Compositions and methods for enhanced payload regulation using nuclear factor of activated t cells (NFAT) response elements

Abstract

Provided herein are nucleic acid constructs comprising one or more nuclear factor of activated T cells (NFAT) response elements and an expressible nucleic acid sequence encoding a first engineered polypeptide monomer, wherein the first engineered polypeptide monomer comprises a regulatable polypeptide payload and at least one drug responsive domain (DRD). The abundance, availability, and / or biological activity of the payload is regulated by interaction of an effective amount of immune cell stimulating agent with the one or more NFAT response element and by the interaction of an effective amount of ligand and the one or more DRDs.

Description

[0001] PATENTAttorney Docket No.: 108407-1462783-053WO1 Client Reference No.: 2095.2300PCT COMPOSITIONS AND METHODS FOR ENHANCED PAYLOAD REGULATION USING NUCLEAR FACTOR OF ACTIVATED T CELLS (NFAT) RESPONSE ELEMENTS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 607,998, filed December 8, 2023, which is incorporated by reference herein in its entirety and for all purposes. REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK This application contains a Sequence Listing submitted electronically and is hereby incorporated by reference in its entirety. The Sequence listing .xml file is entitled “108407- 1462783-053WO1”, is 327,000 bytes in size, and was created on December 3, 2024. BACKGROUND Biological products (biologics) as defined by the U.S. Food and Drug Administration include vaccines, blood and blood components, allergens, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. Cells from the same subject (autologous), from a subject of the same species (homologous or allogenic), or even from a different species (heterologous) can be administered to a subject as a biologic. In adoptive cell therapy (“ACT”), for example, T cells originating from a subject (or sometimes from a different source) are removed, genetically engineered to address the specific needs of the same or different subject, and then transferred back into the same or different subject. There are currently obstacles to widespread adoption and success of biologics such as ACT. Maintaining therapeutically effective levels of a biologic administered to a patient can be difficult, as the biologic may be expressed by an engineered cell at toxic levels. For example, an engineered cell may express biologics for an unacceptably sustained duration. Further, engineered cells lack spatial specificity needed to ensure biologic expression only where antigen density is high, such as at the site of a tumor. There is therefore a need for both temporal and spatial regulation of the biologics for optimization of therapeutic benefits and to facilitate widespread adoption of biologic therapies. BRIEF SUMMARY Provided herein are nucleic acid constructs comprising one or more nuclear factor of activated T cells (NFAT) response elements operably linked to an expressible nucleic acid sequence encoding a first engineered polypeptide monomer comprising a payload operably linked to at least one drug responsive domain (DRD). Also provided are methods, vectors, and cells using the nucleic acid construct to regulate control of payload activity. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 are graphs showing a combination of transcriptional and translational control enables spatiotemporal regulation of payload activity. FIG 2. is a schematic of an embodiment of a nucleic acid construct according to this disclosure. FIG 3. are graphs showing IL-12 expression in nucleic acid constructs comprising one or more NFAT response elements from DSCR1 in the presence and absence of an immune cell stimulation agent and / or ligand. FIG 4. are graphs showing GFP expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.5 are graphs showing GFP expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.6 are graphs showing GFP expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.7 are graphs showing GFP expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.8 are graphs showing GFP expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.9 is a graph showing the percent of IL-12 constructs in a Thy1.2 flow cytometry gate. FIG.10 is a graph showing the percent of IL-12 constructs in a Thy1.2 flow cytometry gate. FIG.11 are graphs showing mbIL-12 expression in nucleic acid constructs following 24-hours of stimulation with either of two immune cell stimulation agents. FIG.12 is a graph showing mbIL-12 expression in nucleic acid constructs following 24-hours stimulation with an immune cell stimulation agent and / or ligand. FIGS.13A and 13B are graphs showing IL-12 expression in the supernatant following either 3 hours (FIG.13A) or 24-hours (FIG.13B) stimulation with an immune cell stimulation agent and / or ligand. FIG.14 are graphs showing IL-12 expression in the supernatant following 24-hours stimulation with an immune cell stimulation agent and / or ligand. FIG.15 are graphs showing IL-12 expression in nucleic acid constructs in the presence and absence of an immune cell stimulation agent and / or ligand. FIG.16 is a schematic illustration of the testing used to determine dual regulation of IL-12 in primary human T cells. FIG.17 is a schematic illustration of the co-culture assay used to determine TH1 skewing in primary human T cells. FIGS.18A and 18B are graphs showing IL-12 expression on the cell surface following stimulation with either of two immune cell stimulation agents in the presence or absence of ligand. In FIG.18A, CD3 / CD28 antibodies are the stimulation agents. In FIG. 18B, phorbol 12-myristate 13-acetate is the stimulation agent. FIGS.19A and 19B are graphs showing IL-12 expression in supernatant following stimulation with either of two immune cell stimulation agents in the presence or absence of ligand. In FIG.19A, CD3 / CD28 antibodies are the stimulation agents. In FIG.19B, phorbol 12-myristate 13-acetate is the stimulation agent. FIG.20 are graphs showing Granzyme B expression on the cell surface following stimulation with an immune cell stimulation agent in the presence or absence of ligand. FIGs.21A and 22B are graphs showing dual regulation of IL-12 according to the system provided herein using different DRD types. FIG.22A and 22B are graphs showing dual regulation of IL-12 in human tumor infiltrating lymphocytes according to the system provided herein. FIG.23 is a graph showing dual regulation of IL-12 in an immunocompetent in vivo murine model using an hDHFR drug responsive domain and NFAT response elements. Mice in the “vector control” group received the mIL12-012 construct, mice in the “NFAT” group received the mIL12-115 construct, and mice in the “NFAT+hDHFR” group received the mIL12-110 construct. FIG.24 is a schematic illustration of the study design used to determine dual regulation of IL-12 in an immunocompetent in vivo murine model. DETAILED DESCRIPTION The present disclosure provides compositions and systems for dual regulatory control of payload activity (“regulation compositions and systems”). Regulation compositions according to this disclosure include nucleic acid constructs, wherein a nucleic acid construct comprises one or more NFAT response elements operably linked to an expressible nucleic acid sequence encoding a first engineered polypeptide monomer comprising a payload operably linked to at least one DRD. Optionally, this disclosure enables both transcriptional and translational control of payload activity, ultimately providing spatiotemporal regulation of payload activity. Optionally, nucleic acid constructs comprising one or more NFAT response elements provide spatial transcriptional control, wherein transcription of the first engineered polypeptide monomer begins after antigen recognition and T cell receptor signaling. Further, optionally, the first engineered polypeptide monomer comprising a payload operably linked to at least one DRD provides translational control of payload activity that is temporally linked to ligand administration. Regulation systems according to this disclosure comprise the regulation compositions, an immune cell stimulation agent, and a stabilizing ligand. The NFAT family of transcription factors are important regulators of T cell activation. NFAT binds NFAT response elements in response to T cell receptor mediated signaling post- antigen recognition. As such, integration of the one or more NFAT response elements into the nucleic acid construct described herein provides spatial, antigen-dependent, regulation of transcription. DRDs are unstable polypeptides that degrade in the absence of their corresponding stabilizing ligand (also referred to as the paired ligand or ligand), but whose stability is rescued by binding to the stabilizing ligand. Because binding of the ligand to the DRD is reversible, later removal of the ligand results in the DRD unfolding, becoming unstable, and ultimately being tagged for degradation by the ubiquitin-proteasome system (“UPS”). Accordingly, it is believed that when a DRD is operably linked to a payload, the entire construct (i.e., DRD plus payload) itself is rendered unstable and degraded by the UPS. However, in the presence of the paired ligand, the construct is stabilized, and the payload remains available for use. In this way, the at least one DRD operably linked to the payload provides a temporal (concurrent with ligand delivery) regulation for translation. Further, it is believed that the conditional nature of DRD stability allows a rapid and non-perturbing switch from stable polypeptide to unstable UPS substrate, and may facilitate regulation of a payload’s activity level, and / or modulation of a payload’s activity level. Multiple DRDs associated together in a polypeptide monomer result in a payload having a lower off-state as compared to the same payload operably linked to a single DRD. That is, in the absence of ligand, increasing the density of DRDs (for example with multiple DRDs) is thought to reduce the abundance or availability of a payload as compared to a payload that is operably linked to only a single DRD. Payloads should be understood to include one or more polypeptides having one or more functions, such as one or more biological activities, desired to be regulated. Payloads include multiple classes of therapeutically important polypeptides (proteins and peptides) such as Type I / II membrane proteins, cytokines, immunomodulatory proteins, intracellular proteins, secreted proteins, CAS9 proteins, and transcription factor proteins. Because the abundance and availability of a payload are related to the activity of a payload, for purposes of this disclosure, the terms “abundance,” “availability,” “activity,” and the phrase “abundance and / or activity” (and similarly “level of abundance,” “level of availability,” “level of activity,” and “level of abundance and / or activity”) are used interchangeably throughout this disclosure and are generally referred to as “activity,” unless explicitly stated otherwise or nonsensical in context. Further, measurements of abundance or availability are used as a proxy for activity level and may be used herein to reflect the activity level. Consequently, changes in the abundance or availability of a payload in the presence of an effective amount of ligand as compared to in the absence of ligand optionally serves as a proxy for measuring changes in activity level. Nucleic Acid Constructs Provided herein are nucleic acid constructs comprising one or more NFAT response elements. The nucleic acid constructs can also include one or more of the expressible nucleic acid sequences encoding one or more engineered polypeptide monomers. The one or more engineered polypeptide monomers comprises a payload operably linked to at least one DRD. The at least one payload has a biological activity, and the biological activity of the payload is regulated by the modulation of expression of the first engineered polypeptide monomer by the interaction of one or more NFAT response elements and an effective amount of an immune cell stimulation agent, and by the interaction of the at least one DRD and an effective amount of the ligand. NFAT Response Elements NFAT is a family of transcription factors important in regulation of T cell activation. NFAT response elements are found in many different proteins, for example, in interleukin-2 (IL-2) promotor (see for example, Chow, et al,, Mol. Cell Biol.19(3): 2300-2307), the Down syndrome critical region 1 (DSCR1) promotor (see for example Lee, M. et al, Hum. Mol. Genet.19(3): 468-479), the interleukin-4 (IL-4) promoter (see for example Burke, et. al.. BBRC, 270(3) 1016-1023) and in transmembrane P24 trafficking protein 10 (TMP21) promoter (see for example Liu, et al., Mol. Neurodegen.6(21): 1-11). The nucleic acid construct described herein comprises one or more (such as 2, 3, 4, 5, 6, or more) NFAT response elements. The NFAT response elements may be those having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% % identity to SEQ ID NOs: 14, 15, 16, 17, or 18, respectively. Optionally, the nucleic acid construct described herein comprises 6 NFAT response elements, for example, comprising the nucleotide sequence of SEQ ID NOs: 14, 15, 16, 17, or 18 repeated 6 times. The NFAT response elements provided herein may be responsive to an immune cell stimulation agent also described herein. Immune Cell Stimulation Agents Immune cell stimulation agents may be any agent that produces a population of activated immune cells under a suitable condition. Immune cell stimulation agents used herein may activate immune cells through an immune cell receptor, for example through a T cell receptor or B cell receptor. By way of example, immune cell stimulation agents include suitable T-cell stimulating agents including, but not limited to, an antibody or functional fragment thereof which targets a T-cell stimulatory or co-stimulatory molecule (e.g., anti- CD2 antibody, anti-CD3 antibody (such as OKT-3), anti-CD28 antibody, or a functional fragment thereof), a cytokine production stimulating reagent, such as phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin, or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), self / foreign peptides presented on MHC(HLA) Class I / II, MHCI / II oligomers (including tetramers), superantigens or a natural ligand to a T-cell-stimulatory or co-stimulatory molecule. Expressible Nucleic Acid Sequences Encoding Engineered Polypeptide Monomers Provided herein are expressible nucleic acid sequences encoding one or more engineered, regulatable polypeptide monomers or regulatable polypeptides as described herein. Expressible nucleic acid sequences provided herein may be regulated by the one or more NFAT response elements via immune-cell receptor mediated signaling post-contact with an immune cell stimulation agent or after contact with an effective amount or selected dose of immune cell stimulation agent. Optionally, the expressible nucleic acid sequences encoding a first engineered polypeptide monomer include at least one DRD and / or at least one payload and optionally comprise additional components, such as oligomerization domains, hinges, linkers, transmembrane domains, tags, and intracellular / cytoplasmic and transmembrane tails as described herein. Optionally, a single expressible nucleic acid sequence comprises nucleic acid sequences that encode all components of a monomer as described herein. For example, the expressible nucleic acid sequence encodes at least a payload, at least one DRD, and an optionally, an oligomerization domain. When expressed multiple times, the monomers can assemble into oligomers, for example, dimers, trimers, tetramers, pentamers or hexamers depending on the oligomerization domain and by way of the oligomerization domain. Optionally, a single expressible nucleic acid sequence encodes multiple copies of the same or different monomers, which, when expressed assemble into an oligomer. Optionally, certain expressible nucleic acid sequences encode an oligomerization domain and at least one payload, and certain nucleic acid constructs encode an oligomerization domain and at least one DRD, such that when expressed they assemble into an oligomer comprising at least one payload and at least one DRDs. The expressible nucleic acid sequences encoding monomers and regulatable polypeptides optionally also encode additional components such as signal sequences and cleavage sites including shedding domains. The expressible nucleic acid sequences optionally further comprise a promoter sequence and other regulatory elements (enhancers, translational control elements (e.g., IRES), and elements that control half-life). Further provided is a plurality of expressible nucleic acid sequences, each expressible nucleic acid sequence encoding one or more monomers or polypeptides described herein. Such a plurality of expressible nucleic acid sequences, upon expression, optionally provide for monomers that oligomerize. The expressible nucleic acid sequences of the plurality can be the same or different. Thus, the expressible nucleic acid sequences of the plurality may encode the same polypeptides or monomers or may encode polypeptides or monomers comprising different DRDs, payloads, and / or additional components. Components of Monomers and Regulatable Polypeptides Example components (building blocks) of the engineered monomers which associate to form regulatable, engineered oligomers, and of the engineered, regulatable polypeptides described herein are referenced throughout this disclosure and provided below. As described herein, monomers include at least a payload and at least one DRD, or a payload, or at least one DRD. Optionally, monomers described herein include an oligomerization domain. As also described herein oligomers and polypeptides include one or more payloads and at least one DRD. The monomers, oligomers, and polypeptides optionally include one or more additional components such as linkers, hinges (e.g., sheddable and non-sheddable hinges), tails (e.g., cytoplasmic tails), and transmembrane domains. As described below in the section related to methods of making, one of skill in the art may select from the various components using guideposts provided below to achieve a desired outcome (e.g., location of the oligomerization domain, payload, or DRD relative the cell in which it is expressed, whether or not the payload is secreted from the cell or tethered to the membrane, or the desired activity of the payload or payloads). DRDs DRDs interact with a ligand such that, when the DRD is operatively linked to a

