Method for control adjustable with composition of cd40l

Abstract

To provide a novel therapy of lower toxicity having more excellent therapeutic effect, longer clinical benefit, and improved safety profile to cancer.SOLUTION: Provided is a controllable biological circuit system, an effector module, and a composition for cancer immunotherapy. Also provided is a method for inducing anti-cancer immune response in a subject. Provided is a novel protein domain showing small molecule-dependent stability. The protein domain is called a drug response domain (DRD). In the absence of a binding ligand of the domain, DRD is unstabilized, decomposition of a payload fused into DRD or a target protein (POI) is caused, an operably connected DRD and payload can be stabilized in the presence of a binding ligand of the domain, and their stability depends on a dosage.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 815,404, filed March 8, 2019, U.S. Provisional Application No. 62 / 835,554, filed April 18, 2019, and U.S. Provisional Application No. 62 / 860,356, filed June 12, 2019, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named 2095_1216PCT_ST25.txt, created on March 4, 2020, with a size of 10.8 MB. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Gene and cell therapy have revolutionized medicine, offering new possibilities for the treatment of previously intractable conditions. However, most current technologies do not allow for titration of the timing or level of target protein induction. This makes many potential gene and cell therapy applications difficult or impossible to deploy safely and effectively.

[0004] Insufficient exogenous and / or endogenous gene control is a significant problem in many gene and cell therapy contexts. This lack of tunability also makes it difficult to safely express proteins that have narrow or uncertain therapeutic windows, or that require more dosed or transient expression.

[0005] One approach to control protein expression or function is the use of drug-responsive domains (DRDs), also known as destabilization domains (DDs). Drug-responsive domains are small protein domains that can be added to target proteins of interest. In the absence of a DRD-binding ligand, the DRD destabilizes the bound protein of interest, and the protein of interest is rapidly degraded by the cellular ubiquitin-proteasome system. However, when a specific small molecule DRD-binding ligand binds to the DRD, the bound protein of interest is stabilized and the protein's function is achieved.

[0006] The role of the immune system in tumor control, particularly T cell-mediated cytotoxicity, is well recognized. There is increasing evidence that T cells can control tumor growth and survival in cancer patients, both at early and late stages of the disease. However, tumor-specific T cell responses are difficult to initiate and sustain in cancer patients.

[0007] T cell pathways that have received the most attention to date include signaling through cytotoxic T lymphocyte antigen-4 (CTLA-4, CD152) and programmed death ligand 1 (PD-L1, also known as B7-H1 or CD274). Recently, however, other molecules that signal through T cell pathways, including CD40 ligand (CD40L), have gained interest as mediators of tumor control.

[0008] CD40L (also known as CD154, CD40 ligand, gp39, or TBAM) is a 33 kDa type II membrane glycoprotein (Swiss-ProtAcc-No P29965). In addition, shorter 18 kDa soluble forms of CD40L exist (also known as sCD40L or soluble CD40L). These soluble forms of CD40L are generated by proteolytic processing of the membrane-bound protein, but the cellular activity of the soluble species is weak in the absence of higher order oligomerization (e.g., trimerization). CD40L binds and activates CD40.

[0009] CD40L is a member of the TNF family of molecules that is expressed primarily on activated T cells (including Th0, Th1, and Th2 subtypes) and forms homotrimers similar to other members of this family. In addition, CD40L has also been found to be expressed on mast cells, activated basophils, and eosinophils. CD40L binds to the receptor CD40 on antigen-presenting cells (APCs), which results in a number of effects depending on the target cell type. In general, CD40L acts as a costimulatory molecule, inducing APC activation in conjunction with T cell receptor stimulation by MHC molecules on APCs. CD40L can also bind to B cells, monocytes, macrophages, platelets, neutrophils, dendritic cells, endothelial cells, and αSMCs (smooth muscle cells). Binding of CD40L to CD40 expressed on dendritic cells can promote dendritic cell (DC) licensing. DCs can be converted to a functional state by antigen-specific T helper cells to activate cytotoxic CD8+ T cells, a process called DC licensing. Engagement of CD40 on DCs results in DC stimulation as evidenced by surface expression of costimulatory molecules and MHC molecules, production of inflammatory cytokines (e.g., IL12 and TNF), and epitope spreading, all immune responses thought to aid in tumor eradication and antitumor effects.

[0010] Despite the great progress made in the past decade in developing strategies to combat cancer and other diseases, patients with advanced, refractory, and metastatic disease have limited clinical options. Chemotherapy, radiation, and high-dose chemotherapy have become dose-limiting toxicities. There remains a substantial unmet need for new, less toxic methods and therapies with better therapeutic efficacy, longer clinical benefit, and improved safety profiles, especially for patients with advanced disease or cancers that are resistant to existing therapies. Summary of the Invention [Means for solving the problem]

[0011] The present disclosure provides novel protein domains that exhibit small molecule-dependent stability. Such protein domains are called drug response domains (DRDs). In the absence of their binding ligand, the DRDs are destabilized, causing degradation of the payload or protein of interest (POI) (used interchangeably herein) fused to the DRD, and in the presence of their binding ligands, the operably linked DRDs and payloads can be stabilized, with the stability being dose-dependent.

[0012] Provided herein is a composition comprising an effector module. The effector module may comprise a stimuli-responsive element (SRE) operably linked to one or more payloads. In various embodiments, the SRE comprises a drug-responsive domain (DRD), or consists essentially of a drug-responsive domain (DRD), or consists of a drug-responsive domain (DRD). As used herein, the SRE may also be referred to as a DRD or DD.

[0013] Exemplary compositions herein include, but are not limited to, compositions comprising an effector module, the effector module comprising a stimulus response element (SRE) operably linked to a first payload, the first payload comprising human CD40L (SEQ ID NO: 3820) or a mutant CD40L comprising one or more mutations selected from Y170G, Y172G, H224G, G226F, G226H, G226W, or G227F relative to SEQ ID NO: 3820, the payload binds, attaches, or binds to the SRE. The compositions set forth above may comprise a DRD, the DRD comprising, in whole or in part, an ER, ecDHFR, FKBP, PDE5, or hDHFR protein, the DRD further comprising one or more mutations in the amino acid sequence of the ER, ecDHFR, FKBP, PDE5, or hDHFR protein.

[0014] Exemplary CD40L payloads described herein may include one or more mutations to SEQ ID NO: 3820, such as, but not limited to, Y170G, Y172G, H224G, G226F, G226H, G226W, or G227F. The first payload may comprise, in whole or in part, human CD40L (SEQ ID NO: 3820). In some embodiments, the first payload may be the entire CD40L (SEQ ID NO: 3820). Non-limiting examples of payloads comprising the entire CD40L may be CD40L(H224G, G226F) (SEQ ID NO: 6598); CD40L(H224G, G226H) (SEQ ID NO: 6600); CD40L(Y172G, G226F) (SEQ ID NO: 6602); CD40L(Y170G, H224G, G226W) (SEQ ID NO: 6604); or CD40L(H125G, G227F) (SEQ ID NO: 6606).

[0015] The SREs described herein can respond to or interact with at least one stimulus, hi one embodiment, the stimulus can be a small molecule.

[0016] The present disclosure provides compositions that include an effector module having an SRE derived in whole or in part from a parent protein, such as ecDHFR, and a first payload that in whole or in part comprises human CD40L (SEQ ID NO: 3820). In one embodiment, the SRE comprises amino acids 2-159 of ecDHFR. In some embodiments, the SRE may comprise one or more mutations compared to the parent protein. The SRE may be, but is not limited to, SEQ ID NO: 6554, 6556, 6558, 6560, 6562, 6564, 6566, 6568, 6570, 6572, 6574, 6576, 6578, 6580, 6582, 6584, 6586, 6588, or 6590.

[0017] The SREs described herein can respond to or interact with at least one stimulus. In one embodiment, the stimulus can be a small molecule, such as, but not limited to, TMP.

[0018] Also provided herein are polynucleotides encoding the compositions described herein, as well as vectors encoding the polynucleotides, and host cells comprising the vectors described herein. The disclosure also provides pharmaceutical compositions comprising the compositions described herein, a pharma- ceutically acceptable excipient, and modified or engineered cells that express the compositions described herein.

