleic acid further comprises a nucleic acid coding for a selectable marker. In some alternatives, a system for inducible expression is provided, wherein the system comprises a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide, which encodes a cytokine, a chemokine receptor, a chimeric cytokine receptor, a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity and b) a second nucleic acid comprising a second promoter, wherein the second nucleic acid is operably linked to third nucleic acid that encodes a transcriptional activator for the first promoter inducible by drug and wherein the system is configured to respond to an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor. In some alternatives, the transcriptional activator is HEA3 or HEA4. In some alternatives, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises mutations that allow selective binding to the drug ligand. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that enhances transcriptional activity in response to a drug. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least three mutations that allow selective binding to the drug ligand, wherein HEA4 comprises an estrogen receptor ligand binding domain. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that enhances transcriptional activity in response to the drug ligand. Mutations in the ER-LBD affect drug to receptor binding. In some alternatives, the HEA3 further comprises an HNF1alpha DNA binding domain. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective binding to the drug ligand, wherein HEA4 comprises an estrogen receptor ligand binding domain, wherein the estrogen receptor ligand binding domain comprises mutations that allow selective binding to the drug ligand. In some alternatives, the HEA4 further comprises an HNF1alpha DNA binding domain. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that enhances transcriptional activity in response to a drug. In some alternatives, HEA4 comprises three mutations in the ER-LBD that enhance sensitivity to drug compared to HEA3. In some alternatives, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives, the variant of a wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives, the wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID No: 1 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). SEQ ID NO: 1 comprises one point mutation in the ER-LBD that ablates binding to endogenous estrogen, but confers nanomolar specificity to 4-OHT, fulvestrant and other estrogen analogs. In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID No: 2 (SEQ ID NO: 2; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADM DFSALLSQISS). SEQ ID NO: 2 comprises a point mutation in HEA3, in the p65 domain which enhances transcriptional activity and is referred to as HEA3 (p65/S536E positions in the RelA protein). In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 3 (SEQ ID NO: 3; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FQSSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). SEQ ID NO: 3 comprises a sequence of HEA3 containing one point mutation (K310Q positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as HEA3(p65/K310Q). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 12, 13, 14, 19, 20, 21, 22 or 122. In some alternatives, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by at least one or more nucleic acid sequences set forth in SEQ ID NO: 15, 16, 17 or 18. In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 8 (SEQ ID NO: 8; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF KSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDF SALLSQISS). SEQ ID NO: 8 comprises the sequence of HEA3 containing three point mutations (L384M, M421G, G521R) in the ER-LBD that ablate binding to endogenous estrogen, and confer nanomolar sensitivity to CMP8 and 4-hydroxytamoxifen. This is referred to as CMP8-HEA3. In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 9 (SEQ ID NO: 9; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF KSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADMDF SALLSQISS). SEQ ID NO: 9 is the sequence of CMP8-HEA3 containing one point mutation (S536E positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as CMP8-HEA3(p65/S536E positions in the RelA protein). In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 10 (SEQ ID NO: 10; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF QSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDF SALLSQISS). SEQ ID NO: 10 is the sequence of CMP8-HEA3 containing one point mutation (K310Q positions in the RelA protein) in the p65 domain that enhances transcriptional activity. This is referred to as CMP8-HEA3(p65/K310Q positions in the RelA protein). In some alternatives, the variant of a wild type HEA3 transcription factor comprises an amino acid sequence set forth in SEQ ID NO: 11 (SEQ ID NO: 11; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFRMMNLQGEEFVCL KSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQ LLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEE TDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYETF QSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPS GQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPV APHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADMDF SALLSQISS). SEQ ID NO: 11 comprises the sequence of CMP8-HEA3 containing two point mutations (K310Q and S536E positions in the RelA protein) in the p65 domain that enhance transcriptional activity. This is referred to as CMP8-HEA3(p65/S536E/K310Q positions in the RelA protein). In some alternatives, the polypeptide that modulates checkpoint signaling inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3. In some alternatives, the second promoter is constitutive or inducible. In some alternatives, the second nucleic acid further comprises a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain, which is a tumor specific molecule, viral molecule, or another molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a polynucleotide coding for a polypeptide spacer, wherein the spacer is optimized, a polynucleotide coding for a transmembrane domain and a polynucleotide encoding an intracellular signaling domain. In some alternatives, the first promoter is in opposite orientation to the second promoter. In some alternatives, the ligand binding domain is an antibody fragment, preferably a binding fragment thereof. In some alternatives, the ligand binding domain is a single chain variable fragment. In some alternatives, the tumor specific molecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In some alternatives, the system employs a synthetic transcriptional activator which, in the presence of a drug binds a synthetic promoter upstream of a transgene to induce expression. In some alternatives, the transcriptional activator is HEA3. In some alternatives, the drug is tamoxifen. In some alternatives, the viral specific molecule is an HIV envelope protein or HIV envelope protein complex, Hepatitis C virus NS3 protein, HbcAg protein, HIV-1 GP120 protein, or Epstein-Barr virus early antigen protein.

Additional changes can be made to the transcriptional activator to increase the properties of the transcription factor including, without limitation, altering one or more amino acids in the estrogen receptor ligand binding domain and/or altering one or more amino acids in the p65 transactivating domain. Altering amino acids in the estrogen receptor binding domain can provide for more specific and higher sensitivity binding of the drug to the transcriptional activator. In some alternatives, HEA4 comprises at least three point mutations. For example, mutations can be made at amino acid position 400, 543, and 544. In some alternatives, the wild type HEA4 comprises the sequence set forth in SEQ ID NO: 4 (SEQ ID NO: 4; MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADM DFSALLSQISS). SEQ ID NO: 4 comprises the sequence of wild-type HEA4 containing three point mutations (G400V, M543A, L544A) in the ER-LBD that ablate binding to endogenous estrogen, and confer higher sensitivity to 4-hydroxytamoxifen, fulvestrant, and estrogen analogs compared to wild-type HEA3. The transcriptional activator with altered sequence has increased affinity for tamoxifen or 4-hydroxytamoxifen, fulvestrant, and estrogen analogs compared to wild-type HEA3. Altering amino acids in the p65 transactivating domain can provide for increased expression of the transgene in the absence of activation of the transduced cells (SEQ ID NO: 5 and SEQ ID NO: 6).