[0002] Feng et al. (2015) Elife 4:e10606, the contents of each of which are hereby incorporated by reference in their entirety. The DRDs, by way of example, can be chosen from FKBP (SEQ ID NO: 8), ecDHFR (SEQ ID NO: 6), hDHFR (SEQ ID NO: 7), ER (SEQ ID NO: 11), PDE5 full length (SEQ ID NO: 10), PDE5 ligand binding domain (SEQ ID NO: 9), CA2 (SEQ ID NO: 2, encoded by the nucleic acid sequence of SEQ ID NO: 1), M1del mutation in CA2 (SEQ ID NO: 5), and L156H mutation in CA2 (SEQ ID NO: 4, encoded by the nucleic acid sequence of SEQ ID NO: 3) or a portion of any of the foregoing that maintains DRD function or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, or 11 or the DRD functional portion thereof. One or more mutations (including truncations, substitutions, and deletions) in the amino acid sequence of FKBP, ecDHFR, hDHFR, ER, PDE5, and CA2, for example, can be advantageous to further destabilize the DRD. Numerous DRD are described herein, but one of skill in the art can identify additional DRDs suitable for use in regulatable compositions according to this disclosure. By way of example, DRDs can be identified using library screening and structure-guided engineering to select the optimal DRD variant with sufficient instability in the absence of the ligand and sufficient stability in the presence of the ligand. A variant library can be generated using random mutagenesis screening by transducing cells (e.g., Jurkat cells) with mutant DRD candidates. To produce an enriched library, cells with the desired characteristics (low basal activity / expression and high dynamic range of activity / expression) are selected by testing polypeptide abundance across a range of concentrations of ligand. Single cell clones are then produced and characterized to identify candidate DRDs. The DRDs described herein are responsive to a paired ligand (also referred to as a “stabilizing ligand” or simply as a “ligand.” Optionally, the DRDs are responsive to a paired ligand that is a small molecule drug, such as an FDA-approved small molecule. However, one of skill in the art can select the DRD and its paired ligand to meet the specific needs of the system. Examples of DRD / ligand pairs are shown in Table 1. Table 1. Listing of DRD and exemplary ligands Optionally, a DRD of the present disclosure may be derived from a carbonic anhydrase, which is a member of a superfamily of metalloenzymes. For example, human carbonic anhydrase (hCA2) can be adapted for use as a DRD. A DRD of the present disclosure may be derived from amino acids 1-260 of CA2 (Uniprot ID: P00918). Optionally, DRDs are derived from CA2 comprising amino acids 2-260 of the parent CA2 sequence (e.g., amino acids 2-260). This is referred to herein as a CA2 M1del mutation (SEQ ID NO: 5). Optionally, a DRD of the present disclosure comprises a region of or the whole human carbonic anhydrase 2, and further comprises one or more mutations relative to the full-length sequence selected from M1del, L156H, and S56N. Optionally, the DRD is selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, or 11. Oligomerization Domains For purposes of this disclosure, oligomerization domains are sequences that promote self-assembly of monomers into oligomers, for example, by non-covalent bonding. Such domains may be found in nature and may be adapted for use in forming oligomers including a payload and at least one DRD as described herein. Examples of oligomerization domains include, but are not limited to, phospholamban, collectin, collagen, VASP, CD40L, TNFSF (e.g., TNFSF14, also referred to herein as LIGHT and 4-1BBL), langerin, DAP12, and NKG2C. Optionally, the oligomerization domain is a phospholamban transmembrane domain, collagen 18, avidin, streptavidin, CD40L extra cellular domain (ECD), langerin ECD, 4-1BBL ECD, VASP tetramer, DAP12, LIGHT extracellular domain (ECD), collectin 7, fungal lectin (5xzk), aspartate transcarbomylase (1EKX), 4-OT trimer, RH3 designed coiled coil (1TGG), HIV-1 gp41, FIV dUTP pyrophosphate, SIV gp41, the Ebola virus gp-2, HTLV-1 gp-21 domain, a foldon domain, a GCN4 domain, a T4 fibritin domain, the yeast heat shock transcription factor, and human collagen VIII, Collagen 18, Collagen 15 and hnRNP. Certain oligomerization domains do not oligomerize with themselves and only oligomerize with a different oligomerization domain, such as NKG2C. Such oligomerization domains are referred to herein as hetero-oligomerization domains. Payloads By way of example, the payload can be any polypeptide having a desired biological function. Such payloads can be modified polypeptides such as glycosylated polypeptides or lipopeptides, which upon expression are modified in a cell by constitutive enzymatic activity or by over-expression of selected enzymes. Payloads include multiple classes of therapeutically important polypeptides or any active portion thereof. For example, payloads include Type I / II membrane proteins such as CD40L, 4-1BBL, and CAR or active portions thereof. Payloads also include cytokines such as IL15, IL12 (SEQ ID NO: 13, encoded by thenucleic acid sequence of SEQ ID NO: 12) L1 IL2, IL7, IL18, IL21, IL23, IL36, TNF ,IFN , IFN , IFN , etc., including membrane-tethered forms thereof or active portionsthereof. Payloads also include intracellular polypeptides such as dnSHP2, T7, RNA polymerase, Cas9, or active portions thereof. Payloads include secreted proteins such as VEGF-trap, and native cytokines, or active portions thereof. And payloads further include transcription factors such as Foxp3, c-Myc, STAT5, and c-Jun, or active portions thereof, or constitutively active versions thereof. Other examples of suitable payloads include cytokine receptors, T-cell receptors (TCR), chimeric antigen receptors (CAR), immunomodulatory proteins in addition to those already exemplified, or any active portion thereof. The payload can also be a gene editing polypeptide or transcription factor in addition to those previously exemplified. The payload can also be a combination of polypeptides having a desired combination of actions, or active portions thereof. Optionally, payloads are therapeutic agents chosen from a cancer therapeutic agent, a therapeutic agent for an autoimmune disease, an immunotherapeutic agent, an anti- inflammatory agent, an anti-pathogenic agent, a gene therapy agent, or combinations thereof. The immunotherapeutic agent may be an antibody or fragments and variants thereof, a TCR, a CAR, a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a co-stimulatory molecule, a cytokine, a mutated version of a cytokine possessing altered receptor binding properties (also called a mutein), a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor, a kinase, a phosphatase, a dominant negative version of a phosphatase (such as SHP-1 or SHP- 2), a dominant negative signaling molecule or receptor (such as a dominant negative Fas Receptor), a dominant negative transcription factor, and a safety switch. Optionally, payloads of the present disclosure may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumornecrosis factors (TNFs), interferons (IFNs), TGF , and chemokines. The interleukins may bechosen from IL15, IL12 (SEQ ID NO: 13, encoded by the nucleic acid sequence of SEQ IDNO: 12), L1 IL2, IL7, IL18, IL21, IL23, IL36, and variants thereof including membrane-bound, secreted, fusion polypeptide, or cytokine mutants with altered receptor binding properties (such as muteins), and bicistronic forms of the interleukins, and combinations thereof. For example, IL12 may include both p35 and p40 subunits encoded by a single nucleic acid that produces a single chain polypeptide. The single chain polypeptide may be generated by placing the p35 subunit at the N terminus or the C terminus of the single chain polypeptide. Similarly, the p40 subunit may be at the N terminus or C terminus of the single chain polypeptide. By way of another example, the payload may be a bicistronic IL12 containing p40 and p35 subunits. Optionally, the payload can be an active portion or variant of a polypeptide with a desired biological function, so long as the payload retains the desired biological function. By way of example, the payload may be the p40 subunit of IL12 or the p35 subunit of IL12 or a variant of IL12 that promotes NK cell survival, regulates NK cell and T cell activation and proliferation, and / or supports NK cell development from hematopoietic stem cells. Optionally, payloads of the present disclosure may be chimeric antigen receptors (CARs) comprising an extracellular targeting domain (e.g., a scFv that recognizes a specific tumor antigen or other tumor cell-surface molecules), a transmembrane domain / region, andan intracellular signaling / activation domain (e.g., the signal region of CD3 , and / or one ormore costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX- 40 (CD134)). Optionally, payloads can be selected that reduce immune responses in a subject. For example, the payload can be an anti-cytokine, such as neutralizing antibodies to tumornecrosis factor (TNF)- or an interleukin. Optionally, payloads of the present disclosuretarget B-cell depletion, such as neutralizing antibodies to CD20, CD22, CD28, CTLA-4, and B-lymphocyte stimulator (BlyS). Optionally, payloads can also be contractile proteins (e.g., actin and myosin), enzymes (e.g., lactase and pepsin), hormones (e.g., insulin, oxytocin, and somatotropin), structural proteins (e.g., keratin, collagen, and elastin), storage proteins (e.g., ovalbumin and ferritin), transport proteins (e.g., hemoglobin), membrane-bound proteins (e.g., class I, II, or III transmembrane proteins; receptors, transporters, and the like). Optionally, payloads of the present disclosure may be one or more components of a gene editing system. In such examples, the oligomer or engineered, regulatable polypeptide regulates activity of the gene editing system and consequently expression of a downstream target protein. A target protein, as used herein, refers to a protein selected for gene editing, including, for example, a protein having a genetic mutation that results in a deleterious effect in a subject or in a cell. For example, payloads of the present disclosure may be a Cas protein (CRISPR-associated protein), including Cas9 and Cas12. The Cas protein may be altered or otherwise modified. For example, the Cas protein may be a deadCas9. Optionally, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein wild-type protein, a Cas9 protein nickase or a nuclease null or nuclease deficient Cas9 protein. Such payloads optionally include nucleases (e.g., Zinc finger nuclease, TALEN (Transcription activator-like effector-based nucleases), or meganucleases) and / or recombinases, such as a Cre recombinase. Payloads useful in the present disclosure also include polypeptides involved in nucleic acid synthesis and replication, for example, DNA and RNA polymerases, transcription factors, primases, helicases, RNases, ligases, topoisomerases, endonucleases, IRES, and telomerases. Linkers Linker sequences (linkers) are known in the art and are described in references cited herein. Linkers include, for example, GS linkers, GSG linkers, and GGSG linkers. These linkers are repeats of the subunit one or more times. Thus, a GS linker is a GSnlinker where n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Similarly, a GSG linker is a GSGn linker wherein n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. A GGSG linker is a GGSGn linker where n is a numerical number being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Hinges and Transmembrane Domains A hinge sequence is a short sequence of amino acids that facilitates flexibility between connected components. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin (e.g., an IgG4 Fc hinge), or theextracellular regions of type 1 membrane proteins such as CD8 CD4, CD28 and CD7, whichmay be a wild type sequence or a derivative thereof. Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. Optionally, the hinge is derived from a transmembrane domain. Transmembrane domains, useful in the engineered regulatable polypeptide constructsof the present disclosure can include, for example, a MHC1 transmembrane domain, a CD8transmembrane domain, a B7-1 transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 Fc region. Intracellular / Cytoplasmic or Transmembrane Tails Optionally, the herein provided monomers or polypeptide constructs comprise an intracellular / cytoplasmic or transmembrane tail. Optionally, the intracellular / cytoplasmic or transmembrane tail is a CD8, CD40L, LIGHT, NKG2C, or B7.1 intracellular tail. The absence of a transmembrane region in a construct can be, for example, designed for a secreted payload or a payload with intracellular or intranuclear activity. Tags Optionally, the monomers or polypeptides described herein include a tag. Such a tag allows for isolation or detection of the monomer or polypeptide or for isolation or detection of the payload. Such tags optionally include fluorescent proteins (e.g., green fluorescent protein), His-tag, HA-tag, Myc tag, FLAG tag, mCherry, CD20, CD34, nerve growth factor receptor (NGFR), truncated NGFR (tNGRR), epidermal growth factor (EGFR), or a truncated EGFR (tEGFR). Polypeptide Monomers, and Engineered, Regulatable Oligomers Provided herein are engineered polypeptide monomers and engineered, regulatable oligomers of at least two engineered polypeptide monomers. Oligomers comprise at least one payload operably linked to at least one DRD. The payload has a biological activity, and the biological activity of the payload is regulated by interaction of the at least one DRD (e.g., 1, 2, 3, 4, or more, which are responsive to the same or different ligands) and an effective amount of the ligand. Optionally, the monomers comprise at least one payload, an oligomerization domain (e.g., a dimerization domain, a trimerization domain, a tetramerization domain, a pentamerization domain, or a hexamerization domain) configured to promote oligomerization of the monomers, and at least one DRD responsive to a ligand, wherein the at least one payload is operably linked to the at least one DRD. In certain examples, the monomers comprise an oligomerization domain; and, at least one payload, or at least one DRD, or at least one payload and at least one DRD, provided that when at least two or more monomers oligomerize to form an oligomer, the resultant oligomer comprises at least one payload operably linked to at least one DRD. Oligomers according to this disclosure are a multimeric association of at least two engineered, regulatable polypeptide monomers. The monomers of the oligomer associate through an oligomerization domain. The oligomer, for example, can be a dimer resulting from association of monomers via a dimerization domain; or it can be a trimer resulting from association of monomers via a trimerization domain; or it can be a tetramer resulting from