[0019] Also exemplified herein are methods for treating a subject having a disease, e.g., cancer, in need of such treatment. An exemplary method of treating cancer according to embodiments of the present disclosure includes a method of reducing tumor burden in a subject, comprising: (a) administering to the subject a therapeutically effective amount of an immune cell disclosed herein, the immune cell comprising an effector module comprising at least one stimuli response element (SRE), the SRE being operably linked to a first payload, the first payload comprising, in whole or in part, human CD40L (SEQ ID NO: 3820), or a variant CD40L thereof; and (b) administering to the subject a therapeutically effective amount of a stimulus to modulate expression of the first payload, thereby reducing tumor burden. In related embodiments, the effector module may comprise a second payload expressed in the immune cell, with or without linkage to the same or different DRD as the first payload. In some related aspects, the second payload is a chimeric antigen receptor (CAR), e.g., a CD19 CAR, as described herein.

[0020] In an illustrative example, the disclosure provides a method of activating dendritic cells in a subject, the method comprising: (a) administering to the subject one or more immune cells, the one or more immune cells comprising an effector module, the effector module comprising at least one stimulus response element (SRE) operably linked to a first payload, the first payload comprising, in whole or in part, human CD40L (SEQ ID NO: 3820), or a variant thereof, and the immune cells being T cells; (b) administering to the subject a therapeutically effective amount of a stimulus to modulate expression of the first payload, the stimulus being a ligand; and (c) measuring IL12, a dendritic cell activation marker, in response to the ligand in the subject to measure dendritic cell activation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The details of one or more embodiments of the present disclosure are described in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and inventions are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the description. In this specification, the singular form includes the plural form unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present description will control. A. Composition The present disclosure provides modified cells, nucleic acid molecules, vectors, and cell and gene therapies that can control the timing or levels of native therapeutic proteins through the administration of oral small molecule drugs. Additionally, the present disclosure provides compositions, systems, and methods for tunable expression of a protein of interest (POI, also referred to herein as "payload" and may be used interchangeably). The compositions relate to systems for tunable expression of a protein of interest in cells, and agents that induce translation of a polynucleotide encoding at least one drug responsive domain (DRD) operably linked to at least one protein of interest. The compositions provided by the present disclosure include nucleic acid molecules, polypeptides, and modified cells associated with the systems for tunable expression of a protein of interest for use in treating disease in the presence of a ligand that stabilizes the DRD. Methods associated with the tunable system for protein expression provided by the present disclosure include methods for producing modified cells, and methods for treating or preventing disease.

[0022] In various embodiments, the present disclosure provides a method for treating a disease in a subject in need thereof, the method comprising: a. providing a population of cells; b. introducing at least one nucleic acid into at least one cell in the cell population, the nucleic acid molecule comprising at least one nucleic acid sequence encoding a protein of interest operably linked to a drug responsive domain (DRD); ​​c. delivering the cells to a subject; and d. administering to the subject a ligand that stabilizes the DRD sufficiently to permit expression of the protein of interest in the at least one cell, wherein expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest in at least one cell in the cell population.

[0023] 1. Tunable Protein Expression System In general, a stimuli response element (SRE) comprising a DRD can be operably linked to a payload construct, which can be any payload (e.g., an immunotherapeutic drug), to form an effector module. When activated by a specific stimulus, a stabilizing ligand or simply a ligand (used interchangeably herein), e.g., a small molecule, the SRE can provide a signal or result in an engineered cell to control the transcription, translation, and / or protein levels of the linked payload. The tunable expression of the payload can be up- or down-regulated by providing or omitting a stabilizing ligand that stabilizes the DRD to provide tunable expression of the operably linked payload. In various embodiments, the present disclosure provides polynucleotides encoding an SRE that tunes the expression levels and activity of any agent used in immunotherapy.

[0024] As used herein, a "biological circuit" or "biological circuit system" includes a stimulus and at least one effector module responsive to the stimulus, where the response to the stimulus results in at least one signal or result within, during, as an indication of, or on a biological system. A biological system is generally understood to be any cell, tissue, organ, organ system, or organism, whether animal, plant, fungus, bacteria, or virus. It is also understood that a biological circuit may be an artificial circuit that uses a stimulus or effector module taught by the present disclosure to produce a signal or result in an acellular environment, such as a diagnostic, reporter system, device, assay, or kit. An artificial circuit may be associated with one or more electronic, magnetic, or radioactive components or parts.

[0025] 2. Effector modules and stimulus response elements (SREs) The disclosed systems, compositions and methods include at least one stimuli response element as a component of the effector module system. As used herein, an "effector module" is a single or multi-component construct or complex that includes at least (a) one or more stimuli response elements and (b) one or more payloads (e.g., payloads of interest or proteins of interest (POIs)). In one embodiment, the DRD of the SRE comprises, in whole or in part, an ER, an ecDHFR, an FKBP, a PDE5, or a hdhdr hDHFR protein, and the DRD further comprises one or more mutations in the amino acid sequence of the ER, an ecDHFR, an FKBP, a PDE5, or a hDHFR protein. In one embodiment, the payload comprises human CD40L (SEQ ID NO: 3820) or a mutant CD40L comprising one or more mutations selected from Y170G, Y172G, H224G, G226F, G226H, G226W, or G227F relative to SEQ ID NO: 3820.

[0026] As used herein, a "stimulus response element (SRE)" is a component of an effector module that is bound, coupled, operably linked, or associated with one or more payloads of the effector module, and is in some cases responsible for the nature of the effector module's responsiveness to one or more stimuli. As used herein, the nature of the "responsiveness" of an SRE to a stimulus may be characterized by covalent or non-covalent interactions, direct or indirect binding, or structural or chemical response to the stimulus. Furthermore, the response of any SRE to a stimulus may be a matter of degree or type. The response may be a partial response. The response may be a reversible response. The response may ultimately result in a controlled signal or outcome. Such outcome signals may be of a relative nature to the stimulus, for example, a modulatory effect between 1% and 100%, or a fold increase or decrease, such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more.

[0027] In some embodiments, the present disclosure provides a method for modulating the expression, function, or level of a protein. In some aspects, the modulation of the expression, function, or level of a protein is at least 20%, e.g., about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40- This refers to modulation of expression, function or levels of: 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100%, or 95-100%.

[0028] 3. Characterization of Ligand-dependent Activity of the Tunable Protein Expression System The ligand-dependent activity of the regulatable protein expression system can be characterized by a variety of methods.

[0029] In some embodiments, the ligand-dependent activity of the regulatable protein expression system is characterized by ligand-dependent regulation of the DRD. In some embodiments, the ligand-dependent activity of the regulatable protein expression system is characterized by ligand dose-dependent regulation of the DRD. The ligand-dependent regulation of the DRD transcription factor polypeptide can be characterized by various methods. In some aspects, the ligand-dependent regulation of the DRD can be evaluated by measuring the level of the DRD, the operably linked protein of interest, or both, such as by an immunoassay aimed at measuring the level of the DRD, the protein of interest, or both.

[0030] In some embodiments, the ligand-dependent activity of the tunable protein expression system is characterized by the ligand-dependent expression of a payload operably linked to the DRD. The expression of the payload can be evaluated in a variety of ways. In some aspects, the expression of the payload is evaluated by measuring protein / polypeptide levels.

[0031] In some embodiments, the tunable protein expression system may be compared to a control tunable protein expression system lacking the DRD. In some embodiments, the ligand-dependent activity of the tunable protein expression system may be analyzed or characterized in comparison to the activity of a tunable protein expression system comprising a payload-containing construct lacking the DRD.

[0032] In various embodiments, the tunable protein expression system provides tunable expression of a protein of interest or payload (used interchangeably herein). In various embodiments, the nucleic acid sequence of the present disclosure comprises a nucleotide sequence that encodes a protein of interest and is operably linked to a nucleotide sequence that encodes a DRD. The nucleotide sequence that encodes a DRD can be located upstream or downstream of the nucleic acid sequence that encodes the payload.

[0033] When a cell or organism containing a DRD is exposed to an exogenous stabilizing ligand, the DRD is stabilized. The stabilized DRD and any polypeptide sequences located upstream or downstream of the DRD are also stabilized and are not transported to the ubiquitin proteasome degradation pathway. In the absence of an exogenous stabilizing ligand, the DRD and any operably linked polypeptide sequences operably linked to the DRD upstream and / or downstream of the DRD are degraded and eliminated within the cell. Thus, both the amount and timing of protein expression can be controlled by administering an exogenous stabilizing ligand to the cell or organism.