In the absence of tamoxifen, TamR-tf is excluded from the nucleus by binding of cytosolic heat-shock protein 90 (HSP90) to the tamoxifen binding active site and transgene expression is in the “OFF” state. Nanomolar concentrations of cytosolic tamoxifen actively outcompete HSP90 for ER-LBD binding, resulting in TamR-tf translocation to the nucleus. Upon nuclear translocation, TamR-tf is readily available to bind its restricted synthetic promoter. In the presence of tamoxifen, binding of TamR-tf to 7×HBD/mE1B promoter induces the “ON” state of transgene expression. In some alternatives, this transcriptional regulator can be modified to provide for varying level of control of transgene expression. Amino acid substitutions in the LBD of TamR-tf (HEA3) permit selective responsiveness to tamoxifen and its metabolites, where 4-hydroxy tamoxifen (4-OHT) is the most pharmacologically active metabolite, with respect to TamR-tf (HEA3) activity, while lacking interaction with endogenous estrogen.

In some alternatives, a system for inducible expression of a transgene is provided. In some alternatives, the transgene encodes a chimeric antigen receptor. In some alternatives, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, is provided. In some alternatives, a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, is provided, the system comprises: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or other molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In some alternatives, the transcriptional activator is HEA3 or HEA4. In some alternatives, HEA3 is a variant of a wild type HEA3 transcription factor. In some alternatives, HEA4 is a variant of a wild type HEA4 transcription factor. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective and high sensitivity binding to the drug ligand. In some alternatives, the HEA3 further comprises an HNF1alpha DNA binding domain. Mutations in the ER-LBD affect drug-to-receptor binding. In some alternatives, HEA3 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that enhances transcriptional activity in response to a drug. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the estrogen receptor ligand binding domain comprises at least one mutation that allows selective and high sensitivity binding to the drug ligand. In some alternatives, HEA4 comprises an estrogen receptor ligand binding domain (ER-LBD) and a RelA (p65) transactivation domain, wherein the RelA (p65) transactivation domain comprises at least one mutation that enhances transcriptional activity in response to a drug. In some alternatives, the HEA4 further comprises an HNF1alpha DNA binding domain. In some alternatives, HEA4 comprises three mutations in the ER-LBD that enhance sensitivity to drug compared to HEA3. Mutations in the ER-LBD affect drug-to-receptor binding. In some alternatives, the variant of the HEA3 or HEA4 chimeric transcription factor comprises p65 phosphomimetic and/or acetylmimetic amino acid substitutions that enhance transcriptional activity in response to the drug ligand. In some alternatives, the variant of the wild type HEA3 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 1-3, 8-11 and/or 121. In some alternatives, the HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 12 (SEQ ID NO: 12; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacaccggccagggcggcctgatccggccagggcggccgacgaggaaccaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcaccatcaactacgacgagttccctaccagggccccgcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 13 (SEQ ID NO: 13; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccagcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 14 (SEQ ID NO: 14; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccagcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 19 (SEQ ID NO: 19; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggatggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcggcgtggagat cttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctg ctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatc accgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcct gagccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctg ctgctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcac ctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtg gagttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatc atgaagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgt gcccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccag cggccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatg gtgtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaaccta cccaggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatag caccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgccc cacacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagc accagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcag cgccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 20 (SEQ ID NO: 20; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggatggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcggcgtggagat cttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctg ctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatc accgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcct gagccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctg ctgctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcac ctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtg gagttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatc atgaagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgt gcccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccag cggccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatg gtgtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaaccta cccaggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatag caccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgccc cacacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagc accagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttcag cgccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 21 (SEQ ID NO: 21; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggatggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcggcgtggagat cttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctg ctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatc accgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcct gagccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctg ctgctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcac ctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtg gagttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcatc atgaagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgt gcccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccag cggccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatg gtgtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaaccta cccaggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatag caccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgccc cacacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagc accagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcag cgccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA3 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 22 (SEQ ID NO: 22; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggatggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcggcgtggagat cttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctg ctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatc accgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcct gagccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctg ctgctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcac ctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtg gagttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcatc atgaagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgt gcccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccag cggccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatg gtgtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaaccta cccaggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatag caccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgccc cacacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagc accagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttcag cgccctgctgtcccagatcagcagc). In some alternatives, the variant of a wild type HEA4 transcription factor comprises at least one or more amino acid sequences set forth in SEQ ID Nos: 4-7. In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 (SEQ ID NO: 15; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcccccccaccatcaactacgacgagttccctaccagggccccgcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 16 (SEQ ID NO: 16; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcccccccaccatcaactacgacgagttccctaccagggccccgcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 17 (SEQ ID NO: 17; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcccccccaccatcaactacgacgagttccctaccagggccccgcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttctccagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the variant of the wild type HEA4 transcription factor is encoded by a nucleic acid sequence set forth in SEQ ID NO: 18 (SEQ ID NO: 18; atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcaggc actcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagctg gccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcaccccccccat cctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccctg gcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaacca gagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcggaa gcagagagaggtggcccagcagtttacacacggccagggcggcctgatccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccacca agaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaaccccag caaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcaggg cctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagctgt ctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagcct gaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagcg aggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggctt cgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagcat ggaacaccccgtgaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggagatc ttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcctgc tgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaagatca ccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatcctg agccacatccggcacatgagcaacaagggaatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacctgct gctcgaggctgccgatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctcacct ggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgtgga gttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcatcatg aagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagcgtgc ccaagcctgccccccagccctaccctttcccccccaccatcaactacgacgagttccctaccagggccccgcg gccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccatggt gtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacctacc caggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaatagca ccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcccca cacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccagcac cagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttcagcg ccctgctgtcccagatcagcagc). In some alternatives, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8.

In some alternatives, the inducible promoter functions in a lentiviral construct and/or in lymphocytes.

Chimeric Antigen Receptors.

A system with an enhanced sensitivity to a drug ligand for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells, comprises: a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from a tumor specific molecule, a viral specific molecule, or any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer, preferably an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a system utilizing a wild type HEA3. In other alternatives, another polynucleotide coding for a chimeric antigen receptor is under the control of a constitutive promoter. In some alternatives, the drug is tamoxifen and/or its metabolites, 4-hydroxytamoxifen, fulvestrant or other estrogen analogs, or CMP8.

Ligand Binding Domain.