association of monomers via a tetramerizing domain; or it can be a pentamer resulting from association of monomers via a pentamerizing domain; or it can be a hexamer resulting from association of monomers via a hexamerizing domain. In each case, the oligomer may result from monomers that are the same or different. Accordingly, the payload or payloads of each of the monomers is the same or different and the at least one DRD of each of the monomer is the same or different. Thus, by way of example, the oligomer can be an association of three monomers, wherein each monomer comprises the same payload, oligomerization domain, and DRD and consequently the oligomer comprises a first, second, and third payload that are the same and a first, second, and third DRD that are responsive to the same ligand. Thus, provided herein is also an engineered, regulatable oligomer comprising at least two polypeptide monomers, wherein each monomer comprises an oligomerization domain and at least one payload and at least one DRD, wherein the oligomer comprises at least two payloads operably linked to at least two DRDs, wherein the at least one payload has a biological activity, wherein the at least one DRD is responsive to a ligand. Optionally, the oligomer is a hetero-oligomer wherein the at least two polypeptide monomers of the hetero- oligomer each comprise an oligomerization domain and wherein in some examples the oligomerization domain of one of the polypeptide monomers comprises a hetero- oligomerization domain. Optionally the DRDs in the hetero-oligomer are the same or different. Optionally, the at least two DRDs are responsive to the same ligand. By way of example, the engineered, regulatable hetero-oligomer comprises a first, a second, and a third polypeptide monomer, wherein the first and second polypeptide monomers are the same, wherein each of the first and second polypeptide monomers comprises a DRD, and wherein the third polypeptide monomer comprises a hetero-oligomerization domain and a payload. Hetero-oligomeric constructs can similarly include a first and second monomers, each including a DRD and payload, and a third monomer including a DRD without a payload. The engineered, regulatable hetero-oligomer, however, can have a 1:1 payload:DRD ratio. Multiple payloads in an oligomer can be the same or different, but in some examples, each of the payload(s) has a biological activity under appropriate conditions. In some such examples, the level of activity of each payload in the oligomer has a range spanning from a basal level of activity in the absence of ligand to a maximum activity in the presence of a saturating amount of ligand, and the basal activity of each payload of the oligomer is less than that of the same payload in a control construct. In some examples, the range of activity of each payload in the oligomer is greater than that of the same payload in a control construct. When expressed, the polypeptide monomers self-assemble via the oligomerization domains and form a multimeric regulatable polypeptide (an engineered regulatable payload composition, which when formed from monomers is also referred to as an oligomer) according to examples of the present disclosure. The degree of multimerization is generally determined by the choice of oligomerization domain. Thus, for example, constructs having CD40L ECD, Collagen 18, Lectin, DAP12, and 41BBl oligomerization domains tend to form trimerized regulatable payload compositions, whereas those with VASP oligomerization domains tend to form tetrameric regulatable payload compositions, and those with phospholamban oligomerization domains tend to form pentameric regulatable payload compositions. Ligands Ligands may be any agent that binds to the DRDs of the engineered, regulatable polypeptides or oligomers described herein, an effective amount of which results in a measurable change in a modification of a characteristic (e.g., abundance, availability, activity) of a payload operably linked to the DRD. Optionally, ligands may be synthetic molecules. Optionally, stabilizing ligands of the present disclosure may be small molecule compounds. Stabilizing ligands are optionally small molecule therapeutic drugs previously approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA). Examples of stabilizing ligands and their corresponding DRDs suitable for use described herein are shown in Table 1, in patents and applications referenced above in the section exemplifying suitable DRDs, and in U.S. Patent No.9,487,787 filed March 33, 2012, U.S. Patent No.10,137,180 filed September 6, 2013, PCT Application No. PCT / US2018 / 037005, filed June 12, 2018, PCT Application No. PCT / US2019 / 036654 filed June 12, 2019, PCT Application No. PCT / US2019 / 057698 filed October 23, 2019, PCT Application No. PCT / US2020 / 021596 filed March 6, 2020, and U.S. Application No.16 / 558,224 filed September 2, 2019, the disclosures of all of the aforereferenced applications are incorporated herein by reference in their entireties. Vectors and Cells Also provided herein are vectors for expressing one or more of the nucleic acid constructs. Such a vector can be chosen from viral vectors and non-viral vectors, plasmids, cosmids, transposons, and artificial chromosomes. By way of example, the vector can be a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno- associated viral vector. The vector optionally comprises nucleic acid sequences that encode transposases and / or nucleases. Non-viral vector examples include physical vectors such as electroporation and chemical vectors such as lipid nanoparticles. Cells containing one or more nucleic acid constructs or vectors as described herein are provided. The cell provides an expression system or a therapeutic target for the monomers, oligomers, or polypeptides described herein. Suitable cells include somatic cells, such as immune cells, epithelial cells, stem cells, or germline cells. Optionally, the immune cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMC), PBMC collected after stimulation with G-CSF, bone marrow, or umbilical cord blood. Optionally, the immune cells are tumor infiltrating lymphocytes (TIL), for example collected from a tumor. The immune effector cells may also be NK cells, Tcells, iNKT cells, T cells, macrophages, B cells, dendritic cells, myeloid derived progenitor cells, eosinophils, basophils, neutrophils, or Tregs. Optionally, the stem cells are hematopoietic stem cells, human embryonic stem cells, or iPSCs. The cells provided herein are optionally mammalian cells, or, more specifically, human cells. Methods of Making The present disclosure provides methods of making nucleic acid constructs and vectors encoding monomers and polypeptides of this disclosure, methods of making the monomers and polypeptides of this disclosure by expression of nucleic acid constructs, and methods of making cells comprising the nucleic acid constructs or vectors described herein. For example, a NFAT response element is chosen for regulation of the expressible nucleic acid sequence encoding a first engineered polypeptide monomer. In some examples, the NFAT response element is chosen such that it may be truncated and operably linked to a minimal promotor to regulate payload activity. One of skill in the art, using this disclosure, can select the appropriate NFAT response elements and order them within the construct to achieve the desired outcome. By way of example, a DRD / ligand pair is chosen for regulation of the desired payload. Optionally, a DRD is chosen having a ligand pair that is an FDA-approved small molecule drug. In some examples, the DRD / FDA-approved small molecule drug combination is chosen such that it is clinically tractable (e.g., the ligand is capable of regulating the abundance of or regulating the activity of the payload within the approved FDA dose range of the ligand). Thereafter, the nucleic acid or vector is designed to encode the building blocks / components of the polypeptide or monomers described herein, which components include in the case of polypeptides at least a payload and at least one of the chosen DRDs, and in the case of a monomer, at least one of the chosen DRDs, or at least one payload, or at least one payload and at least one of the chosen DRDs. The nucleic acid or vector may be further designed to encode additional building block / components such as transmembrane domains, linkers, hinges, and tags. One of skill in the art, using this disclosure, can select the appropriate components and order them within the construct to achieve the desired outcome. For example, the order of building blocks influences whether the DRD regulates the payload at the N- or C-terminus. As another example, linker and linker length may influence “constitutive” activity level (i.e., basal activity in the absence of ligand) and optionally, the specific linker and length is chosen to maximize the on state (e.g., maximum activity level) while maintaining low basal activity level and ligand (e.g., drug) responsiveness. As yet another example, the specific hinge may allow for conformational changes and thereby influence ligand responsiveness and is thus chosen to result in a sufficient dynamic range to obtain a desired range of payload abundance and biologic activity (i.e., an acceptable payload activity range that corresponds to variation in ligand from zero or minimal to maximum saturation). By way of example, if the goal is to create a membrane-tethered cytokine like IL15 with a sufficient dynamic range, the polypeptide optionally includes from the N-terminus the payload (IL15), a linker, a hinge, a transmembrane region, a tail, and a DRD. Optionally, the nucleic acid sequences encoding polypeptides or monomers may be engineered to include one or more additional components, such that, when expressed in a cell, result in the payload localizing internal to the cell membrane (optionally, tethered to the membrane or released into the cytoplasm) and, in certain examples, external to the cell membrane (optionally, tethered to the membrane or released extracellularly (i.e., shed or cleaved off of the membrane). Similarly, the nucleic acid constructs are optionally configured to encode polypeptide monomers that oligomerize by way of association of an intracellular, transmembrane, or extracellular oligomerization domain. Thus, all or a portion of the at least one DRD or optionally, an oligomerization domain, can be engineered to reside intracellularly, within the cell membrane, or on the surface of the cell (e.g., tethered to the cell surface). Optionally, the nucleic acid sequences encoding polypeptide monomer constructs or regulatable payload compositions may be engineered to include one or more of these additional components such that, when expressed in a cell, the regulatable payload composition is engineered with a signal sequence to transport part or all of the engineered regulatable polypeptide construct into the secretory pathway. After designing the construct for proper function including localization, appropriate components such as promoters, enhancers, multicistronic expression, translation control and half-life control elements are selected to achieve the desired control of payload abundance or activity. Additionally, nucleic acid constructs are designed for cistronic or multicistronic expression as required for the desired expression of various engineered components. Additionally, vehicles / vectors are chosen and designed to deliver the nucleic acid constructs into the desired cell. For example, the vehicles may be chosen from those previously described including viral vectors (such as lentiviral vectors, retroviral vectors, and adeno- associated vectors), plasmids, cosmids, and artificial chromosomes. Such vectors can be designed to encode transposases, nucleases, and elements that control translation (e.g., IRES). The choice of vector may also influence the choice of various building block components. For example, vectors which demand smaller constructs may require using smaller DRDs. Cells for engineering can be isolated from any biological sample, including for example, blood (e.g., umbilical cord blood or peripheral blood), bone marrow, fetal tissue (human embryonic stem cells), or tumors. Cells can be modified cells, such as a stem cell modified to be pluripotent (e.g., an induced pluripotent stem cell (iPSC)) or CAR T cells, prior to transduction with the described nucleic acids and vectors. By way of example, isolated T cells or tumor infiltrating lymphocytes (TIL) can be isolated from a biological sample, transduced, and, optionally, expanded in culture. Such expansion can be performed, for example, by contacting the transduced cell with IL2, feeder cells, recombinant antigen, or an antibody that stimulates cell expansion. The cell to which the nucleic acid is delivered is selected based, at least in part, on the ability of the cell to allow expression of the polypeptides or monomers disclosed herein and to allow payload activity in a sufficient dynamic range. Optionally, the cell expresses little or no payload in the absence of the provided nucleic acid or vector. In certain examples, one of skill in the art would select a cell in need of an increase in payload activity or abundance in a cell that expresses the payload. In certain examples, one of skill in the art would select a cell in need of gene editing. Optionally, the cell is selected as an effector cell, for example, an immune effector cell. A person of ordinary skill in the art applying knowledge from this disclosure can build a variety of monomers, polypeptides, and oligomers, as well as nucleic acids and vectors encoding them within scope of this disclosure beyond those explicitly exemplified herein. For example as described, payloads, DRDs, oligomerization domains, and additional components such as linkers, tails, transmembrane domains, signal sequences, hinges, and the like can be selected to create a variety of polypeptides, oligomers, and nucleic acids encoding them upon considering such factors as desired therapeutic outcome, whether the payload is membrane-bound or secreted, whether the payload acts intracellularly or extracellularly, and whether the oligomerization domain should be located intracellularly, extracellularly, or transmembrane. Accordingly, the example embodiments are non-limiting and help provide guidance for a person of skill in the art to implement other embodiments within the scope of this disclosure. Methods of Use Disclosed herein are methods of regulating payload function, for example methods of regulating abundance, availability and / or activity (“activity”) of a payload. Optionally, the method of regulation is a method of modifying the activity of the payload comprising contacting a cell comprising the nucleic acid construct with an immune cell stimulation agent and a selected dose of ligand. Optionally, the cell is contacted with