[0034] In various embodiments, the DRD and the protein of interest are typically operably linked or may be separated by one or more intervening nucleotide, peptide, polypeptide, or protein sequences, such as a linker, a signal sequence, a leader sequence, a transmembrane domain, a domain within the tail of a cleavage site. In various embodiments, the first polynucleotide may include a first nucleic acid sequence encoding a drug responsive domain (DRD) and a second nucleic acid sequence encoding a protein of interest. In such embodiments, the protein of interest described herein within the cell is operably linked to the DRD. In addition, the cell may also include any other nucleotide, peptide, polypeptide, or protein sequence that may be operably linked to the DRD, the protein of interest, or both.

[0035] In some embodiments, the vector comprises a polynucleotide described herein. In some embodiments, the vector comprises at least a first polynucleotide, which may comprise a first nucleic acid sequence encoding a payload and a second nucleic acid sequence encoding a drug responsive domain (DRD) operably linked to the payload. Optionally, in some embodiments, the first polynucleotide and / or the vector may comprise additional components of a tunable protein expression system, e.g., an IRES sequence and a cleavage site, for monocistronic and / or bicistronic expression of one or more payloads and one or more DRDs, with optional intervening peptide or polypeptide sequences located upstream or downstream of the payload, or upstream or downstream of the DRD.

[0036] In some embodiments, the vector also has an origin of replication (ori) that allows for the amplification of the vector, for example in bacteria. Additionally or alternatively, the vector includes selectable markers, such as antibiotic resistance genes, genes for color markers, and suicide genes, as well as other regulatory sequences that allow for cloning and / or expression in bacteria, viruses, and eukaryotic cells.

[0037] 4. Drug-responsive domains (DRDs) A drug-responsive domain (DRD or DD) is a protein domain that is unstable and degraded in the absence of a ligand, but whose stability is rescued by binding to a corresponding DRD-binding ligand (also referred to herein as a stimulatory or stimulatory ligand or simply a ligand). The term drug-responsive domain (DRD) is interchangeable with the term destabilizing domain (DD). A drug-responsive domain (DRD) can be added to a polypeptide or protein of interest and can destabilize the bound polypeptide or protein in the absence of a DRD-binding ligand. The DRD provides destabilizing properties to the bound polypeptide or protein through proteolysis. Without wishing to be bound by a particular theory, it is believed that in the absence of a DRD-binding ligand, the attached or operably linked polypeptide or protein is rapidly degraded by the cellular ubiquitin-proteasome system. A ligand that binds to or interacts with the DRD can modulate the stability of the DRD and any attached or operably linked polypeptide or protein upon such binding or interaction. When the ligand binds to the DRD of interest, the instability is reversed and the function of the attached polypeptide or protein is restored. The conditional nature of DRD stability allows for rapid and unwavering switching from a stable protein to an unstable substrate for degradation, and its dependence on ligand concentration further provides tunable control of the degradation rate.

[0038] In some embodiments, the DRD of the present disclosure can be derived from a known polypeptide that can regulate protein post-translation. In some embodiments, the DRD of the present disclosure can be developed or derived from a known protein. A region or part or domain of a wild-type protein can be used as a DRD in whole or in part. They can be combined or rearranged to create new peptides, proteins, regions, or domains, any of which can be used as a starting point for the design of a DRD or further DRDs.

[0039] In some embodiments, the DRD may be derived from a parent protein or a mutant protein having one, two, three or more amino acid mutations compared to the parent protein. In some embodiments, the parent protein may be selected from, but is not limited to, FKBP; human protein FKBP; human DHFR (hDHFR); E. coli DHFR (ecDHFR); PDE5 (phosphodiesterase 5); and ER (estrogen receptor). Examples of proteins that can be used to develop DRDs and their ligands are listed in Table 1.

[0040] Table 1: DRDs and their binding ligands [Table 1-1] [Table 1-2]

[0041] In some embodiments, the sequence of the protein used to develop the DRD may include all, a portion, or a region of the protein sequence in Table 1. In some embodiments, the protein that may be used to develop the DRD includes isoforms of the proteins listed in Table 1.

[0042] In some embodiments, the DRD of the present disclosure is derived from hPDE5. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform 2. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform 3. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform X1.

[0043] In some embodiments, the DRD of the disclosure is derived from a human dihydrofolate reductase (hDHFR) protein, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or a fragment or variant thereof.

[0044] In some embodiments, the DRD may be derived from an hDHFR protein and may contain at least one mutation. In some embodiments, the DRD may be derived from an hDHFR protein and may contain more than one mutation. In some embodiments, the DRD may be derived from an hDHFR protein and may contain two, three, four, or five mutations.

[0045] In some embodiments, the DRD of the present disclosure is derived from E. coli dihydrofolate reductase (ecDHFR). In some embodiments, the DRD may be derived from an ecDHFR protein and may contain at least one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and may contain more than one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and may contain two, three, four, or five mutations.

[0046] In some embodiments, the DRD of the present disclosure is derived from FK506 binding protein (FKBP) protein or a fragment or variant thereof. In some embodiments, the DRD can be derived from FKBP protein and can include at least one mutation. In some embodiments, the DRD can be derived from FKBP protein and can include more than one mutation. In some embodiments, the DRD can be derived from FKBP protein and can include two, three, four, or five mutations.

[0047] In some embodiments, the DRD of the present disclosure is derived from estrogen receptor (ER) protein or a fragment or variant thereof. In some embodiments, the DRD may be derived from ER protein and may contain at least one mutation. In some embodiments, the DRD may be derived from ER protein and may contain more than one mutation. In some embodiments, the DRD may be derived from ER protein and may contain two, three, four, or five mutations.

[0048] The amino acid sequence of a DRD encompassed by this disclosure has at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89%, or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the parent protein from which it is derived.

[0049] Examples of DRDs of the present disclosure include those derived from human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. Suitable DRDs, which may be referred to as drug-responsive domains or ligand-binding domains, are also known in the art. For example, WO2018 / 161000;WO2018 / 231759;WO2019 / 241315;US8,173,792;US8,530,636;WO201 8 / 237323;WO2017 / 181119;US2017 / 0114346;US2019 / 0300864;WO2017 / 156238;Miyazaki et al., J Am Chem Soc, 134:3942 (2012);Banaszynski et al. (2006) Cell 126:995-1004;Stankunas, K. et al. (2003) Mol. Cell 12:1615-1624;Banaszynski et al. (2008) Nat. Med. 14:1123-1127;Iwamoto et al. (2010) Chem. Biol. 17:981-988;Armstrong et al. (2007) Nat. Methods 4:1007-1009;Madeira da Silva et al. (2009) Proc. See Natl. Acad. Sci. USA 106:7583-7588; Pruett-Miller et al. (2009) PLoS Genet. 5:e1000376; and Feng et al. (2015) Elife 4:e10606.

[0050] In some embodiments, the DRD of the disclosure is derived from a human dihydrofolate reductase (hDHFR) protein, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or a fragment or variant thereof.

[0051] In some embodiments, the DRD may be derived from an hDHFR protein and may contain at least one mutation. In some embodiments, the DRD may be derived from an hDHFR protein and may contain more than one mutation. In some embodiments, the DRD may be derived from an hDHFR protein and may contain two, three, four, or five mutations.

[0052] In some embodiments, the DRD of the present disclosure is derived from E. coli dihydrofolate reductase (ecDHFR). In some embodiments, the DRD may be derived from an ecDHFR protein and may contain at least one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and may contain more than one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and may contain two, three, four, or five mutations.

[0053] In some embodiments, the DRD of the present disclosure is derived from FK506 binding protein (FKBP) protein or a fragment or variant thereof. In some embodiments, the DRD can be derived from FKBP protein and can include at least one mutation. In some embodiments, the DRD can be derived from FKBP protein and can include more than one mutation. In some embodiments, the DRD can be derived from FKBP protein and can include two, three, four, or five mutations.

[0054] In some embodiments, the DRD of the present disclosure is derived from estrogen receptor (ER) protein or a fragment or variant thereof. In some embodiments, the DRD may be derived from ER protein and may contain at least one mutation. In some embodiments, the DRD may be derived from ER protein and may contain more than one mutation. In some embodiments, the DRD may be derived from ER protein and may contain two, three, four, or five mutations.

[0055] The amino acid sequence of a DRD encompassed by this disclosure has at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89%, or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the parent protein from which it is derived.