In some alternatives, the chimeric receptor nucleic acid comprises a polynucleotide coding for a ligand binding domain. In some alternatives, the ligand binding domain specifically binds to a tumor a viral molecule, or other molecule expressed on a target or selected cell population, and it may be humanized. In some alternatives, a ligand binding domain, includes without limitation, receptors or portions thereof, small peptides, peptidomimetics, substrates, cytokines, and the like. In some alternatives, the ligand binding domain is an antibody or fragment thereof, preferably a binding fragment thereof, any of which may be humanized. A nucleic acid sequence coding for an antibody or antibody fragment can readily be determined. In a specific alternative, the polynucleotide codes for a scFv that specifically binds CD19. In other specific alternatives, the polynucleotide codes for a single chain Fv that specifically binds HER2, CE7, hB7H3, or EGFR and, optionally, said polynucleotide encodes a humanized version thereof. The sequences of these antibodies and binding domains thereof are known to or can readily be determined by those of skill in the art. In some alternatives, the polynucleotide codes for a single scFv that specifically binds with CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC or VAR2CSA.

Tumor antigens are proteins that are produced by tumor cells that elicit an immune response. The selection of the ligand binding domain of the invention will depend on the type of cancer to be treated, and can target tumor antigens or other tumor cell surface molecules. A tumor sample from a subject can be characterized for the presence of certain biomarkers or cell surface markers. For example, breast cancer cells from a subject can be positive or negative for each of Her2Neu, Estrogen receptor, and/or the Progesterone receptor. A tumor antigen or cell surface molecule that is found on the individual subject's tumor cells is selected. Tumor antigens and cell surface molecules are well known in the art and include, for example, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1, CE7, hB7H3, ROR1, mesothelin, c-Met, GD-2, and/or MAGE A3 TCR. In some alternatives, the tumor antigen or tumor specific molecule is selected from the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR and/or any combinations thereof. In some alternatives, a target molecule is a cell surface molecule that is found on tumor cells and is not substantially found on normal tissues, or restricted in its expression to non-vital normal tissues.

In some alternatives, the target is CD19, CD20, CD22, EGFRvIII, EGFR, EphA2, IL13Ra2, L1CAM, oaGD2, GD2, Her2, B7H3, CD33, Mesothelin, ROR-1, FITC, and/or VAR2CSA.

In one alternative, the target molecule on the tumor comprises one or more epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for T cell receptor or chimeric receptor mediated recognition. Other target molecules belong to the group of cell transformation-related molecules such as the oncogene HER-2/Neu/ErbB2. In some alternatives, the tumor antigen is selectively expressed or overexpressed on the tumor cells as compared to control cells of the same tissue type. In other alternatives, the tumor antigen is a cell surface polypeptide.

Once a tumor cell surface molecule that may be targeted with a chimeric receptor is identified, an epitope of the target molecule is selected and characterized. Antibodies that specifically bind a tumor cell surface molecule can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule. Phage display libraries of human antibodies are also available. In some alternatives, antibodies specifically bind to a tumor cell surface molecule and do not cross react with nonspecific components such as bovine serum albumin or other unrelated antigens. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.

Antibodies or antigen binding fragments include all or a portion of polyclonal antibodies, a monoclonal antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a bispecific antibody, a minibody, and a linear antibody. “Antibody fragments” has its plain and ordinary meaning when read in light of the specification, and may be used, for example, to described a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody and can readily be prepared. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In some alternatives, the antibody fragments are Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; or multispecific antibodies formed from antibody fragments. Any of such aforementioned antibodies or antibody fragments can be humanized and used with the compositions and methods described herein.

In some alternatives, a number of different antibodies that bind to a particular tumor cell surface molecule can be isolated and characterized. In some alternatives, the antibodies are characterized based on epitope specificity of the targeted molecule. In addition, in some cases, antibodies that bind to the same epitope can be selected based on the affinity of the antibody for that epitope. In some alternatives, an antibody has an affinity of at least 1 mM, and preferably <50 nM. In some alternatives, the antibody has an affinity of 50 nM, 100 nM, 200 nM, 300 nM 400 nM, 500 nM, 1 uM, 100 uM, 200 uM, 300 uM, 400 uM, 500 uM, 600 uM, 700 uM, 800 uM, 900 uM or 1 mM or an affinity within a range defined by any two of the aforementioned values. In some alternatives, an antibody is selected that has a higher affinity for the epitope, as compared to other antibodies. For example, an antibody is selected that has at least a 2 fold, at least a 5 fold, at least a 10 fold, at least a 20 fold, at least a 30 fold, at least a 40 fold, or at least a 50 fold greater affinity than a reference antibody that binds to the same epitope or an affinity that is greater than a reference antibody within a range defined by any two of the aforementioned values.

In some alternatives, target molecules are CD19, CD20, CD22, CD23, CE7, hB7H3, EGFR, CD123, CS-1, ROR1, mesothelin, Her2, c-Met, PSMA, GD-2, or MAGE A3 TCR or any combination thereof. In some alternatives, the antibody or binding fragment there of specific for these target molecules is humanized.

In specific alternatives, the target antigen is CD19, CD20, CD22, EGFRvIII, EphA2, L13Ra2, oaGD2, GD2, CD33, Mesothelin, ROR-1, and/or VAR2CSA. A number of antibodies specific for CD19, CD20, CD22, EGFRvIII, EphA2, L13Ra2, oaGD2, GD2, CD33, Mesothelin, ROR-1 and/or VAR2CSA are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity. The disclosure also contemplates variable regions that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to that of the scFv for CD19, CD20, CD22, EGFRvIII, EphA2, IL13Ra2, oaGD2, GD2, CD33, Mesothelin, ROR-1 and/or VAR2CSA.

“Humanized antibodies,” has its plain and ordinary meaning when read in light of the specification, and may be used, for example, and may refer to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of “humanization” can be applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). Humanization can be desirable when the process of developing a specific antibody involves utilization of a non-human immune system (such as that in mice). The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients. Humanized antibodies are distinct from chimeric antibodies, in that they have the protein sequences made more similar to human antibodies but can carry a larger stretch of non-human protein. A derivative of a humanized antibody can refer to a segment of an antibody or sequence that is derived from a humanized antibody. In some alternatives, the ligand binding domain comprises a humanized antibody or portion thereof. In some alternatives, the ligand binding domain comprises a scFv. In some alternatives, the scFv is a humanized scFv.