a selected dose of the immune cell stimulation agent. Optionally, the activity of the payload (e.g., corresponding to the abundance and / or availability of the payload) is reduced as compared to the activity of a payload in a control cell, for example, a cell not containing one or more NFAT response elements and not engineered to express the payload independent of a DRD. Optionally, the payload has a biological activity level ranging from a basal activity level in the absence of ligand to a maximum activity in the presence of a saturating amount of ligand, and the range from basal to maximum activity level of the payload is greater than that of the same payload in a control nucleic acid lacking the one or more NFAT response elements. Optionally, the method is a method of modulating the activity of a payload comprising contacting a cell comprising the nucleic acid construct with a selected dose of an immune stimulation agent and a selected dose of ligand such that the activity of the payload is increased relative to the basal activity level. Optionally, the method comprises contacting the cell with a selected amount of ligand, wherein the selected amount of ligand results in a selected activity level of the payload. In certain examples, the method comprises alternatively contacting the cell with varying selected amounts of ligand, to achieve varying selected activity levels ranging from the basal level to the maximum level. Optionally, contacting the cell with the immune cell stimulation agent occurs at the same time as contacting the cell with ligand. Optionally, contacting the cell with the immune cell stimulation agent occurs at least 24 hours prior to contacting the cell with the ligand. Optionally, contacting the cell with the immune cell stimulation agent is for a duration between 6 and 24 hours. The contacting step can occur in vitro, ex vivo, or in vivo. The contacting step is optionally performed to achieve a continuous selected activity of the payload (i.e., to achieve a continuous on-state of the payload) or to achieve intermittent activity of the payload (i.e., to provide a pulsed delivery of the payload between an on-state and an off-state). “Off-state” means the payload activity is the basal activity level. “On-state” means a selected activity level in the presence of an effective amount of ligand, which is greater than the off-state. Continuous activity of the payload can be achieved by continuous contact of the at least one DRDs with an effective amount of ligand or by providing a subsequent contacting step or steps of the at least one DRDs with the ligand, wherein the subsequent contacting step or steps is / are performed before the activity level of the payload from the previous contacting step reaches the basal activity level. Each contacting step can be varied with regard to the amount of ligand such that, when more ligand is used, more payload activity results, and, when less ligand is used, less payload activity results. Thus, the amount of ligand can be varied with subsequent contacting steps to tune up or tune down the amount and / or activity of the payload over time. Each contacting step can also be varied with respect to frequency in order to achieve a desired pattern of activity level. Also disclosed are methods of delivering a payload to a subject, for example a therapeutically effective payload to a subject in need thereof, whereby a nucleic acid construct or a vector as described herein is administered to the subject. The method results in expression, for example in target cells of the subject, of one or more polypeptides or monomers, which monomers self-assemble to form oligomers as described herein. The methods may further comprise controlling the dose or duration of administration of a payload to a subject. For example, the method optionally comprises administering to the subject a selected amount of an immune cell stimulation agent, paired ligand, or both to deliver a selected activity of the payload to the subject. The ligand can be delivered to achieve continuous or intermittent payload activity in the subject. Continuous payload activity may be a substantially consistent level of activity, or the level of activity may be modulated. Intermittent activity, between the off-state and on-state includes modulating activity between the off-state and a substantially consistent on-state, or between the off-state and varying on- state activity levels. A higher dose or longer duration of administration of the ligand is administered when more activity of the payload is desired, and reduction or elimination of the ligand dose is chosen when less activity is desired. The dose and duration of ligand administration and the resulting activity of the payload may be selected to avoid unacceptable or undesired side effects or toxicity in the subject. Dosages of ligand and schedules for administering the dosages of ligand may be determined empirically by one skilled in the art based on the amount of resulting payload, the activity of the payload, or based on one or more signs of the effect of the payload activity. The ranges for administration of the ligand range from any amount above zero to a saturating dose and the resulting payload activity ranges from a basal level to a maximal level, optionally with a sufficient dynamic range that allows for the desired dose-response to the ligand and concomitant activity range for the payload (e.g., for a given ligand and payload, the range of difference in off-state and maximum payload activity would result from at least a 10 fold range of ligand). This sufficient dynamic range allows for fine tuning and a dose response curve that is not unacceptably steep. In embodiments, the dosage or frequency of administration of ligand and resulting abundance and activity of payload is chosen to avoid, mitigate against, or limit unacceptable or undesired adverse side effects and will vary with the age, condition, and / or sex of the subject, and type of condition being treated, the extent of the condition, or, and whether other therapeutic agents are included in the treatment regimen. Guidance can be found in the literature for appropriate dosages for given classes of ligands. By way of further example, for a subject with cancer, a nucleic acid construct or vector comprising a nucleic acid construct according to this disclosure is provided to a subject, wherein the nucleic acid construct encodes a payload operably linked to at least one DRD and wherein the payload targets a tumor cell or an immune cell that in turn targets the tumor cell. Such a method optionally comprises administering to the subject a nucleic acid construct or vector comprising a nucleic acid construct according to this disclosure encoding a payload operably linked to at least one DRD, wherein the payload is an immune checkpoint inhibitor, a cytokine, CAR, or TCR. Also provided herein are methods of delivering a payload operably linked to at least one DRD to a subject by administering to the subject a cell containing a nucleic acid or vector comprising a nucleic acid construct described herein. Such a method can further comprise isolating cells from a subject, transducing the isolated cells with the nucleic acids or vectors comprising a nucleic acid construct encoding the monomers (which when expressed assemble into regulatable oligomers) or regulatable polypeptides described in this disclosure, expanding the cells in vitro, and providing the cells to the same or different subject. The payload is selected to treat the subject receiving the cells. The subject may have cancer, an autoimmune disease, a genetic mutation, a deficiency in an essential polypeptide or the like. The transduced cells can be, by way of example, immune effector cell (e.g., an NK cell, iNKT, T cell, T cell, tumor infiltrating lymphocyte (TIL), macrophages, B cells), or stem cells). The cells can be isolated from the same subject (autologous source) that receives the transduced cells or the cells can be isolated from a different subject (e.g., an allogeneic source). By way of example, when the subject has cancer, a T cell or TIL can be isolated from the subject and engineered to express a cytokine, a CAR, and / or a TCR. For example, the cell can be an anti-CD19 CAR-T cell. The cell is optionally expanded, and the cell or the expanded population of cells is administered to the same or different subject. In certain examples, the T cells, CAR-T cells, NK cells, or TIL, are administered in an amount from about 1000 cells / injection to up to about 10 billion cells / injection, such as 1×1010, 1×109, 1×108, 1×107, 5×107, 1×106, 5×106, 1×105, 5×105, 1×104, 5×104, 1×103, 5×103cells per injection, or any ranges between any two of the numbers, end points inclusive. Optionally, from 1×108to 1×1010cells are administered to the subject. Optionally, the cells are administered one, two, three, or four times as needed. In the treatment methods, the ligand and / or immune cell stimulation agent dosage regimen including the selected amount of ligand and / or immune cell stimulation agent dosage for administration to the subject and frequency of administration of the selected amount of ligand and / or immune cell stimulation agent dosage is chosen to result in regulation of the payload and / or a desired outcome for the subject. The subject is optionally monitored for the outcome. Thus, for example, the number of malignant cells in a sample, the circulating tumor DNA in a sample, or the size of a solid tumor upon imaging can be detected. If the desired end point is achieved (e.g., showing successful treatment of cancer), the ligand and immune cell stimulation agent dosage can be reduced or discontinued so as to reduce or eliminate the payload, for example to reduce the abundance, availability and / or activity of the payload below a pre-determined threshold to eliminate or mitigate against unwanted or undesired side effects. Similarly, if the subject develops a cytokine storm, an allergic reaction, or other adverse effect from the payload, the ligand and immune cell stimulation agent can be reduced or discontinued. Also disclosed herein are methods of regulating expression of a downstream target protein of a gene editing process. Optionally, such method comprises engineering a cell to express an oligomer or engineered, regulatable polypeptide comprising a payload such as a CAS9 protein or transcription factor protein operably linked to a at least one DRD. Optionally, for a subject with a genetic mutation, a nucleic acid construct or vector comprising a nucleic acid construct according to this disclosure is provided to deliver a payload that provides a nucleic acid editing polypeptide or system operably linked to at least one DRD. Target cells in the subject are transduced with the nucleic acid construct or vector comprising a nucleic acid construct to allow the gene editing payload to modify the nucleic acid (e.g., genomic DNA or RNA) of the transduced cell. The activity level of the payload and therefore expression of the target protein can be regulated by administration of ligand and / or immune cell stimulation agent dosage to the subject. Systems for nucleic acid editing are known in the art, such as CRISPR / Cas 9 systems, TALENs, retrotransposons, and the like. The nucleic acid construct or vector can be engineered to encode one or more components of any one of the systems, such that one or more nucleic acids in the transduced cell of the subject is edited to provide a desired nucleic acid modification. Definitions The terms “about” and “approximate,” when used to refer to a measurable value such as an amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, position, length and the like, is meant to account for variations due to experimental error, which could encompass variations of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, concentration, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, weight, position, length and the like. All measurements or numbers are implicitly understood to be modified by the word about, even if the measurement or number is not explicitly modified by the word about. In instances in which the terms “about” and “approximate” are used in connection with the location or position of regions within a reference polypeptide, these terms encompass variations of ± up to 20 amino acid residues, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± up to 3 amino acid residues, ± up to 2 amino acid residues, or even ± 1 amino acid residue. Reference is made herein to a “basal activity level.” Basal level as used herein can be zero, near zero, or any amount in the absence of exogenous ligand. Basal activity may occur because of endogenous levels of the same or different ligand or may occur because of a resting level of payload production in the absence of exogenous ligand. Reference to “biological activity” is understood to mean under appropriate conditions even if not so stated. As used herein “contacting” is understood to mean providing an agent (such as an immune cell stimulation agent or a ligand) to a target (such as a DRD) such that the agent and target may come into contact with one another. Contacting of the agent and target can be in vitro, ex vivo or in vivo, whereby the agent is added to cell culture medium or administered to the patient. As a non-limiting example, contacting also includes providing the ligand to a cell, wherein the DRD is located intracellularly, such that the ligand reaches the cytoplasm or the nucleus or other cellular organelles. Similarly, a cell can be contacted in vivo, by administering an immune cell stimulation agent or a ligand to a subject such that the ligand reaches a cell or the DRD contained on the surface or in the interior of the cell. In each case, the agent needs to reach a minimum intracellular concentration to exert its stabilizing effect on the target. As used herein, the term “control construct” refers to a construct similar to the test engineered, regulatable oligomer or engineered regulatable polypeptide (“test construct”) except the control construct lacks the one or more NFAT response elements and the at least one DRD operably linked to a payload. “NFAT response element” is understood to mean a region of DNA upstream of a minimal promotor which NFAT transcription factors, optionally contained in a protein complex, may bind to and induce transcription. An “immune cell stimulation agent” may be any agent that produces a population of activated immune cells under a suitable condition. “DRD” is understood to mean a domain responsive to a ligand, even if not so stated. A “hetero-oligomerization domain” as described herein is a first oligomerization domain on a first monomer that promotes oligomerization through a second oligomerization domain on a second monomer, wherein the second oligomerization domain is different than the first and wherein the first