[0056] Examples of DRDs of the present disclosure include those derived from human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. Suitable DRDs, which may be referred to as drug-responsive domains or ligand-binding domains, are also known in the art. For example, WO2018 / 161000;WO2018 / 231759;WO2019 / 241315;US8,173,792;US8,530,636;WO201 8 / 237323;WO2017 / 181119;US2017 / 0114346;US2019 / 0300864;WO2017 / 156238;Miyazaki et al., J Am Chem Soc, 134:3942 (2012);Banaszynski et al.(2006) Cell 126:995-1004;Stankunas, K. et al. (2003) Mol. Cell 12:1615-1624;Banaszynski et al. (2008) Nat. See Med. 14:1123-1127; Iwamoto et al. (2010) Chem. Biol.17:981-988; Armstrong et al. (2007) Nat. Methods 4:1007-1009; Madeira da Silva et al. (2009) Proc. Natl.Acad.Sci.USA 106:7583-7588; Pruett-Miller et al. (2009) PLoS Genet. 5:e1000376; and Feng et al. (2015) Elife 4:e10606.

[0057] In one embodiment, the SRE is derived from a region of a parent protein or a mutant protein. The parent protein regions are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 , 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 31 6, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 41 63, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409 , 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0058] In one embodiment, the SRE is derived from a parent or variant protein and comprises a region of the parent protein. The SRE comprises 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30~35%, 35~40%, 40~45%, 45~50%, 50~55%, 55~60%, 60~65%, 65~70%, 70~75%, 75~80%, 80~85%, 85~90%, 90~95%, 95~100%, 10~20%, 20~30%, 30~40%, 40~50%, 50~60%, 60~70%, 70~80%, 80~90 %, 90~100%, 10~30%, 20~40%, 30~50%, 40~60%, 50~70%, 60~80%, 70~90%, 80~100%, 10~40%, 20~50%, 30~60%, 40~70%, 50~80%, 60~90%, 70~100%, 10~50%, 20~60%, 30~70%, 40~80%, 50~90%, It may comprise a region of the parent protein that is 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75%, or 75-100%.

[0059] In one embodiment, the SRE is derived from a parent or variant protein and is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 5-10%, 10-20%, or at least 20-30%, or at least 30-40%, or at least 40-50%, or at least 50-60%, or at least 60-70%, or at least 70-80%, or at least 80-90%, or at least 90-100%, or at least 100-100%, or at least 100-200%, or at least 100-300%, or at least 100-400%, or at least 100-5 ... ~15%, 15~20%, 20~25%, 25~30%, 30~35%, 35~40%, 40~45%, 45~50%, 50~55%, 55~60%, 60~65%, 65~70%, 70~75%, 75~80%, 80~85%, 85~90%, 90~95%, 95~100%, 10~20%, 20~30%, 30~40%, 40~50%, 50~60% , 60~70%, 70~80%, 80~90%, 90~100%, 10~30%, 20~40%, 30~50%, 40~60%, 50~70%, 60~80%, 70~90%, 80~100%, 10~40%, 20~50%, 30~60%, 40~70%, 50~80%, 60~90%, 70~100%, 10~50%, 20~60%, 30~70%, It may have 40-80%, 50-90%, 60-100%, 10-60%, 20-70%, 30-80%, 40-90%, 50-100%, 10-70%, 20-80%, 30-90%, 40-100%, 10-80%, 20-90%, 30-100%, 10-90%, 20-100%, 25-50%, 50-75%, or 75-100% identity.

[0060] In one embodiment, an effector module and / or SRE of the present disclosure may comprise at least one drug responsive domain (DRD). An effector module and / or SRE may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DRDs. When more than one DRD is present, each DRD may be derived from the same parent protein (e.g., PDE5), different parent proteins (e.g., PDE5 and hDHFR), may be a fusion of two different parent proteins, or may be artificial.

[0061] In one embodiment, an effector module and / or SRE of the disclosure may comprise two DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise three DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise four DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise five DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise six DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise seven DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise eight DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise nine DRDs. In one embodiment, an effector module and / or SRE of the disclosure may comprise ten DRDs. The DRDs may be derived from any parent protein known in the art and / or described herein. In some embodiments, the DRDs are derived from the same parent protein (e.g., PDE5). In some embodiments, the DRDs are derived from different regions of the same parent protein (e.g., amino acids 535-860 and amino acids 590-836 of PDE5). In some embodiments, the DRDs are derived from different parent proteins (e.g., PDE5 and hDHFR).

[0062] 5. Drug-responsive domain (DRD) from human dihydrofolate reductase (hDHFR) In one embodiment, the SRE may comprise at least one drug responsive domain (DD) derived from a human dihydrofolate reductase (hDHFR) protein, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or a fragment or variant thereof. As a non-limiting example, the SRE comprises at least one DRD derived from hDHFR1. As a non-limiting example, the SRE comprises at least one DRD derived from hDHFR2.

[0063] In some embodiments, the DRD of the present disclosure may be derived from human dihydrofolate reductase (hDHFR). hDHFR is a small (18 kDa) enzyme that catalyzes the reduction of dihydrofolate and plays an important role in various anabolic pathways. Dihydrofolate reductase (DHFR) is an essential enzyme that converts 7,8-dihydrofolate (DHF) to 5,6,7,8,tetrahydrofolate (THF) in the presence of nicotinamide adenine dihydrogen phosphate (NADPH). Antifolates that bind more tightly to DHFR than to its natural substrate DHF, such as methotrexate (MTX), a structural analogue of folate, disrupt folate metabolism primarily by inhibiting dihydrofolate reductase, suppressing the synthesis of purine and pyrimidine precursors. Other hDHFR inhibitors, such as folic acid, TQD, trimethoprim (TMP), epigallocatechin gallate (EGCG), and ECG (epicatechin gallate), also bind to hDHFR and regulate its stability.

[0064] In one embodiment, the SRE comprises a region of the hDHFR protein. The regions of the hDHFR protein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 2 10, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 17, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 , 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0065] In one embodiment, the DRD may be derived from an hDHFR protein and may contain at least one mutation. Non-limiting examples of mutations include M1del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17V, K19E, N20D, G21E, G21T, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31D, E31G, F32M, R33G, R33S, F35L, Q36F, Q36K, Q36R, Q36S, R37G, M38T, M38V, T40A, V44A, K47R, N49D, N49S, M53T, G54R, K56E, K56R, T5 7A, F59S, I61T, E63G, K64R, N65A, N65D, N65F, N65S, L68S, K69E, K69R, R 71G, I72A, I72T, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F 89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102 G, Q103R, P104S, E105G, M112T, M112V, V113A, W114R, I115L, I115V, V116 I, G117D, V121A, Y122C, Y122D, Y122I, Y122N, A107T, A107V, N108D, K10 9E, K109R, V110A, D111N, K123E, K123R, A125F, M126I, N127R, N127S, N12 7Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135L, F135P, F135S, F1 35V, V136M, T137R, R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D1 42G, F143L, F143S, E144G, D146G, T147A, F148L, F148S, F149L, P150L, ​​E 151G, I152V, D153A, D153G, E155G, K156R, Y157C, Y157R, K158E, K158R, L 159P, L160P, E162G, Y163C, V166A, N168D, S168C, D169G, V170A, Q171R, E172G, E173A, E173G, K174R, I176A, I176F, I176T, K177E, K177R, Y178C,Examples of the nucleotide sequences include Y178H, F180L, E181G, V182A, Y183C, Y183H, E184G, E184R, K185del, K185E, K185R, N186D, N186S, D187G, and D187N.