In some alternatives, the scFv binds specifically to CD19, CD22, CD20, B7H3 receptor, L1CAM, EGFR, EGFRVIII, EphA2, FITC(E2), GD2, Her2, IL13R2a (hu08) VlVh, IL13Ra2, IL13Ra2, oaGD2, ROR1, CD33, Mesothelin, and/or VAR2CSA.

Humanization can be desirable in some alternatives for reducing the immunogenicity of monoclonal antibodies that are derived from xenogeneic sources, such as, for example, rodents. Humanization is also desirable in some alternatives so as to improve the interaction of the antibody or a fragment thereof with the human immune system. Due to the development of hybridoma technology, a large number of xenogeneic antibodies are highly immunogenic in humans, which can ultimately limit their clinical applications especially when administration may need to be repeated. Additionally, they can be rapidly removed from the circulation and can cause systemic inflammatory effects as well. Therefore, humanization strategies are desirable in some alternatives to circumvent these situations. Techniques for antibody humanization are known to those skilled in the art.

In some alternatives, a polynucleotide coding for a ligand binding domain is operably linked to a polynucleotide coding for a spacer region. In some alternatives, the polynucleotide coding for a ligand binding domain can also have one or more restriction enzyme sites at the 5′ and/or 3′ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide with another polynucleotide coding for a ligand binding domain coding for a different antigen or that has different binding characteristics. For example, a restriction site, NheI, is encoded upstream of the leader sequence; and a 3′ RsrII located within the hinge region allows subcloning of any desirable scFv into a chimeric receptor vector. In some alternatives, the polynucleotide is codon optimized for expression in mammalian cells, such as humans. Subcloning into a restriction site of a desired vector is a technique that is appreciated by those skilled in the art.

In some alternatives, the polynucleotide coding for a ligand binding domain is operably linked to a signal peptide. In some alternatives, the signal peptide is a signal peptide for granulocyte colony stimulating factor. Polynucleotides coding for other signal peptides such as CD8 alpha can be utilized. In some alternatives, the polynucleotide codes for CD8 alpha.

In some alternatives, the polynucleotide coding for a ligand binding domain is operably linked to a promoter. A promoter is selected that provides for expression of the chimeric antigen receptor in a mammalian cell, such as a human cell. In a specific alternative, the promoter is an inducible promoter.

Spacer

In some alternatives, the chimeric receptor nucleic acid comprises a polynucleotide coding for a spacer region. Typically, a spacer region is found between the ligand binding domain and the transmembrane domain of the chimeric receptor. In some alternatives, a spacer region provides for flexibility of the ligand binding domain and allows for high expression levels in lymphocytes. For example, a CD19-specific chimeric receptor having a spacer domain of 229 amino acids had less antitumor activity than a CD19-specific chimeric receptor with a short spacer region comprised of the modified IgG4 hinge only. In some alternatives, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor.

In some alternatives, a spacer region has at least 10 to 229 amino acids, 10 to 200 amino acids, 10 to 175 amino acids, 10 to 150 amino acids, 10 to 125 amino acids, 10 to 100 amino acids, 10 to 75 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 20 amino acids, or 10 to 15 amino acids, or a length within a range defined by any two of the aforementioned lengths. In some alternatives, a spacer region has 12 amino acids or less, 119 amino acids or less, or 229 amino acids or less but greater than 1 or 2 amino acids. In some alternatives, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor.

In some alternatives, the spacer region is derived from a hinge region of an immunoglobulin molecule. In some alternatives, a spacer region comprises all or a portion of the hinge region from a human IgG1, human IgG2, a human IgG3, or a human IgG4, and can contain one or more amino acid substitutions. In some alternatives, a portion of the hinge region includes the upper hinge amino acids found between the variable heavy chain and the core, and the core hinge amino acids including a polyproline region. In some alternatives, the spacer is optimized for increased T cell proliferation and/or cytokine production in response to the ligand as compared to a reference chimeric receptor.

In some alternatives, a short spacer region has 12 amino acids or less and comprises all or a portion of a IgG4 hinge region sequence or variant thereof, an intermediate spacer region has 119 amino acids or less and comprises all or a portion of a IgG4 hinge region sequence and a CH3 region or variant thereof, and a long spacer has 229 amino acids or less but not zero and comprises all or a portion of a IgG4 hinge region sequence, a CH2 region, and a CH3 region or variant thereof. In some alternatives, a short spacer region has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids or a size within a range defined by any two of the aforementioned amino acid lengths. In some alternatives, a medium spacer region has 13, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 119 amino acids or a size within a range defined by any two of the aforementioned amino acid lengths. In some alternatives, a spacer region has 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 219 amino acids or a size within a range defined by any two of the aforementioned amino acid lengths.

A polynucleotide coding for a spacer region can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In some alternatives, a polynucleotide coding for a spacer region is operably linked to a polynucleotide coding for a transmembrane region. In some alternatives, the polynucleotide coding for the spacer region can also have one or more restriction enzyme sites at the 5′ and/or 3′ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide with another polynucleotide coding for a different spacer region. In some alternatives, the polynucleotide coding for the spacer region is codon optimized for expression in mammalian cells, such as human cells.

In an alternative, the spacer region is a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 or a portion thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH2 region or variant thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH3 region or variant thereof, and a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of a CH2 region or variant thereof, and/or a CH3 region or variant thereof. In some alternatives, a short spacer region is a modified IgG4 hinge sequence having 12 amino acids or less but greater than one or two amino acids, an intermediate sequence is a IgG4 hinge sequence with a CH3 sequence having 119 amino acids or less but greater than one or two amino acids; or a IgG4 hinge sequence with a CH2 and CH3 region having 229 amino acids or less but greater than one or two amino acids.

Transmembrane Domain.

In some alternatives, the chimeric receptor nucleic acid comprises a polynucleotide coding for a transmembrane domain. The transmembrane domain provides for anchoring of the chimeric receptor in the membrane.

In an alternative, the transmembrane domain that naturally is associated with one of the domains in the chimeric receptor is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain can be derived either from a natural or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

Transmembrane regions in some alternatives comprise at least the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and/or CD154.

A transmembrane domain can be synthetic or a variant of a naturally occurring transmembrane domain. In some alternatives, synthetic or variant transmembrane domains comprise predominantly hydrophobic residues such as leucine and valine, for example. In some alternatives, a transmembrane domain can have at least 80%, 85%, 90%, 95%, or 100% amino acid sequence identity with a transmembrane domain as set forth in SEQ ID NO: 110-113 or an amino acid sequence identity that is within a range defined by any two of the aforementioned values. Variant transmembrane domains preferably have a hydrophobic score of at least 50 as calculated by Kyte Doolittle.