oligomerization domain does not promote oligomerization with an oligomerization domain that is the same. The terms “ligand,” “paired ligand,” and “stabilizing ligand” are used interchangeably and mean the same thing when used in reference to a drug responsive domain (“DRD”). As used herein, “operably linked” means that, in the presence of a paired ligand, the at least one DRD is linked to the payload directly or indirectly so as to alter a measurable characteristic of the payload (e.g., alters the level of activity of the payload as compared to the level of activity in the absence of the paired ligand). Optionally, the measured level of amount and / or activity of the payload increases in the presence of an effective amount of ligand as compared to the measured level of expression or activity in the absence of ligand. An effective amount of ligand means the amount of ligand needed to see an increase in the measure of the amount or activity of the payload. Optionally, the effective amount is not so great as to produce unacceptable toxicity or off-target effects. Optionally, the measurable characteristic is a therapeutic outcome, an amount of the payload in a sample, or a biological activity level of the payload (for which measuring the amount of payload can serve as a proxy). The term “payload” refers to the agent whose abundance, activity, availability, expression, function or other characteristic is desired to be regulated by a DRD. Wherever the phrase “linked” or “bound” or the like is used, the phrase “directly or indirectly” is understood to follow unless explicitly stated otherwise or nonsensical in context. Thus, the phrase “a DRD linked to another DRD” or “two linked DRDs” means in both cases that a first DRD is directly or indirectly linked to a second DRD. For example, “two linked DRDs” and the like covers the situation wherein two DRDs are not directly connected to one another but rather are in association with one another because each is connected to an oligomerization domain, and the respective oligomerization domains are linked, e.g., via non-covalent bonds. The details of one or more embodiments of the present disclosure are set forth in the description and accompanying drawings. It is to be understood that other embodiments may be utilized and structural or process changes made without departing from the scope of the disclosure. In other words, illustrative embodiments and aspects are described below. But it will be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions may be made to achieve the developer’s specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it will be appreciated that such development effort might be complex and time-consuming but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The examples below are intended to further illustrate certain aspects of the methods and compositions described herein and are not intended to limit the scope of the claims. EXAMPLES Example 1. Combining transcriptional and translational regulation Regulated expression of payload activity is crucial for preventing toxicity in biologics administration. The nucleic acid construct described herein provides both transcriptional and translational control of payload activity that results in spatiotemporal regulation. For example, nucleic acid constructs comprising one or more NFAT response elements provide spatial transcriptional control of an immune cell, such as a T cell, wherein transcription of the first engineered polypeptide monomer begins after recognition of a cognate antigen of the immune cell receptor, such as a T cell receptor. As a further example, the first engineered polypeptide monomer comprising a payload operably linked to at least one DRD provides translational control of payload activity that is temporally linked to ligand administration. The dual control system described herein reduces the need for a cellular kill switch by providing additional control if either the transcriptional or translational control mechanisms malfunction, for example, because of a mutation in the DRD. Additionally, the dual control system described herein reduces the overall basal abundance of the payload. The dual control system provided herein enables spatiotemporal regulation of payload activity (FIG.1). In response to an immune cell stimulation agent and ligand, IL-12 expression increased in T cells transduced with construct IL12-277 (SEQ ID NO: 44), which contained a functional DRD and NFAT-responsive minimal promoter. The constructs used in this example are shown in Table 2. On day 0, primary human T cells were stimulated with CD3 / 28 dynabeads using a 3:1 bead to T-cell ratio in Roswell Park Memorial Institute (RPMI) media with 10% fetal bovine serum (FBS) and plated at a density of 1e6cells / ml in 500 μl media. On day 1, activated T cells were transduced with lentiviruses for each of constructs IL12-273, 277 and 256 (SEQ ID NOs: 43, 44, and 38, respectively) by spin- infection at 800g for 1 hour at 32°C. On day 6, a fraction of the cells were used for assessing transduction efficiency via flow cytometry for Thy1.2, and the rest of the cells were rested after removal of beads for 24 hours. On day 7, 100,000 transduced cells were plated in a 96- well plate and treated with ligands (25 M ACZ or an equivalent volume of Dimethyl sulfoxide (DMSO) vehicle) either with or without re-stimulation with PMA / Ionomycin (Biolegend) in a final volume of 200 μl for 6 hours. The cells were then stained for membrane-bound (mb) IL-12 and Thy1.2 expression. As shown in FIG.1, integration of two signals was needed for expression of IL12, evinced by IL12-273 (SEQ ID NO: 43), which was the only construct in this test set with a functional DRD and a NFAT-responsive minimal promoter. Both activation and drug were required to express IL-12. Contrastingly, IL12-277 (SEQ ID NO: 44) required only activation as it was coupled with wildtype CA2, which does not function as a DRD. In IL12-256 (SEQ ID NO: 38), a DRD controls IL-12 abundance under the control of a constitutive strong eukaryotic translation elongation factor 1 alpha 1 (EF1a) promoter. As expected, IL12-256 (SEQ ID NO: 38) responds primarily to presence of ACZ by showing robust induction in the presence of ligand. Interestingly in this experiment, Thy1.2 was under the control of the NFAT responsive minimal promoter linked via the ribosomal skip sequence P2A. As a result, even though Thy 1.2 could not adequately be used as a transduction marker, Thy 1.2 could be used to gauge the effect of PMA / Ionomycin activation independent of the main cargo IL-12. FIG.2 shows a construct depiction having 6 NFAT response elements, a minimal promotor from either YB-TATA or IL-2, the payload (IL-12, in this case) linked to a DRD, a P2A sequence, and a THY1.2 sequence. The construct depicted in FIG.2 is the design of the constructs used in this Example 1, with YB-TATA used as the minimal promoter. Table 2. Constructs used in Example 1. Example 2. Replacing ARRE1 with alternative NFAT response elements The NFAT response elements found on the IL-2 gene is known as the ARRE1 circuit. The disclosure herein provides alternative NFAT response elements from DSCR1, which is a negative regulator of NFAT signaling. DSCR1 has 3 NFAT response elements, NRE1, NRE2, and NRE3 (SEQ ID NOs: 16, 17, and 18, respectively). NFAT response elements from DSCR1 were used in constructs IL12-284, 285, 286, 287, and 288 (SEQ ID NOs: 47, 48, 49, 50, and 51, respectively) and provided transcriptional and translational control of IL- 12 payload in transduced cells contacted with immune cell stimulation agent PMA / Ionomycin (Biolegend) for 6 hours and with 25 μM ACZ (FIG.3). Constructs used in this example are shown in Table 3. On day 0 primary human T cells were stimulated with CD3 / 28 dynabeads using a 3:1 bead to T-cell ratio in RPMI media with 10% FBS and plated at a density of 1e6cells / ml in 500 μl media. On day 1, activated T cells were transduced with lentiviruses for each of constructs IL12-284, 285, 286, 287, and 288 (SEQ ID NOs: 47, 48, 49, 50, and 51, respectively) by spin-infection at 800g for 1 hour at 32°C. On day 6, a fraction of the cells were used for assessing transduction efficiency via flow cytometry for Thy1.2, the rest of the cells were rested after removal of beads for 24 hours. On day 7, 100,000 transduced cells were plated in a 96-well plate and treated with ligands (25 M ACZ or an equivalent volume of DMSO vehicle) either with or without re- stimulation with PMA / Ionomycin (Biolegend) in a final volume of 200 l for 6 hours. The cells were then stained for mbIL-12 and Thy1.2 expression. Constructs IL12-285, IL12-286 and IL12-287 (SEQ ID NOs: 48, 49, and 50, respectively) each used 6 copies of a single kind of NFAT response element from DSCR1. NRE3 (SEQ ID NO: 18) was used in IL12-285, NRE2 (SEQ ID NO: 17) was used in IL12- 286, and NRE1 (SEQ ID NO: 16 was used in IL12-287. The other two constructs IL12-284 (SEQ ID NO: 47) and IL12-288 (SEQ ID NO: 51) used mixtures of NFAT response elements. As previously observed, integration of two signals was needed for expression of IL12. That is, both activation and drug were required to express IL12. However, as shown in FIG.3, only NRE3, used in IL12-285 (SEQ ID NO: 48), and NRE1, used in IL12-287 (SEQ ID NO: 50), were able to actuate IL12 production post activation and in the presence of drug. NRE2 in IL12-286 (SEQ ID NO: 49) was inactive. The NRE1, 2 and 3 mixtures were intermediate in their responsiveness. Interestingly in this experiment, Thy1.2 was under the control of the NFAT responsive minimal promoter linked via the ribosomal skip sequence P2A. As a result, even though Thy 1.2 could not adequately be used as a transduction marker, Thy 1.2 could be used to gauge the effect of PMA / Ionomycin activation independent of the main cargo IL-12. Table 3. Constructs used in Example 2. Example 3. Testing non-T cell specific promotors Non-T cell specific promotors which could regulate payload activity using NFAT response elements from DSCR1 were tested using green fluorescence protein (GFP) as the payload (FIGS.4-8). On day 0, primary human T cells were stimulated with CD3 / 28 dynabeads using a 3:1 bead to T cell ratio in RPMI media with 10% FBS and plated at a density of 1e6cells / ml in 500 μl media. On day 1, activated T cells were transduced with lentiviruses for each of the constructs AcGFP-033, 034, 035, 036, 037, 038, 038m, 039, 040, 041, 042, 043, 044, 045, 046, 047, 048, 049, and 031 (SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 19, respectively) by spin infection at 800g for 1 hours at 32°C. On day 6, a fraction of the cells were used for assessing transduction efficiency via flow cytometry for mCherry, the rest of the cells were rested after removal of beads for 24 hours. On day 7, 100,000 transduced cells were plated in a 96-well plate and treated with ligands (50 M ACZ or DMSO vehicle) either with or without re-stimulation with PMA / Ionomycin (1:500, Biolegend) in a final volume of 200 l for 6 hours. The cells were then checked for GFP and mCherry expression. FIG.4 shows the results for constructs AcGFP-033, 034, 035, and 036 (SEQ ID NOs: 20, 21, 22, and 23, respectively). FIG.5 shows the results for constructs AcGFP-037, 038, 039, and 040 (SEQ ID NOs: 24, 25, 27, and 28). FIG.6 shows the results for constructs AcGFP-041, 042, 043, 038m, and 031 (SEQ ID NOs: 29, 30, 31, 26, and 19, respectively). FIG.7 shows the results for constructs AcGFP-044, 045, 046, and 047 (SEQ ID NOs: 32, 33, 34, and 35, respectively). FIG.8 shows the results for constructs AcGFP-048, 049, and 031 (SEQ ID NOs: 36, 37, and 19, respectively). In the experiments shown in FIGs.4-8, the aim was to determine usable minimal promoters and sequence boundaries of such minimal promoters. Success in this experiment was defined as no promoter activity in the resting (non-stimulated) state and activity when contacted by stimulating agents. Another aim was to ensure non-T-cell promoters could respond to the recruitment of NFAT proteins by the NFAT response elements. For simplicity, the previously-described ARRE-1 elements from IL-2 were used in these constructs. Tissue specific promoters were derived from various tissues including the muscle, lung, eye and B- cells. NFAT response elements were inserted within the boundaries of the non-T-cell specific promoters to change the responsiveness of the minimal promoters. The constructs used in this example are shown in Table 4. Even though the signal-to-noise ratio of this experiment was low, minimal promoters that met the above criteria were found from endothelin-1, as used in AcGFP-046, and from Integrin B6, used in AcGFP-048 and AcGFP-049. Table 4. GFP constructs used in Example 3. Next, IL-12 was tested as a payload in a subset of promotors of interest, shown in Table 5 below, which had the good activation using GFP as payload. On day 0, primary human T cells were stimulated with CD3 / 28 dynabeads using a 3:1 bead to T-cell ratio in RPMI media with 10% FBS and plated at a density of 1e6cells / ml in 500 μl media. On day 1 activated T cells were transduced with lentiviruses for each of IL12-297, 298, 300, 301, 302, 303, 304, 305, 306, 307, and 308 (SEQ ID NOs: 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, and 68, respectively) by spin-infection at 800g for 1 hour at 32°C. On day 6, a fraction of the cells were used for assessing transduction efficiency via flow cytometry for Thy1.2, the rest of the cells were rested after removal of beads for 24 hours. On day 7, 100,000 transduced cells were plated in a 96-well plate and treated with ligands (50 M ACZ or an equivalent volume of DMSO vehicle) either with or without re-stimulation with PMA / Ionomycin (Biolegend) in a final volume of 200 μl for 6 hours. The cells were then stained for mbIL-12 and Thy1.2 expression. Table 5. IL-12 constructs used in Example 3. FIG.9 shows the percent of IL-12 constructs out of IL12-300, 301, 302, 303, 304, 305, 297, and 298 (SEQ ID NOs: 60, 61, 62, 63, 64, 65, 57, and 58, respectively) in the THY1.2 positive gate assessed via flow cytometry. FIG.10 shows the percent of IL-12 constructs out of IL12-306, 307, 308, and 297 (SEQ ID NOs: 66, 67, 68, and 57, respectively) in the THY1.2 positive gate assessed via flow cytometry. FIG.11 shows mbIL-12 expression in constructs IL12-291, 292, 297, 298, 302, 305, 306, 307, and 308 (SEQ ID NOs: 52, 53, 57, 58, 62, 65, 66, 67, and 68, respectively) following 24 hours of stimulation with human Immunocult soluble CD3 / CD28 reagent from StemCell Technologies or PMA from Biolegend. FIG.12 shows quantitation of mbIL-12 expression in constructs IL12-291, 297, 302, 305, 306, 307, and 308 (SEQ ID NOs: 52, 53, 57, 58, 62, 65, 66, 67, and 68, respectively) following 24 hours of stimulation with PMA. FIGS.13A and 13B shows quantitation of IL-12 expression in the supernatant following either 3 hours (FIG.15A) or 24 hours (FIG.15B) of stimulation with PMA, with or without ACZ, in constructs IL12-291, 297, 298, 299, 302, 305, 306, 307, and 308 (SEQ ID NOs: 52, 57, 58, 59, 62, 65, 66, 67, and 68, respectively) as well as in untransduced cells. Accumulated IL-12 in the culture supernatants was measured using using human IL-12p70 (and / or human interferon-gamma) MSD V-plex assay kits (Meso Scale Discovery). Fold- change of IL-12 within a restimulation condition was calculated as the concentration of IL12 with ligand added divided by the concentration without ligand for a given construct. FIG.14 shows quantitation of IL-12 expression in the supernatant following 24 hours of stimulation with human Immunocult soluble CD3 / CD28, with or without ACZ, in constructs IL12-291, 297, 298, 299, 302, 305, 306, 307, and 308 (SEQ ID NOs: 52, 57, 58, 59, 62, 65, 66, 67, and 68, respectively), as well as in untransduced cells. Accumulated IL-12 in the culture supernatants were measured using using human IL-12p70 (and / or human interferon-gamma) MSD V-plex assay kits (Meso Scale Discovery). Fold-change of IL-12 within a restimulation condition was calculated as the concentration of IL12 with ligand added divided by the concentration without ligand for a given construct. Testing of non T-cell-expressed, tissue-specific promoters used in this example shows the minimal promoters selected based on AcGFP constructs continued to perform as expected with a different cargo, IL-12. Minimal promoters based on surfactant protein B (SPPR), myosin heavy chain, G-protein coupled receptor kinase 1 (GRK1), integrin B6, and endothelin-1 (ET1) all showed responsiveness to T-cell activation. Since all the test constructs used in this example had a functional DRD, full IL-12 expression and stabilization also required presence of saturating quantities of ACZ. The constructs were compared to IL12-291 (SEQ ID NO: 52), which is the same form of membrane tethered flexi-IL12 payload but under the control of only the DRD, and to IL12-297 (SEQ ID NO: 57) and IL12- 299 (SEQ ID NO: 59), which are based on ARRE1 NFAT-responsive constructs, in which the NFAT response elements and the minimal promoters are derived from IL-2. IL12-297 (SEQ ID NO: 57) differs from IL12-299 (SEQ ID NO: 59) in that IL12-297 (SEQ ID NO: 57) has a membrane tethered IL-12 of the form also present in tested constructs IL12-300, 301, 302, 303, 304, 305, 306, 307, and 308 (SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, and 68, respectively), while IL12-299 (SEQ ID NO: 59) has secreted flexi-IL12. The construct most relevant for comparison of a spatiotemporal regulation phenotype is therefore IL12-297 (SEQ ID NO: 57). As observed in FIG.10, the same phenotype was recapitulated in IL12- 307 (SEQ ID NO: 67). Further, as shown in FIGS.11-12, the ET-1-based promoter, in IL12- 307 (SEQ ID NO: 67) shows a higher “ON” state than seen in IL12-297 (SEQ ID NO: 57). Only one promoter, that from the myosin heavy chain used in construct IL12-302 (SEQ ID NO: 62), was found to be leaky (FIGS.11-12). Leakiness was manifested by the ability of ACZ to stabilize the expressed protein and the appearance of IL-12 on the cell surface in the non-activated state. This regulation pattern was not only seen in the membrane staining for IL-12, but also in the supernatant by MSD assay kits detecting shed IL-12. Further, as shown in FIGS.13A, 13B, and 14, different activation conditions could produce similar results. Example 4. Combining NFAT response elements and promotor from Endothelin-1 provides more robust induction than control constructs. Constructs used in this example are shown in Table 7. On day 0 primary human T cells were stimulated with CD3 / 28 dynabeads using a 3:1 bead to T-cell ratio in RPMI media with 10% FBS and plated at a density of 1e6cells / ml in 500 μl media. On day 1 activated T cells were transduced with lentiviruses for each of IL12-311, 312, 297, and 307 (SEQ ID NOs: 69, 70, 57, and 67, respectively) by spin-infection at 800g for 1 hour at 32°C. On day 6, a fraction of the cells were used for assessing transduction efficiency via flow cytometry for Thy1.2, the rest of the cells were rested after removal of beads for 24 hours. On day 7, 100,000 transduced cells were plated in a 96-well plate and treated with ligands (50 μM ACZ or an equivalent volume of DMSO vehicle) either with or without re-stimulation with PMA / Ionomycin (Biolegend) in a final volume of 200 μl for 6 hours. The cells were then stained for mbIL-12 and Thy1.2 expression. FIG.15 shows a more robust induction of IL-12 in constructs IL12-311, and 312 (SEQ ID NOs: 69 and 70, respectively) compared to the existing ARRE1 construct with IL-2 minimal promoter, IL12-297 (SEQ ID NO: 57) or with ET-1 minimal promoter, IL12-307 (SEQ ID NO: 67). The robust NFAT response elements from DSCR1 shown in Example 2 and ET-1 minimal promoter shown in Example 3 were combined in IL12-311 and 312 (SEQ ID NOs: 69 and 70, respectively). IL12-311 (SEQ ID NO: 69) combined NRE3 from DSCR1 with the ET-1 minimal promoter and IL12-312 (SEQ ID NO: 70) combined NRE1 from DSCR1 with the ET-1 minimal promoter to create two new activation responsive casettes. As shown in FIG.15, the induction becomes more robust using NRE1 and NRE3 as NFAT response elements. Table 6. IL-12 constructs used in Example 4. See FIG.16 for an overview of the testing schematic used to determine dual regulation of IL-12 in primary human T cells. On day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3 / CD28) at a 3:1 bead: cell ratio in media containing 10% fetal bovine serum (FBS). The next day, any one of the constructs using IL- 12 as a payload were packaged as lentivirus. On day 2, the cells were diluted 1:2 with fresh 10% FBS media. On day 3, the cells were expanded 1:4 with fresh medium. On day 6, the cells were assessed for transduction efficiency using flow cytometry. As a readout of cell transduction, Thy1.2 was detected using anti-Thy1.2. The Dynabeads were removed using magnetic selection and the cells split 1:2 into fresh media. On day 8, a subset of cells were used to test mbIL-12 regulation while the remaining cells were diluted 1:2 with fresh media. To test mbIL-12 regulation, a normalized number of transduced cells were plated in a 96-well plate and treated with ligands (25 M ACZ 50 M TMP or equivalent volume of DMSO vehicle) and re-stimulation with immune cell stimulation agents (human Immunocult soluble CD3 / CD28 reagent from StemCell Technologies or PMA / Ionomycin from Biolegend) for a final volume of 200 l. After overnight incubation, mbIL-12 expression in transduced T cells was analyzed by flow cytometry using live / dead discrimination dye, anti-Thy1.2, anti-IL- 12p70, and anti-CD3. The geometric mean fluorescent intensities (GMFIs) of IL-12p70 in transduced cells treated with ligand were normalized against the GMFIs of mbIL-12 from identical cells with not treated with ligand for each given construct. Cytokines that accumulated in the overnight culture supernatants were measured using human IL-12p70 and / or human interferon-gamma MSD V-Plex assay kits (Meso Scale Discovery). Fold change of IL-12 within a stimulation condition was calculated as the concentration of IL-12 within a restimulation condition was calculated as the concentration of IL-12 with ligand added divided by the concentration without ligand for a given construct. Cells not used for testing IL-12 regulation were pelleted and rested in fresh media on day 9. A co-culture assay with rested cells was performed on day 10 to assess regulation of TH1 skewing in GFP-labeled cells. IL-12 signaling causes T cells to acquire a TH1 phenotype after 3-5 days. FIG.17 describes the procedures for testing the functional off-state and regulation of IL-12 in transduced T cells. On day 0, 100,000 cells transduced with IL-12 were co-cultured in a 48- well plate with 200,000 GFP labeled cells with or without ACZ and / or CD3 / 28 stimulation in 500 l of media. After 4 days, the cells were plated in a 96-well plate with or without ACZ and / or CD3 / 28 stimulation for 24 hours. In the final 3 hours, Brefeldin A was added to allow for intracellular cytokine accumulation prior to staining. Cells were then stained for TH1 markers including IFNy, GranzymeB, perforin, IL-12, CD107a, and TNFa and analyzed via flow cytometry to visualize TH1 skewing in bystander cells by gating on GFP+ versus IL-12- producing cells. As shown in FIGS.18A and 18B, NFAT response element regulation coupled with at least one DRDs allowed for more robust dual control of IL-12 expression on the cell surface with cells in either group being activated through CD3 / 28 (FIG.18A) or PMA stimulation (FIG.18B) coupled with ACZ ligand for DRD stabilization. As shown in FIGS.19A and 19B, NFAT response element regulation coupled with at least one DRDs allowed for more robust dual control of IL-12 expression in the supernatant with cells in either group being activated through CD3 / 28 (FIG.19A) or PMA stimulation (FIG.19B) coupled with ACZ ligand for DRD stabilization. Stimulation of tissue-specific promoters coupled with NFAT response elements (e.g., constructs IL12-305, 311, 312, 316, and 317; SEQ ID NOs: 65, 69, 70, 74, and 75, respectively) achieved similar off-states and higher fully ON states than the ARRE1 NFAT response element (construct IL12-298; SEQ ID NO: 58) with the IL-2 promoter (FIGS.18-19). The functional ON and OFF states were also evaluated in a co- culture assay (FIG.17) with IL-12 naïve, GFP labelled cells. The TH1 marker Granzyme B in the co-cultured cells was regulated by both CD3 / 28 stimulation and ACZ administration during the co-culture with an undetectable effect of IL-12 in the OFF-state for several constructs (FIG.20). This two-stage control was not constrained to CA2 (SEQ ID NO: 2, encoded by the nucleic acid sequence of SEQ ID NO: 1) acting as the DRD, as similar or better control was also observed with hDHFR (SEQ ID NO: 7) acting as the DRD (FIGS. 21A and 21B). In FIG.21A, cells were activated with CD3 / CD28 antibodies for 18 hours, and in FIG.21B, cells were activated with PMA for 18 hours. Example 5. Assessing IL-12 regulation in human tumor infiltrating lymphocytes Human tumor infiltrating lymphocytes were isolated from tumor fragments and engineered with lentivirus vectors containing a secretable IL-12 under the control of NFAT promoter (IL12-313); a membrane-bound IL-12 (IL12-318, IL12-319, and IL12-320) under the dual control of NFAT promoters and an hDHFR DRD; or a membrane-bound IL-12 underthe control of a EF1 promoter and hDHFR DRD (IL12-321) before initiating a rapidexpansion protocol (REP). Tumor infiltrating lymphocytes received soluble IL2 to support cell growth during the REP. At the end of expansion, cells were cryopreserved, then thawed and rested for 2 days with cytokines before initiating in vitro assays. One hundred thousand (100,000) transduced cells were normalized for total cell number with empty vector cells, plated in a 96-well plate and treated with ligands (10 M Trimethoprim (TMP) or equivalent volume of dimethyl sulfoxide (DMSO) vehicle) and re-stimulated with PMA(phorbol myristic acetate) / Ionomycin from Biolegend, Inc. (San Diego, CA) for a final volume of 200 l. After overnight (~18 hours) incubation, mbIL-12 expression in transduced T-cells was analyzed by flow cytometry using a live dead discrimination dye, anti-Thy1.2, anti-IL- 12p70, and anti-CD3. The fold-change of mbIL-12 within a restimulation condition was calculated as the gMFI of mbIL-12 with ligand added divided by the gMFI without ligand for a given construct. Both secreted IL-12 and shed IL-12 were assessed by measuring IL-12 accumulated in the culture supernatants using human IL-12p70 (and / or human interferon- gamma) MSD V-plex assay kits (Meso Scale Discovery). Fold-change of IL-12 within a restimulation condition (PMA / Ionomycin) was calculated as the concentration of IL-12 with ligand added divided by the concentration without ligand for a given construct. IL-12 regulation using the dual regulation system described herein was demonstrated in human TIL (FIGs.22A-22B). Soluble (shed) IL-12 was regulatable with both PMA activation and TMP ligand in engineered TIL following 24-hour in vitro culture (FIG.22A). Similarly, membrane-bound (mbIL-12) was regulatable with both PMA activation and TMP ligand in engineered TIL (FIG.22B). Example 6. In vivo regulation of IL-12 activity using NFAT response elements and a drug responsive domain (DRD) The study design for this Example is depicted in FIG.24. Syngeneic B16-F10 melanoma cells were implanted subcutaneously in C57BL / 6J mice. Animals (n=8 per treatment group) were randomized and lymphodepleted with cyclophosphamide(200 mg / kg) on Day 8 post-tumor implant. T-cells responsive to the premelanosome protein (PMEL) melanoma antigen present on B16 tumors were engineered with retroviruses containing constructs mIL12-012, mIL12-115, and mIL12-110 and expanded for 6 days. Cells were infused intravenously after the cyclophosphamide treatment. No transfer, vector control, and NFAT response element and hDHFR domain responsive domain groups were dosed with either TMP daily via oral gavage at 500 mg / kg or vehicle (polyethylene glycol (PEG) 400) from the day of infusion.100 L blood was collected via submandibular bleed on Day 7 post-adoptive cell transfer. Plasma cytokines including interferon gamma (IFN ) were measuredusing a Meso Scale Discovery V-plex assay. IFN is a downstream cytokine associated withIL-12 activity. Table 7. IL-12 constructs used in Example 6. IL-12 regulation using the dual hDHFR DRD and NFAT response element systemdescribed herein and, accordingly, IFN regulation was demonstrated in vivo in animmunocompetent murine model. As shown in FIG. 23, IL-12 activity and, thus, IFN wasregulatable in vivo when both signals, i.e., T-cell activation (via antigen binding) and TMP ligand were provided. More specifically, murine constructs with the ARRE1 IL2 NFAT system described herein in combination with hDHFR drug responsive domain regulationdemonstrated hDHFR-TMP based regulation of IL-12 by controlling plasma IFN – adownstream cytokine associated with IL-12 activity in vivo.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid construct comprising (a) one or more nuclear factor of activated T cells (NFAT) response elements and (b) an expressible nucleic acid sequence encoding a first engineered polypeptide monomer, wherein the first engineered polypeptide monomer comprises (1) a regulatable polypeptide payload and (2) at least one drug responsive domain (DRD), wherein the NFAT response element is operably linked to the expressible nucleic acid sequence, wherein the regulatable polypeptide payload has a biological activity and is operably linked to the at least one drug responsive domain (DRD), and wherein the at least one DRD is responsive to a ligand.