[0066] In one embodiment, the DRD may be derived from hDHFR protein and may contain more than one mutation. Any of the mutations listed herein may be included in the DRD. Non-limiting examples of double mutations include C7R, Y163C; A10V, H88Y; I17V, Y122I; G21T, Y122I; Q36K, Y122I; M53T, R138I; T57A, I72A; E63G, I176F; L74N, Y122I; V75F, Y122I; L94A, T147A; V121A, Y122I; Y122I, A125F; Y1 22I, N127Y; Y122I, M140I; H131R, E144G; T137R, F143L; E162G, I176F; Y178H, E181G; Y183H, K185E; M1del, I17V; M1del, Y122I; M1del, K185E; M1del, N127Y; M1del, N168D; and M1del, M140I. Non-limiting examples of triple mutations include I8V, K133E, Y163C; V9A, S93R, P150L; K19E, F89L, E181G; G21E, I72V, I176T; L23S, V121A, Y157C; E31D, F32M, V116I; Q36F, N65F, Y122I; Q36K, N65F, Y122I; Q36F, Y122I, A125F; N49D, F59S, D153G; V110A, V136M, K177R; Y122I, H131R, E144G; M1del, I17V, Y122I; M1 del, G21T, Y122I; M1del, G21T, Y122N; M1del, Q36K, Y122I; M1del, M53T, R138I; M1del, L74N, Y122I; M1del, V75F, Y122I; M1del, L94A, T147A; M1del, V121A, Y122I; M1del, Y122I, A125F; M1del, Y122I, M140I; M1del, Y122I, N127Y; M1del, H131R, E144G; and M1del, E162G, I176F. Non-limiting examples of the four mutations include: G54R, I115L, M140V, S168C; M1del, E31D, F32M, V116I; M1del, Q36F, N65F, Y122I; M1del, Q36F, Y122I, A125F; M1del, Q36K, N65F, Y122I;and M1del, Y122I, H131R, E144G. Non-limiting examples of the five mutations include V2A, R33G, Q36R, L100P, K185R; D22S, F32M, R33S, Q36S, N65S; and M1del, D22S, F32M, R33S, Q36S, N65S. Non-limiting examples of more than five mutations include I17N, L98S, K99R, M112T, E151G, E162G, E172G; G16S, I17V, F89L, D96G, K123E, M140V, D146G, K156R; K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E; R138G, D142G, F143S, K156R, K158E, E162G, V1 66A, K177E, Y178C, K185E, N186S; N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E 173A, F180L;F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, I139V, M140I, D142G, D146G, E173G, D187G;L 28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N, L134P, F135V, T147A, I152V, K158R, E172G, V 182A, E184R; V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, V110A, I115V, Y122D, L132P, F135 S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S; L100P, E102G, Q103R, P104S, E105G, N108D, V113A, W114R, Y122C, M1 26I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S;and A10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, M112V, W114R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N.

[0067] In some embodiments, the DRD derived from hDHFR can include amino acids 2-187 of the parent hDHFR sequence, which is referred to herein as the M1del mutation.

[0068] In one embodiment, the stimulus is a small molecule that binds to the SRE to regulate post-translational protein levels. In one aspect, the DHFR ligands: trimethoprim (TMP) and methotrexate (MTX) are used to stabilize hDHFR mutants.

[0069] A non-limiting list of hDHFR-derived drug responsive domains (amino acid and nucleic acid sequences) are listed in Table 2. For hDHFR-derived DRDs, the positions of the mutated amino acids listed in Table 2 are relative to human DHFR of SEQ ID NO: 30 (Uniprot ID: P00374) (referred to as "WT" in Table 2). In Table 2, "del" means that the mutation is a deletion of an amino acid at that position compared to the wild type sequence. In Table 2, a DHFR-derived drug responsive domain that includes amino acids 1-187 of the parent hDHFR sequence is shown as hDHFR with the identification of the mutation in brackets, e.g., hDHFR(Y122I).

[0070] Table 2: Drug-responsive domains (DDs) derived from hDHFR. [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

[0071] In one embodiment, the SRE is selected from the group consisting of hDHFRDD-1, hDHFRDD-2, hDHFRDD-3, hDHFRDD-4, hDHFRDD-5, hDHFRDD-6, hDHFRDD-7, hDHFRDD-8, hDHFRDD-9, hDHFRDD-10, hDHFRDD-11, hDHFRDD-12, hDHFRDD-13, hDHFRDD-14, hDHFRDD-15, hDHFRDD-16, hDHFRDD-17, hDHFRDD-18, hDHFRDD-19, hDHFRDD-20, hDHFRDD-21, hDHFRDD -22, hDHFRDD-23, hDHFRDD-24, hDHFRDD-25, hDHFRDD-26, hDHFRDD-27, hDHFRDD-28, hDHFRDD-29, hDHFRDD-30, hDHFRDD-31, hDHFRDD-32, hDHFRDD -33, hDHFRDD-34, hDHFRDD-35, hDHFRDD-36, hDHFRDD-37, hDHFRDD-38, hDHFRDD-39, hDHFRDD-40, hDHFRDD-41, hDHFRDD-42, hDHFRDD-43, hDHFRDD -44, hDHFRDD-45, hDHFRDD-46, hDHFRDD-47, hDHFRDD-48, hDHFRDD-49, hDHFRDD-50, hDHFRDD-51, hDHFRDD-52, hDHFRDD-53, hDHFRDD-54, hDHFRDD -55, hDHFRDD-56, hDHFRDD-57, hDHFRDD-58, hDHFRDD-59, hDHFRDD-60, hDHFRDD-61, hDHFRDD-62, hDHFRDD-63, hDHFRDD-64, hDHFRDD-65, hDHFRDD hDHFRDD-66, hDHFRDD-67, hDHFRDD-68, hDHFRDD-69, hDHFRDD-70, hDHFRDD-71, hDHFRDD-72, hDHFRDD-73, hDHFRDD-74, hDHFRDD-75, hDHFRDD-76, hDHFRDD-77, hDHFRDD-78, hDHFRDD-79, hDHFRDD-80, hDHFRDD-81, hDHFRDD-82, hDHFRDD-83, hDHFRDD-84, hDHFRDD-85, and hDHFRDD-86,It may contain at least one drug-responsive domain (DD) derived from hDHFR.

[0072] 6. Drug-responsive domain (DRD) from E. coli dihydrofolate reductase (ecDHFR) In one embodiment, the SRE may comprise at least one drug responsive domain (DRD) derived from E. coli dihydrofolate reductase (ecDHFR) protein, or a fragment or variant thereof.

[0073] In one embodiment, the SRE comprises a region of the ecDHFR protein. The regions of the ecDHFR protein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 1 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 2 10, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 17, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 , 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0074] In one embodiment, the DRD may be derived from an ecDHFR protein and may contain at least one mutation, non-limiting examples of which include M1del, R12Y, R12H, G27S, Y100I, and E129K.

[0075] In one embodiment, the DRD may be derived from ecDHFR protein and may contain more than one mutation. Any of the mutations listed herein may be included in the DRD. Non-limiting examples of double mutations include R12Y and Y100I. Non-limiting examples of triple mutations include M1del, R12Y, Y100I; M1del, R12Y, E129K; and M1del, R12H, E129K. Non-limiting examples of four mutations include M1del, R12Y, G27S, Y100I.

[0076] In some embodiments, the DRD derived from ecDHFR can include amino acids 2-159 of the echDHFR sequence, which is referred to herein as the M1del mutation.

[0077] In one embodiment, the stimulus is a small molecule that binds to the SRE to regulate post-translational protein levels. In one aspect, the ecDHFR ligands: trimethoprim (TMP) and methotrexate (MTX) are used to stabilize ecDHFR mutants.

[0078] A non-limiting list of drug responsive domains (amino acid and nucleic acid sequences) from ecDHFR are listed in Table 3. The positions of the mutated amino acids listed in Table 3 correspond to the E. The sequences are based on Coli DHFR (Uniprot ID: P0ABQ4) (referred to as "WT" in Table 3). In Table 3, "del" means that the mutation is a deletion of an amino acid at that position compared to the wild type sequence. In Table 3, a drug responsive domain derived from ecDHFR that includes amino acids 1-159 of the parent ecDHFR sequence is shown as ecDHFR with the identification of the mutation in brackets, e.g., ecDHFR(R12Y, Y100I).

[0079] Table 3: Drug-responsive domains (DDs) derived from ecDHFR. [Table 3-1] [Table 3-2] [Table 3-3]

[0080] In one embodiment, the SRE may comprise at least one ecDHFR-derived drug responsive domain (DD), such as, but not limited to, ecDHFRDD-1, ecDHFRDD-2, ecDHFRDD-3, ecDHFRDD-4, ecDHFRDD-5, ecDHFRDD-6, ecDHFRDD-7, ecDHFRDD-8, ecDHFRDD-9, ecDHFRDD-10, ecDHFRDD-11, ecDHFRDD-12, ecDHFRDD-13, ecDHFRDD-14, ecDHFRDD-15, ecDHFRDD-16, ecDHFRDD-17, ecDHFRDD-18, ecDHFRDD-19, ecDHFRDD-20, ecDHFRDD-21, ecDHFRDD-22, ecDHFRDD-23, ecDHFRDD-24, and ecDHFRDD-25.

[0081] Drug-responsive domain (DRD) from FK506-binding protein (FKBP)

[0082] In one embodiment, the SRE may comprise at least one drug responsive domain (DRD) derived from an FK506 binding protein (FKBP) protein, or a fragment or variant thereof.