A polynucleotide coding for a transmembrane domain can be readily prepared by synthetic or recombinant methods. In some alternatives, a polynucleotide coding for a transmembrane domain is operably linked to a polynucleotide coding for an intracellular signaling region. In some alternatives, the polynucleotide coding for a transmembrane domain can also have one or more restriction enzyme sites at the 5′ and/or 3′ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide coding for a transmembrane domain with another polynucleotide coding for a different transmembrane domain. In some alternatives, the polynucleotide coding for a transmembrane domain is codon optimized for expression in mammalian cells, such as human cells.

Intracellular Signaling Domain.

In some alternatives, the chimeric receptor nucleic acid comprises a polynucleotide coding for an intracellular signaling domain. The intracellular signaling domain provides for activation of one function of the transduced cell expressing the chimeric receptor upon binding to the ligand expressed on tumor cells. In some alternatives, the intracellular signaling domain contains one or more intracellular signaling domains. In some alternatives, the intracellular signaling domain is a portion of and/or a variant of an intracellular signaling domain that provides for activation of at least one function of the transduced cell.

Examples of intracellular signaling domains for use in a chimeric receptor of the disclosure include the cytoplasmic sequences of the CD3 zeta chain, and/or co-receptors that act in concert to initiate signal transduction following chimeric receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation and provide a T cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs, which are known as receptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences suitable for use with alternatives described herein include those derived from CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and/or CD66d.

In a preferred alternative, the intracellular signaling domain of the chimeric receptor can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). For example, the intracellular signaling domain of the chimeric receptor can comprise a CD3zeta chain and a costimulatory signaling region.

The costimulatory signaling region refers to a portion of the chimeric receptor comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for a response of lymphocytes to an antigen. Examples of such molecules suitable for use in the alternatives described herein include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, zeta chain associated protein kinase (ZAP70), and/or a ligand that specifically binds with CD83.

The intracellular signaling sequences of the chimeric receptor can be linked to each other in a random or specified order. In some alternatives, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, can form the linkage. In one alternative, the intracellular signaling domains comprises all or a portion of the signaling domain of CD3-zeta or variant thereof and all or a portion of the signaling domain of CD28 or a variant thereof. In another alternative, the intracellular signaling domain comprises all or a portion of the signaling domain of CD3-zeta or variant thereof and all or a portion of the signaling domain of 4-1BB or variant thereof. In yet another alternative, the intracellular signaling domain comprises all or a portion of the signaling domain of CD3-zeta or variant thereof, all or a portion of the signaling domain of CD28 or variant thereof, and all or a portion of the signaling domain of 4-1BB or variant thereof. In a specific alternative, the amino acid sequence of the intracellular signaling domain comprises a variant of CD3zeta and a portion of the 4-1BB intracellular signaling domain.

In an alternative, a polynucleotide coding for an intracellular signaling domain comprises a 4-1BB intracellular domain linked to a portion of a CD3zeta domain. In other alternatives, a 4-1BB intracellular domain and a CD28 intracellular domain are linked to a portion of a CD3 zeta domain.

In some alternatives, the intracellular domain is encoded by a sequence set forth in SEQ ID NO: 114. In some alternatives, the intracellular domain is encoded by a sequence set forth in SEQ ID NO: 116. In some alternatives, the intracellular domain is encoded by a sequence set forth in SEQ ID NO: 118. In some alternatives, the intracellular domain comprises an amino acid sequence set forth in SEQ ID NO: 115. In some alternatives, the intracellular domain comprises an amino acid sequence set forth in SEQ ID NO: 117. In some alternatives, the intracellular domain comprises an amino acid sequence set forth in SEQ ID NO: 119.

A polynucleotide coding for an intracellular signaling domain can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In some alternatives, the polynucleotide coding for an intracellular signaling domain can also have one or more restriction enzyme sites at the 5′ and/or 3′ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide coding for an intracellular signaling domain with another polynucleotide coding for a different intracellular signaling domain. In some alternatives, the polynucleotide coding for an intracellular signaling domain is codon optimized for expression in mammalian cells. In some alternatives, the mammalian cells are human cells.

Marker Sequences.

In some alternatives, the system further comprises one or more marker sequences under the control of an inducible promoter. A marker sequence can provide for selection of transduced cells, and/or identification of transduced cells. In some alternatives, the marker sequence is for a selection of transduced cells and/or identification of transduced cells. In some alternatives, the marker sequence is operably linked to a polynucleotide sequence coding for a linker sequence. In some alternatives, the linker sequence is a cleavable linker sequence. In some alternatives, the linker is 2A linker such as, for example, T2A, P2A or F2A. In some alternatives, the linker is a cleavable T2A linker.

A number of different marker sequences can be employed. Typically, a marker sequence has a functional characteristic that allows for selection of transduced cells and/or detection of transduced cells. In some alternatives, the marker sequence is compatible with transduction of human lymphocytes. In some alternatives, the marker sequence allows for selection of transduced cells and/or detection of transduced cells.

The positive selectable marker can be a gene, which upon being introduced into the host cell, expresses a dominant phenotype permitting positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5, which codes for resistance to the antibiotic G418, the double mutant of the dihydrofolate reductase (DHFR) gene, which provides resistance to methotrexate, DHFR dm. Transduced cells cultured in the presence of these agents will survive and be selected.

In an alternative, a first nucleic acid further comprises a polynucleotide coding for a marker sequence. In an alternative, the marker sequence is a truncated epidermal growth factor receptor (EGFRt). In some alternatives, the marker sequence is a truncated Her2 sequence (Her2t). Selectable marker sequences that can be used in the alternatives set forth in this disclosure are provided below.