2. The nucleic acid construct of claim 1, wherein the biological activity of the regulatable polypeptide payload is regulated by modulation of expression of the first engineered polypeptide monomer by the one or more NFAT response elements and by the interaction of the at least one DRD and an effective amount of the ligand.

3. The nucleic acid construct of claim 1 or 2, wherein the first engineered polypeptide monomer further comprises an oligomerization domain configured to promote oligomerization of the first engineered polypeptide monomer with at least a second engineered polypeptide monomer.

4. The nucleic acid construct of claim 3, wherein the oligomerization domain is chosen from a dimerization domain, a trimerization domain, a tetramerization domain, a pentamerization domain, and a hexamerization domain.

5. The nucleic acid construct of any one of claims 1-4, wherein the biological activity of the regulatable polypeptide payload ranges from a basal activity level in the absence of the ligand to a maximum activity level in the presence of a saturating amount of the ligand and wherein the range from basal to maximum activity level of the regulatable polypeptide payload is greater than that of the same regulatable polypeptide payload in a control nucleic acid construct lacking the one or more NFAT response elements.

6. The nucleic acid construct of any one of claims 1-5, wherein the regulatable polypeptide payload has a basal activity level in the absence of ligand that is lower than that of the sameregulatable polypeptide payload in a control nucleic acid construct lacking the one or more NFAT response elements.