[0083] In one embodiment, the SRE comprises a region of an FKBP protein. The regions of the FKBP protein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 12 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15 0, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2 11, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 3 64, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 4 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0084] In one embodiment, the DRD may be derived from an FKBP protein and may contain at least one mutation, non-limiting examples of which include M1del, E32G, F37V, R72G, K106E, and L109P.

[0085] In one embodiment, DRD can be derived from FKBP protein and can include more than one mutation. Any of the mutations described herein can be included in DD. Non-limiting examples of double mutations include M1del, F37V; F37V, L107P. Non-limiting examples of triple mutations include M1del, F37V, L107P. Non-limiting examples of four mutations include E32G, F37V, R72G, K106E. Non-limiting examples of five mutations include M1del, E32G, F37V, R72G, K106E.

[0086] In some embodiments, the DRD derived from FKBP can include amino acids 2-108 of the parent FKBP sequence, which is referred to herein as the M1del mutation.

[0087] In one embodiment, the stimulus is a small molecule that binds to the SRE to regulate post-translational protein levels. In one aspect, the FKBP ligand shield-1 is used to stabilize the FKBP mutant.

[0088] A non-limiting list of drug responsive domains (amino acid and nucleic acid sequences) from FKBP proteins are listed in Table 4. The positions of the mutated amino acids listed in Table 4 are relative to FKBP of SEQ ID NO: 37 (Uniprot ID: P62942) (referred to as "WT" in Table 4). In Table 4, "del" means that the mutation is a deletion of an amino acid at that position compared to the wild type sequence. In Table 4, a drug responsive domain from FKBP that includes amino acids 1-108 of the parent FKBP sequence is shown as FKBP with the identification of the mutation in parentheses, e.g., FKBP(F37V).

[0089] Table 4: Drug-responsive domains (DRDs) from FKBPs. [Table 4]

[0090] In one embodiment, the SRE may comprise a drug responsive domain (DD) from at least one FKBP, such as, but not limited to, FKBPDD-1, FKBPDD-2, FKBPDD-3, FKBPDD-4, FKBPDD-5, FKBPDD-6, and FKBPDD-7.

[0091] 7. Drug-responsive domain (DRD) from human phosphodiesterase (hPDE) In one embodiment, the SRE is selected from the group consisting of human phosphodiesterase 1A (hPDE1A), human phosphodiesterase 1B (hPDE1B), human phosphodiesterase 1C (hPDE1C), human phosphodiesterase 1D (hPDE1D), human phosphodiesterase 2A (hPDE2A), human phosphodiesterase 3A (hPDE3A), human phosphodiesterase 3B (hPDE3B), human phosphodiesterase 4A (hPDE4A), human phosphodiesterase 4B (hPDE4B), human phosphodiesterase 4C (hPDE4C), human phosphodiesterase 4D (hPDE4D), human phosphodiesterase 6A (hPDE6A), human phosphodiesterase 7A (hPDE7B), human phosphodiesterase 8A (hPDE8C), human phosphodiesterase 9A (hPDE9A), human phosphodiesterase 10A (hPDE9A), human phosphodiesterase 11A (hPDE9A), human phosphodiesterase 12A (hPDE9A), human phosphodiesterase 13A (hPDE9A), human phosphodiesterase 14A (hPDE9A), human phosphodiesterase 15A (hPDE9A), human phosphodiesterase 16A (hPDE9A), human phosphodiesterase 17A (hPDE9A), human phosphodiesterase 18A (hPDE9A), human phosphodiesterase 19A (hPDE9A), human phosphodiesterase 20A (hPDE20A), human phosphodiesterase 21A (hPDE9A), human phosphodiesterase 22A (hPDE2A), human phosphodiesterase 23A (hPDE9A), human phosphodiesterase 24A (hPDE9A), human The SRE may comprise at least one drug-responsive domain (DRD) derived from a human phosphodiesterase (hPDE) protein, such as, but not limited to, human phosphodiesterase 6B (hPDE6B), human phosphodiesterase 6C (hPDE6C), human phosphodiesterase 7A (hPDE7A), human phosphodiesterase 7B (hPDE7B), human phosphodiesterase 8A (hPDE8A), human phosphodiesterase 8B (hPDE8B), human phosphodiesterase 9A (hPDE9A), human phosphodiesterase 10A (hPDE10A), and human phosphodiesterase 11A (hPDE11A), or a fragment or variant thereof. As a non-limiting example, the SRE comprises at least one DD derived from hPDE1A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE1B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE1C. As a non-limiting example, the SRE comprises at least one DD derived from hPDE1D. As a non-limiting example, the SRE comprises at least one DD derived from hPDE2A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE3A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE3B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE4A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE4B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE4C.As a non-limiting example, the SRE comprises at least one DD derived from hPDE4D. As a non-limiting example, the SRE comprises at least one DD derived from hPDE6A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE6B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE6C. As a non-limiting example, the SRE comprises at least one DD derived from hPDE7A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE7B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE8A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE8B. As a non-limiting example, the SRE comprises at least one DD derived from hPDE9A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE10A. As a non-limiting example, the SRE comprises at least one DD derived from hPDE11A.

[0092] In one embodiment, the SRE comprises a region of an hPDE protein. The regions of the hPDE protein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 12 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15 0, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2 11, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 3 64, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 4 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0093] In one embodiment, the DRD may be derived from a PDE protein and may contain at least one mutation.

[0094] In one embodiment, the DRD may be derived from a PDE5 protein and may contain at least one mutation. Non-limiting examples of mutations include E535D, E536G, Q541R, S542L, V548M, P549S, S550F, L554R, K555R, I556S, F559L, S560G, F561L, S562G, F564L, F564S, F564I, L569M, T571S, T571I, L573M, C574Y, V585A, V585M, N587S, Q586L, Q586P, Q589E, K591E, H592R, V594I, I599V, K604R, K604E, N605D, N605Y, R607W, R60 7Q, K608E, N609H, N609Y, A611T, Y612F, Y612W, Y612A, H613L, H613R, W6 15R, H617L, N620S, M625I, K630R, K633E, N636S, I648V, H653A, D656L, R6 58H, G659A, N661S, N662Y, S663Y, S663P, Q666H, L675P, Y676D, Y676N, C6 77R, H678R, I680S, E682A, D687A, H685L, M691I, L693P, I700F, I706N, E7 08V, Y709H, K710N, T711A, T712S, T712M, I715K, A722V, T723S, T723R, D 724Y, D724A, D724N, D724G, L727P, Y728L, K730E, R732L, R732G, R732A, R 732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R 732E, R732Q, R732N, R732M, R732C, R732K, F735L, F736A, F736G, F736L, F 736M, F736R, F736W, F736K, F736Q, F736E, F736S, F736P, F736V, F736C, F736Y, F736H, F736I, F736N, F736D, F736T, L738H, N742S, Q743L, F744L, L746S, F755L, F755Y, M758I, M760I, A762T, A762S, D764N, D764G, D764V, D764A, S766F, A767E, I768N, K770Q, W772C, A779T, L781P, T784I, F787V,F787A, K795E, E795R, K795N, E796G, E796D, L797F, I799L, I799T, T802P, D803N, L804P, E808G, S815C, M816A, M816T, F820I, I821A, A823T, I824T, Y829A, T833S, C839S, F840S, R847G, R847T, K848N, K852E, W853F, E858V, and Q859R.

[0095] In one embodiment, DRD can be derived from PDE5 protein and can include more than one mutation. Any of the mutations listed herein can be included in DD. Non-limiting examples of double mutations include Y612A, R732L; Y612F, R732L; Y612W, R732L; Y709H, F787V; Y728L, D764N; L569M, T833S; D724A, R732L; D724G, D764G; D724G, K848N; E682A, R732L; F736A, D764N; G659A, T784I; H617L, A 722V; H653A, R732L; I556S, E796D; I700F, E796G; K770Q, K848N; L573M, F735L; N605Y, I715K; N609Y, I799L; R732L, D764A; R732L, D764N; R732L, F736A; S550F, L554R; V548M, D803N; V548M, F820I. Non-limiting examples of triple mutations include A722V, F755L, M760I; F561L, G659A, T784I; H613L, D724Y, F755Y; L554R, Q589E, A823T; L554R, Q589E, M691I; S542L, E708V, W772C; S562G, L727P, R847T; T571S, V585M, T723S; T712M, M758I, Q859R. Non-limiting examples of four mutations include: L554R, Q586P, K710N, K730E; Q586L, S663Y, A762T, E808G; T571I, K604R, I706N, E795R; W615R, T723R, A762T, E808G. Non-limiting examples of five mutations include: F564I, N662Y, H685L, L693P, F736I; P549S, F564I, R658H, A779T, R847G.