Selectable Marker Sequences

EGFRt nucleotide sequence:

(SEQ ID NO: 30)

Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagca

ttcctcctgatcccacgcaaagtgtgtaacggaataggtattggtgaattt

aaagactcactctccataaatgctacgaatattaaacacttcaaaaactgc

acctccatcagtggcgatctccacatcctgccggtggcatttaggggtgac

tccttcacacatactcctcctctggatccacaggaactggatattctgaaa

accgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaac

aggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc

aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatcc

ttgggattacgctccctcaaggagataagtgatggagatgtgataatttca

ggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgttt

gggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagc

tgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgc

tggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggc

agggaatgcgtggacaagtgcaaccttctggagggtgagccaagggagttt

gtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggcc

atgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcc

cactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatg

ggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgc

cacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaa

ggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtg

ggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatg

tga;

EGFRt amino acid sequence:

(SEQ ID NO: 31)

M;

Her2t nucleotide sequence:

(SEQ ID NO: 32)

Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagca

ttcctcctgatcccatgccaccctgagtgtcagccccagaatggctcagtg

acctgttttggaccggaggctgaccagtgtgtggcctgtgcccactataag

gaccctcccttctgcgtggcccgctgccccagcggtgtgaaacctgacctc

tcctacatgcccatctggaagtttccagatgaggagggcgcatgccagcct

tgccccatcaactgcacccactcctgtgtggacctggatgacaagggctgc

cccgccgagcagagagccagccctctgacgtccatcatctctgcggtggtt

ggcattctgctggtcgtggtcttgggggtggtctttgggatcctcatctg

a;

Her2t amino acid sequence:

(SEQ ID NO: 33)

PAEQRASPLTSITSAVVGILLVVVLGVVFGILI;

Her2tG nucleotide sequence:

(SEQ ID NO: 34)

Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagca

ttcctcctgatcccatgccaccctgagtgtcagccccagaatggctcagtg

acctgttttggaccggaggctgaccagtgtgtggcctgtgcccactataag

gaccctcccttctgcgtggcccgctgccccagcggtgtgaaacctgacctc

tcctacatgcccatctggaagtttccagatgaggagggcgcatgccagcct

tgccccatcaactgcacccactcctgtgtggacctggatgacaagggctgc

cccgccgagcagagagccagcccgttaacgggtggaggcagcggaggtggc

tccatcatctctgcggtggttggcattctgctggtcgtggtcttgggggtg

gtattgggatcctcatctga;

Her2tG amino acid sequence:

(SEQ ID NO: 35)

PAEQRASPLTGGGSGGGSIISAVVGILLVVVLGVVFGILI;

CD19t nucleotide sequence:

(SEQ ID NO: 36)

Atgccacctcctcgcctcctcttcttcctcctcttcctcacccccatggaa

gtcaggcccgaggaacctctagtggtgaaggtggaagagggagataacgct

gtgctgcagtgcctcaaggggacctcagatggccccactcagcagctgacc

tggtctcgggagtccccgcttaaacccttcttaaaactcagcctggggctg

ccaggcctgggaatccacatgaggcccctggccatctggcttttcatcttc

aacgtctctcaacagatggggggcttctacctgtgccagccggggcccccc

tctgagaaggcctggcagcctggctggacagtcaatgtggagggcagcggg

gagctgttccggtggaatgtttcggacctaggtggcctgggctgtggcctg

aagaacaggtcctcagagggccccagctccccttccgggaagctcatgagc

cccaagctgtatgtgtgggccaaagaccgccctgagatctgggagggagag

cctccgtgtgtcccaccgagggacagcctgaaccagagcctcagccaggac

ctcaccatggcccctggctccacactctggctgtcctgtggggtaccccct

gactctgtgtccaggggccccctctcctggacccatgtgcaccccaagggg

cctaagtcattgctgagcctagagctgaaggacgatcgcccggccagagat

atgtgggtaatggagacgggtctgttgttgccccgggccacagctcaagac

gctggaaagtattattgtcaccgtggcaacctgaccatgtcattccacctg

gagatcactgctcggccagtactatggcactggctgctgaggactggtggc

tggaaggtctcagctgtgactttggcttatctgatcttctgcctgtgttcc

cttgtgggcattcttcatcttcaaagagccctggtcctgaggaggaaaaga

taa;

CD19t amino acid sequence:

(SEQ ID NO: 37)

DHFRdm nucleotide sequence:

(SEQ ID NO: 38)

Atggttggttcgctaaactgcatcgtcgctgtgtcccagaacatgggcatc

ggcaagaacggggacttcccctggccaccgctcaggaatgaatccagatat

ttccagagaatgaccacaacctcttcagtagaaggtaaacagaatctggtg

attatgggtaagaagacctggttctccattcctgagaagaatcgaccttta

aagggtagaattaatttagttctcagcagagaactcaaggaacctccacaa

ggagctcattttctttccagaagtctagatgatgccttaaaacttactgaa

caaccagaattagcaaataaagtagacatggtctggatagttggtggcagt

tctgtttataaggaagccatgaatcacccaggccatcttaaactatttgtg

acaaggatcatgcaagactttgaaagtgacacgttttttccagaaattgat

ttggagaaatataaacttctgccagaatacccaggtgttctctctgatgtc

caggaggagaaaggcattaagtacaaatttgaagtatatgagaagaatga

t;

and

DHFRdm amino acid sequence:

(SEQ ID NO: 39)

EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND.

DNA spacer sequence:

(SEQ ID NO: 120)

Gctagcgtttaaacttaagcttggtaccgagctcggatccgccacc.

The DNA spacer sequence may be described also as

the 7xHBD/mE1B_DNA spacer.

In some alternatives herein, the DNA spacer, a 46 base pair sequence, may reside in between the 7×HBD/mE1B promoter and inducible transgene. The DNA spacer which comprises SEQ ID NO: 120, allows enhanced gene expression of the transgene. In some alternatives herein, a vector construct comprising a DNA spacer between a promoter and a transgene showed an increase in transcription of the transgene as compared to a vector that had the promoter and the transgene without a DNA spacer. In some alternatives herein, the DNA spacer enhanced transgene expression as compared to a vector that did not have a DNA spacer between the promoter and the transgene. In some alternatives, a DNA spacer is between the mE1B promoter and the transgene.

In some alternatives, the polynucleotide coding for the marker sequence is operably linked to a polynucleotide coding for a linker sequence. In some alternatives, the linker is a 2A linker, such as, for example, T2A, P2A, E2A or F2A.

A polynucleotide coding for marker sequence can be readily prepared by synthetic or recombinant methods from the amino acid sequence. In some alternatives, a polynucleotide coding for a marker sequence is operably linked to a polynucleotide coding for an intracellular signaling domain. In some alternatives, the polynucleotide coding for a marker sequence can also have one or more restriction enzyme sites at the 5′ and/or 3′ ends of the coding sequence in order to provide for easy excision and replacement of the polynucleotide coding for a marker sequence with another polynucleotide coding for a different marker sequence. In some alternatives, the polynucleotide coding for a marker sequence is codon optimized for expression in mammalian cells, preferably humans.