7. The nucleic acid construct of any one of claims 1-6, wherein the at least one DRD is selected from the group consisting of CA2 (SEQ ID NO:2), ecDHFR (SEQ ID NO:6), hDHFR (SEQ ID NO:7), FKBP (SEQ ID NO:8), PDE5 ligand binding domain (SEQ ID NO:9), PDE5 (SEQ ID NO:10), and ER (SEQ ID NO:11), and variants thereof, wherein the variants of CA2, ecDHFR, hDHFR, FKBP, PDE5 ligand binding domain, PDE5, and ER have at least 85% amino acid sequence identity to SEQ ID NOs: 2, 6, 7, 8, 9, 10, or 11.

8. The nucleic acid construct of any one of claims 1-7, wherein the regulatable polypeptide payload is selected from the group consisting of a cytokine, a cytokine receptor, a TCR, a CAR, an immunomodulatory proteins, and combinations thereof.

9. The nucleic acid construct of claim 8, wherein the regulatable polypeptide payload is an IL-12 or IL-15 cytokine.

10. The nucleic acid construct of any one of claims 1-9, wherein the nucleic acid construct comprises 1-6 NFAT response elements.

11. The nucleic acid construct of any one of claims 1-10, wherein the one or more NFAT response elements have at least 85% nucleic acid identity to SEQ ID NOs:14, 15, 16, 17, or 18.

12. A nucleic acid construct comprising one or more NFAT response elements with at least 85% nucleic acid identity to a NFAT response element selected from the group consisting of SEQ ID NO:14, 15, 16, 17 and 18.

13. A vector comprising one or more nucleic acid constructs of any one of claims 1-12.

14. A cell comprising one or more nucleic acid constructs of any one of claims 1-12.

15. The cell of claim 14, wherein the cell is a human cell.

16. The cell of claim 14 or 15, wherein the cell is an immune cell.

17. A method of regulating a biological activity level of a regulatable polypeptide payload encoded by the nucleic acid construct of any one of claims 1-12 comprising: (a) contacting a cell comprising the nucleic acid construct with an immune cell stimulation agent; and (b) contacting the cell with a selected dose of the ligand, wherein the selected doses of the immune cell stimulation agent and the ligand results in a selected biological activity level of the regulatable polypeptide payload.

18. The method of claim 17, wherein contacting is performed in vivo.

19. The method of claim 17 or 18, wherein the immune cell stimulation agent is phorbol 12- myristate 13-acetate (PMA).

20. The method of claim 19, wherein the immune cell stimulation agent further comprises ionomycin.

21. The method of claim 17 or 18, wherein the immune cell stimulation agent is an antibody that binds CD3, an antibody that binds CD28, or a combination thereof.

22. The method of any one of claims 17-21, wherein contacting the cell with the immune cell stimulation agent occurs at least 24 hours prior to contacting the cell with the ligand.

23. The method of any one of claims 17-22, wherein the ligand comprises acetazolamide (ACZ).

24. A method of regulating a biological activity level of a regulatable polypeptide payload encoded by the nucleic acid construct of any one of claims 1-12 comprising contacting the cell with a selected dose of the ligand, wherein the selected dose of the ligand results in a selected biological activity level of the regulatable polypeptide payload.

25. The method of claim 24, wherein the contacting is performed in vivo.

26. A method of delivering a regulatable polypeptide payload to a subject comprising administering to the subject the nucleic acid construct of any one of claims 1-12 or the vector of claim 12.

27. A method of delivering a regulatable polypeptide payload to a subject comprising administering to the subject the cell of any one of claims 14-16.

28. The method of claim 26 or 27, further comprising administering to the subject a selected amount of the ligand, a selected amount of an immune cell stimulation agent, or both to deliver a selected activity of the regulatable polypeptide payload to the subject.

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