[0096] In some embodiments, the DRD derived from PDE5 can include amino acids 2-875 of the parent PDE5 sequence, which is referred to herein as the M1del mutation.

[0097] In some embodiments, the DRD is derived from a region of the PDE5 protein. As a non-limiting example, this region is amino acids 535-860 of hPDE5 (SEQ ID NO: 43). As a non-limiting example, this region is amino acids 535-830 of hPDE5 (SEQ ID NO: 44). As a non-limiting example, this region is amino acids 535-836 of hPDE5 (SEQ ID NO: 45). As a non-limiting example, this region is amino acids 567-860 of hPDE5 (SEQ ID NO: 46). As a non-limiting example, this region is amino acids 590-836 of hPDE5 (SEQ ID NO: 47). As a non-limiting example, this region is amino acids 590-860 of hPDE5 (SEQ ID NO: 48).

[0098] In one embodiment, the stimulus is a small molecule that binds to the SRE to regulate post-translational protein levels. In one embodiment, the PDE5 ligand sildenafil, vardenafil, or tadalafil is used to stabilize the PDE5 mutant.

[0099] A non-limiting list of drug responsive domains (amino acid and nucleic acid sequences) from PDE5 are listed in Table 5. The positions of the mutated amino acids listed in Table 5 are based on PDE5 (Uniprot ID: O76074) of SEQ ID NO: 1. In Table 5, "del" means that the mutation is a deletion of the amino acid at that position compared to the wild type sequence.

[0100] Table 5: Drug-responsive domains (DRDs) from PDE5 [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

[0101] In one embodiment, the SRE is PDE5DD-1, PDE5DD-2, PDE5DD-3, PDE5DD-4, PDE5DD-5, PDE5DD-6, PDE5DD-7, PDE5DD-8, PDE5DD-9, PDE5DD-10, PDE5DD-11, PDE5DD-12, PDE5DD-13, PDE5DD-14, PDE5DD-15, PDE5DD-16, PDE5DD-17, PDE5DD-18, PDE5DD-19, PDE5DD-20, PDE5DD-21, PDE5DD-22, PDE5DD-23, PDE5DD-24, PDE5DD-25, PDE5DD-26, PDE5DD-27, PDE5DD-28, PDE5DD-29, PDE5DD-30, PDE5DD-31, PDE5DD-32, PDE5DD-33, PDE5DD-34, PDE5DD-35, PDE5DD-36, PDE5DD-37, PDE5DD-38, PDE5DD-39, PDE5DD-40, PDE5DD-41, PDE5DD-42, PDE5DD-43, PDE5DD-44, PDE5DD-45, PDE5DD-46, PDE5DD-47, PDE5DD-48, PDE5DD-49, PDE5DD-50, PDE5DD-51, PDE5DD-52, PDE5DD-53, PDE5DD-54, PDE5DD-55, PDE5DD-56, PDE5DD-57, PDE5DD-58, PDE5DD-59, PDE5DD-60, PDE5DD-61, PDE5DD-62, PDE5DD-63, PDE5DD-64, PDE5DD-65, PDE5DD-66, PDE5DD-67, PDE5DD-68, PDE5DD-69, PDE5DD-70, PDE5DD-71, PDE5DD-72, PDE5DD-73, PDE5DD-74, PDE5DD-75, PDE5DD-76, PDE5DD-77, PDE5DD-78, PDE5DD-79, PDE5DD-80, PDE5DD-81, PDE5DD-82, PDE5DD-83, PDE5DD-84, PDE5DD-85, PDE5DD-86, PDE5DD-87, PDE5DD-88, PDE5DD-89, PDE5DD-90, PDE5DD-91, PDE5DD-92, PDE5DD-93, PDE5DD-94, PDE5DD-95, PDE5DD-96, PDE5DD-97, PDE5DD-98, PDE5DD-99,PDE5DD-100、PDE5DD-101、PDE5DD-102、PDE5DD-103、PDE5DD-104、PDE5DD-105、PDE5DD-106、PDE5DD-107、PDE5DD-108、PDE5DD-109、PDE5DD-110、PDE5DD-111、PDE5DD-112、PDE5DD-113、PDE5DD-114、PDE5DD-115、PDE5DD-116、PDE5DD-117、PDE5DD-118、PDE5DD-119、PDE5DD-120、PDE5DD-121、PDE5DD-122、PDE5DD-123、PDE5DD-124、PDE5DD-125、PDE5DD-126、PDE5DD-127、PDE5DD-128、PDE5DD-129、PDE5DD-130、PDE5DD-131、PDE5DD-132、PD、 E5DD-133、PDE5DD-134、PDE5DD-135、PDE5DD-136、PDE5DD-137、PDE5DD-138、PDE5DD-139、PDE5DD-140、PDE5DD-141、PDE5DD-142、PDE5DD-143、PDE5DD-144、PDE5DD-145、PDE5DD-146、PDE5DD-147、PDE5DD-148、PDE5DD-149、PDE5DD-150、PDE5DD-151、PDE5DD-152、PDE5DD-153、PDE5DD-154、PDE5DD-155、PDE5DD-156、PDE5DD-157、PDE5DD-158、PDE5DD-159、PDE5DD-160、PDE5DD-161、PDE5DD-162、PDE5DD-163、PDE5DD-164、PDE5DD-165、PDE5DD-166、PDE5DD-167、PDE5DD-168、PDE5DD-169、PDE5DD-170、PDE5DD-171、PDE5DD-172、PDE5DD-173、PDE5DD-174、PDE5DD-175、PDE5DD-176、PDE5DD-177、PDE5DD-178、PDE5DD-179、PDE5DD-180、PDE5DD-181、PDE5DD-182、PDE5DD-183、PDE5DD-184、PDE5DD-185、PDE5DD-186、PDE5DD-187、PDE5DD-188、PDE5DD-189、PDE5DD-190、PDE5DD-191、PDE5DD-192、PDE5DD-193、PDE5DD-194、PDE5DD-195、PDE5DD-196、PDE5DD-197、PDE5DD-198、PDE5DD-199、PDE5DD-200、PDE5DD-201、PDE5DD-202、PDE5DD-203、PDE5DD-204、PDE5DD-205、PDE5DD-206、PDE5DD-207、PDE5DD-208、PDE5DD-209、PDE5DD-210、PDE5DD-211、PDE5DD-212、PDE5DD-213、PDE5DD-214、PDE5DD-215、PDE5DD-216、PDE5DD-217、PDE5DD-218、PDE5DD-219、PDE5DD-220、PDE5DD-221、PDE5DD-222、PDE5DD-223、PDE5DD-224, PDE5DD-225, PDE5DD-226, PDE5DD-227, PDE5DD-228, PDE5DD-229, PDE5DD-230, PDE5DD- 231, PDE5DD-232, PDE5DD-233, PDE5DD-234, PDE5DD-235, PDE5DD-236, PDE5DD-237, PDE5DD-238, PDE5 The drug may include at least one drug responsive domain (DD) from PDE5, such as, but not limited to, PDE5DD-239, PDE5DD-240, PDE5DD-241, PDE5DD-242, PDE5DD-243, PDE5DD-244, PDE5DD-245, PDE5DD-246, PDE5DD-247, PDE5DD-248, and PDE5DD-249.

[0102] 8. Drug-responsive domain (DD) derived from PPAR gamma (PPARg) In one embodiment, the SRE may comprise at least one drug responsive domain (DD) derived from a PPAR gamma (PPARg) protein or a fragment or variant thereof.

[0103] In one embodiment, the SRE comprises a region of the PPARg protein. The region of the PPARg protein may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 120, 121, 122, 130, 131, 132, 133, 134, 135, 136 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413 , 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0104] 9. Drug-responsive domain (DRD) derived from estrogen receptor (ER) In one embodiment, the SRE may comprise at least one drug responsive domain (DRD) derived from an estrogen receptor (ER) protein or a fragment or variant thereof.

[0105] In one embodiment, the SRE comprises a region of the ER protein. The regions of ER proteins are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 1 51, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318 , 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 , 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 41 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, or more than 450 amino acids in length. The region of the parent protein may be 5-50, 25-75, 50-100, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225-275, 250-300, 275-325, 300-350, 325-375, 350-400, 375-425, or 400-450 amino acids in length.