In some alternatives, two or more marker sequences can be employed. In some alternatives, a first marker sequence is under control of a constitutive promoter and provides for an indication that the transduced cell is expressing the transgene. In other alternatives, a second marker sequence is under the control of the inducible promoter and provides an indication that the transgene expression has been induced. In some alternatives, the marker under the control of the inducible promoter can be used to select for cells in which noninduced or basal expression is much lower than in other cells by selecting cells that have a lower expression of the marker sequence under the control of the inducible promoter and expand those cells for further applications. In some alternatives, the transgene encodes a polypeptide that regulates apoptosis, a polypeptide that modulates the extracellular environment, a polypeptide that modulates checkpoint signaling, a microRNA product that regulates growth, a microRNA product that regulates survival, or a microRNA product that regulates cytolytic capacity. In some alternatives, the transgene encodes a protein that modulates T cells. In some alternatives, the transgene encodes a chimeric antigen receptor.

Other Genetic Components Under Control of Inducible Promoter.

In some alternatives, the first nucleic acid comprises a polynucleotide sequence coding for genes that promote survival and proliferation, genes that prevent apoptosis, and/or genes that inhibit negative checkpoint signaling under the control of an inducible promoter. Such genes include genes encoding IL-2, IL-7, IL-15, chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, PD-1-IFNalpha and/or PD-1CD28 chimeras. These genes are also placed under the control of an inducible promoter as described herein. In some alternatives, the genes encode IL-2, IL-7, IL-15, chemokine receptors, chimeric cytokine receptors, Bcl2, CA-Akt, dn-TGFbetaRII, dn-SHP1/2, PD-1-IFNalpha, and/or PD-1CD28 chimeras. In some alternatives, the gene that modulates checkpoint signaling encodes a polypeptide that inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3.

In some alternatives, a first nucleic acid comprises a first inducible promoter linked to a polynucleotide coding for a cytokine, chemokine receptor, or chimeric cytokine receptor. chemokines, also referred to as chemotactic cytokines, are a group of structurally related proteins that regulate cell trafficking of lymphocytes. In some alternatives, the chemokines are homeostatic or inflammatory. Chemokine receptors include CCR2, CCR7, or CCR15. Cytokines include interleukins such as IL2, IL-12, IL-7, and/or Il-15, interferons, such as interferon 6, tumor necrosis factor, and a TLR4 agonist. Chimeric cytokine receptors can also include CCR(CD122). In some alternatives, the chemokine receptors comprise CCR2, CCR7, and/or CCR15. In some alternatives, the chemokine receptors include CCR2, CCR7, or CCR15. In some alternatives, the cytokines include interleukins, wherein the interleukins are 1L2, IL-12, IL-7 and/or 11-15 or interferons, wherein the interferons comprise interferon 6, tumor necrosis factor, or a TLR4 agonist.

In some alternatives, a first nucleic acid comprises a first inducible promoter linked to a polynucleotide coding for a polypeptide that regulates apoptosis. In some alternatives, genes that inhibit apoptosis include, for example, Bcl2, and/or CA-Akt. In some alternatives, the polypeptide is Bcl2 or CA-Akt. In some alternatives, genes that inhibit apoptosis include, for example, Survivin, Bcl2, CA-Akt, and/or dnCaspase3. In some alternatives, the polypeptide is Survivin, Bcl2, or CA-Akt and/or dnCaspase 3. In some alternatives, Survivin is encoded by a sequence set forth in SEQ ID NO: 124 (Atgggtgccccgacgttgccccctgcctggcagccctttctcaaggaccaccgcatctctacattcaagaactggcccttcttggag ggctgcgcctgcaccccggagcggatggccgaggctggcttcatccactgccccactgagaacgagccagacttggcccagtgttt cttctgcttcaaggagctggaaggctgggagccagatgacgaccccatagaggaacataaaaagcattcgtccggttgcgctttccttt ctgtcaagaagcagtttgaagaattaacccttggtgaatttttgaaactggacagagaaagagccaagaacaaaattgcaaaggaaac caacaataagaagaaagaatttgaggaaactgtgaagaaagtgcgccgtgccatcgagcagctggctgcaatggat). In some alternatives, Survivin comprises the amino acid sequence set forth in SEQ ID NO: 123 (MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQC FFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAK ETNNKKKEFEETVKKVRRAIEQLAAMD).

In some alternatives, a first nucleic acid comprises a first inducible promoter linked to a polynucleotide coding for a polypeptide that modulates checkpoint signaling. Such genes include dn-TGFbetaRII, dn-SHP1/2, PD-1-IFNalpha, and/or PD-1CD28 chimeras. In some alternatives, the polypeptide is dn-TGFbetaRII, dn-SHP1/2, PD-1-IFNalpha, and/or PD-1CD28 chimeras. In some alternatives, the polypeptide that modulates checkpoint signaling inhibits negative checkpoint regulators. In some alternatives, the negative checkpoint regulator comprises PD-1, VISTA, LAG-3 and/or TIM3.

Any number of nucleic acids can be placed under the control of an inducible promoter including those coding for chimeric antigen receptor, a marker sequences, a cytokine, a chemokine, an inhibitor of apoptosis, and/or an inhibitor of negative checkpoint signaling. In some alternatives, one or more inducible promoters can be utilized to provide for an adequate expression level of each of the nucleic acids. In some alternatives, constructs can be prepared with a gene such as a cytokine under the control of an inducible promoter and a construct comprising a chimeric antigen receptor under the control of a constitutive promoter. Such constructs are useful to provide for cell survival and proliferation of transduced cells, for example lymphocytes expressing a chimeric antigen receptor.

In the systems provided herein, the systems are inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor or a wild type HEA4 chimeric transcription factor.

In some alternatives, HEA3 comprises two amino acid mutations, wherein the sequence is encoded by a sequence set forth in SEQ ID NO: 122 (atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcagg cactcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagct ggccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcacccccccc atcctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccc tggcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaac cagagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcgg aagcagagagaggtggcccagcagtttacacacgccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccac caagaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaacccc agcaaagaggaacgggagacactggtggaagagtgcaaccgggccgagtgcatccagagaggcgtgagcccttctcaggctcag ggcctcggcagcaatctggtcaccgaagtgcgggtgtacaattggttcgccaaccggcggaaagaggaagccttccggcacaagct gtctgctggcgatatgagagccgccaacctgtggcccagccccctgatgatcaagcggagcaagaagaacagcctggccctgagc ctgaccgccgatcagatggtgtccgctctgctggacgccgagccccctatcctgtacagcgagtacgaccccaccagacccttcagc gaggccagcatgatgggcctgctgaccaacctggccgaccgggagctggtgcacatgatcaactgggccaagcgggtgcccggc ttcgtggacctgaccctgcacgaccaggtccacctgctggaatgtgcctggctggaaatcctgatgatcggcctcgtgtggagaagc atggaacaccccggcaagctgctgttcgcccccaacctgctcctggaccggaaccagggaaagtgcgtggagggcatggtggaga tcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaggaattcgtgtgcctgaagtccatcatcct gctgaacagcggcgtgtacaccttcctgtcatccaccctgaagtccctggaagagaaggaccacatccaccgggtgctggacaaga tcaccgacaccctgatccacctgatggccaaggctggcctgacactccagcagcagcaccagagactggcccagctgctgctgatc ctgagccacatccggcacatgagcaacaagcggatggaacacctgtacagcatgaagtgcaagaacgtggtgcccctgtacgacct gctgctcgagatgctggatgcccacagactgcacgcccctacaagcagaggcggagccagcgtggaggaaaccgaccagtctca cctggccaccgccggcagcacaagcagccacagcctgcagaagtactacatcaccggcgaggccgagggattccctgccaccgt ggagttccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattccagagcat catgaagaagtcccccttcagcggccccaccgatcccagacccccccctagaagaatcgccgtgcccagcagatctagcgccagc gtgcccaagcctgccccccagccctaccctttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttcccca gcggccagatctctcaggcctctgctctggcacctgctccacctcaggtgctgcctcaggcccctgctccagccccagcccctgccat ggtgtctgcactggcccaggctccagctcctgtgcctgtgctggcccctggacctcctcaggctgtggcccctcctgcccctaaacct acccaggccggggagggaacactgtctgaggccctgctgcagctccagttcgacgacgaggatctgggagcactgctgggcaata gcaccgaccccgccgtgtttaccgacctggcctccgtggacaacagcgagttccagcagctcctcaaccagggcatccctgtcgcc ccacacaccaccgagcccatgctgatggaataccccgaggccatcaccagactggtcacaggcgcccagaggcctccagatccag caccagctccactgggagcccctggcctgcctaatgggctgctgtctggcgacgaggacttcgagagcattgccgacatggacttca gcgccctgctgtcccagatcagcagc). In some alternatives, the HEA3 comprises an amino acid sequence set forth in SEQ ID NO: 121 (MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGEL AELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWR VAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVR KQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNP SKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPF SEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVC LKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLA QLLLILSHIRHMSNKRMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVE ETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVEFQYLPDTDDRHRIEEKRKRTYET FQSSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFP SGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIP VAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFESIADM DFSALLSQISS). This sequence comprises the sequence of HEA3 containing two point mutations (K310Q and S536E, positions in the RelA protein) in the p65 domain that enhance transcriptional activity. This is referred to as HEA3(p65/S536E/K310Q). K310Q in RelA corresponds to position 621 in HEA3; S536E corresponds to position 846 in HEA3.

Constitutive Promoter Systems.

In other alternatives, a system comprises a second nucleic acid that comprises a constitutive promoter or a second inducible promoter linked to a transcriptional activator. In other alternatives, a system comprises a second nucleic acid that comprises a promoter linked to a transcriptional activator. In some alternatives, the promoter is a constitutive promoter or an inducible promoter. In some alternatives, a constitutive promoter includes EF1α promoter, actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In some alternatives, viral promoters such as the CMV promoter are excluded. In some alternatives, the constitutive promoter can be linked to one or more of a polynucleotide coding for marker, or a chimeric antigen receptor as described herein.

Constitutive Promoters.

A constitutive promoter provides for continuous gene expression of the gene under the control of the promoter. In some alternatives, the constitutive promoter is a promoter that provides for gene expression in a lentiviral construct and/or in lymphocytes. In some alternatives, the promoter is not derived from a xenogenic source such as a plant or a virus.

In a specific alternative, the constitutive promoter comprises EF1α promoter, actin promoter, the myosin promoter, the hemoglobin promoter, and/or the creatine kinase promoter. In some alternatives, viral promoters such as the CMV promoter are excluded.

Transcriptional Activators.

In some alternatives, the constitutive promoter is operably linked to a transcriptional activator. In some alternatives, the transcriptional activator activates an inducible promoter in the presence of the inducer (e.g. drug).

In some alternatives, an inducible promoter is induced in the presence of a transcriptional activator. In some alternatives, the transcriptional activator preferentially binds to the promoter in the presence of the drug. In some alternatives, the transcriptional activator is TamR-tf (HEA3 or HEA4). Modification of the transcriptional activator can be made in the amino acid sequence that can affect the ability of an activator to bind to the drug, the promoter, or both. For example, binding in the ER ligand binding domain would affect the binding of the drug to the transcriptional activator. In some alternatives, HEA3 comprises two amino acid mutations, wherein the sequence is encoded by a sequence set forth in SEQ ID NO: 122 (atggtgtccaagctgtcccagctgcagacagaactgctggcagcactgctggaaagcggcctgagcaaagaggccctgattcagg cactcggcgaacctggaccttatctgctcgctggcgaaggccctctggataagggcgagagctgtggcggaggaagaggagagct ggccgagctgcctaacggcctgggcgagacaagaggcagcgaggacgagacagacgacgacggcgaggacttcacccccccc atcctgaaagagctggaaaacctgagccccgaggaagccgcccaccagaaagccgtggtggagacactgctgcaggaagatccc tggcgggtcgccaagatggtcaagagctacctgcagcagcacaacatcccccagcgggaggtggtggacaccaccggcctgaac cagagccacctgagccagcacctgaacaagggcacccccatgaaaacccagaagagagccgccctgtacacttggtacgtgcgg aagcagagagaggtggcccagcagtttacacacgccggccagggcggcctgatcgaggaacctaccggcgacgagctgcccac caagaagggcagacggaaccggtttaagtggggccctgcatctcagcagatcctgttccaggcctacgagcggcagaagaacccc agcaaagaggaacgggagacactgg