[0106] In one embodiment, the DRD may be derived from an ER protein and may contain at least one mutation, non-limiting examples of which include K303R, N304S, T371A, L384M, M421G, N519S, G521G, and Y537S.

[0107] In one embodiment, the DRD can be derived from ER protein and can include more than one mutation. Any of the mutations listed herein can be included in the DRD. Non-limiting examples of the four mutations include L384M, M421G, G521R, Y537S. Non-limiting examples of the six mutations include T371A, L384M, M421G, N519S, G521R, Y537S. Non-limiting examples of the eight mutations include K303R, N304S, T371A, L384M, M421G, N519S, G521R, Y537S.

[0108] In some embodiments, the DRD derived from ER can include amino acids 2-595 of the parent ER sequence, referred to herein as the M1del mutation.

[0109] In one embodiment, the stimulus is a small molecule that binds to an SRE and regulates post-translational protein levels.

[0110] In some embodiments, the DRD derived from ER can include amino acids 2-875 of the parent ER sequence, referred to herein as the M1del mutation.

[0111] In some embodiments, the DD is derived from a region of the ER protein. As a non-limiting example, this region is amino acids 305 to 549 of ER (SEQ ID NO: 76).

[0112] A non-limiting list of ER drug responsive domains (amino acid and nucleic acid sequences) is listed in Table 6. The positions of the mutated amino acids listed in Table 6 are based on ER (Uniprot ID: P03372.2) of SEQ ID NO: 42. In Table 6, "del" means that the mutation is a deletion of the amino acid at that position compared to the wild type sequence.

[0113] Table 6: Drug-responsive domains (DDs) derived from the ER [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0114] In one embodiment, the SREs include ERDD-1, ERDD-2, ERDD-3, ERDD-6, ERDD-7, ERDD-8, ERDD-9, ERDD-10, ERDD-11, ERDD-12, ERDD-13, ERDD-14, ERDD -15, ERDD-16, ERDD-17, ERDD-18, ERDD-19, ERDD-20, ERDD-21, ERDD-22, ERDD-23, ERDD-24, ERDD-25, ERDD-26, ERDD-27, ERDD-2 8, ERDD-29, ERDD-30, ERDD-31, ERDD-32, ERDD-33, ERDD-34, ERDD-35, ERDD-36, ERDD-37, ERDD-38, ERDD-39, ERDD-40, ERDD-41, ERDD-42, ERDD-43, ERDD-44, ERDD-45, ERDD-46, ERDD-47, and ERDD-48.

[0115] In some embodiments, the ERDD is N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N413I, N413E, N413L, N413P, N413F, N413Y, N413G, The mutations may include one or more mutations selected from, but not limited to, Q502D, Q502H, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q502I, Q502G, Q502P, Q502M, Q502C, L384M, M421G, G521R, Y537S, K303R, N304S, S305N, R335G, T371A, T431I, N519S, E523G, A546T, and G442V.

[0116] The present disclosure provides compositions that include an effector module having an SRE derived in whole or in part from a parent protein, such as PDE5, and a first payload that includes in whole or in part human CD40L (SEQ ID NO: 3820) or a variant thereof. In one embodiment, the SRE includes amino acids 535-860 of PDE5. In some embodiments, the SRE can include one or more mutations compared to the parent protein. The SREs are selected from the group consisting of SEQ ID NOs: 294, 296, 298, 300, 302, 306, 308, 313, 315, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407 7, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 375, 378, 381, 384, 387, 389, 391, 394, 397, 400, 403, 406, 409, 412, 415, 417, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 4 58, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 6406, 6408, 6410, 6412, 6414, 6416, 6418, 6420, 6422, 6424, 6426, 6428,6430, 6432, 6434, 6436, 6438, 6440, 6442, 6444, 6446, 6448, 6450, 6452, 6454, 6456, 6458, 6460, 6462, 6464, 6466, 6468, 6470, 6472, 6474, 6476, 6478, 6480, 6482, 64 The number of ribs may include, but is not limited to, 84, 6486, 6488, 6490, 6492, 6494, 6496, 6498, 6500, 6502, 6504, 6506, 6508, 6510, 6512, 6514, 6516, 6518, 6520, 6522, 6524, 6526, 6528, or 6530.

[0117] In one embodiment, the SRE comprises the mutation R732L, and the SRE may comprise the amino acid sequence of SEQ ID NO:308.

[0118] The SRE described herein can respond to or interact with at least one stimulus.In one embodiment, the stimulus can be a small molecule, such as but not limited to vardenafil, tadalafil, or sildenafil.In one embodiment, the small molecule is vardenafil.

[0119] In some aspects, the effector module may include a linker. Such a linker can operably link the SRE to the first payload. In some embodiments, the linker can be a glycine and serine-containing linker or any one or more of the following publications in the art, for example: WO2018 / 161000; WO2018 / 231759; WO2019 / 241315; WO2018 / 160993; WO2018 / 237323; and WO2018 / 161038. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6532.

[0120] In one embodiment, the effector module may be SEQ ID NO:6534.

[0121] Also provided herein are polynucleotides encoding the compositions described herein, and vectors encoding the polynucleotides. The present disclosure also provides pharmaceutical compositions comprising the compositions described herein, a pharma- ceutically acceptable excipient, and immune cells that express the compositions described herein.

[0122] Also provided herein is a method of reducing tumor burden in a subject. The method may include administering a therapeutically effective amount of immune cells to the subject. The immune cells may include or express a composition comprising a stimulus response element (SRE) operably linked to a first payload. The first payload may include, in whole or in part, human CD40L (SEQ ID NO: 3820) or a variant thereof. The immune cells may also express a pharmaceutical composition comprising a composition described herein. The method also includes administering a therapeutically effective amount of a stimulus to the subject. In one embodiment, the stimulus is a ligand. The ligand can regulate the expression of the first payload to reduce tumor burden. In some embodiments, the ligand is vardenafil, tadalafil, sildenafil, shield-1, or trimethoprim.

[0123] The present disclosure also provides a method for activating dendritic cells in a subject. The method may include administering a therapeutically effective amount of immune cells to the subject. The immune cells may include or express a composition comprising a stimulus response element (SRE) operably linked to a first payload. In some embodiments, the immune cells are T cells. The first payload may include, in whole or in part, human CD40L (SEQ ID NO: 3820) or a variant thereof. The immune cells may also express a pharmaceutical composition comprising a composition described herein. The method also includes administering a therapeutically effective amount of a stimulus to the subject. In one embodiment, the stimulus is a ligand. The method may further include measuring a dendritic cell activation marker IL12 in the subject in response to the ligand to determine activation of the dendritic cells. In one embodiment, the dendritic cells may be myeloid dendritic cells, plasmacytoid dendritic cells, CD14+ dendritic cells, Langerhans cells, or microglia. In one aspect, the dendritic cells are myeloid dendritic cells.

[0124] The compositions described herein may further comprise a CAR. The CAR may comprise (a) an extracellular targeting portion, (b) a transmembrane domain, (c) an intracellular signaling domain, and (d) optionally one or more costimulatory domains. The extracellular targeting portion of the CAR may be an scFv. In one embodiment, the scFv may be a CD19 scFv. In some embodiments, a costimulatory domain may be present.

[0125] 10. Stimulation of Tunable Protein Expression Systems The regulatable protein expression system of the present disclosure is capable of responding to a stimulus.

[0126] In some embodiments, the stimulus is a ligand. The ligand may be nucleic acid-based, protein-based, lipid-based, organic, inorganic, or any combination of the above. In some embodiments, the ligand may be a synthetic molecule. In some embodiments, the ligand may be a small molecule therapeutic compound. In some embodiments, the ligand may be a small molecule drug previously approved by a regulatory agency such as the FDA.

[0127] As described in this disclosure, the tunable protein expression system can exhibit ligand-dependent activity. The ligand can bind to the DRD and stabilize the attached or operably linked protein of interest. Ligands known to bind to the candidate DRD can be tested for their effect on the activity of the tunable protein expression system.

[0128] In some embodiments, the ligand is cell-permeable, hi some embodiments, the ligand may be designed to be lipophilic to improve cell permeability.

[0129] In some embodiments, the ligand is a small molecule. Small molecule ligands may be clinically approved to be safe and have suitable pharmacokinetics and distribution.

[0130] In some embodiments, the ligand may be complexed with or bound to one or more other molecules, such as, but not limited to, another ligand, a protein, a peptide, a nucleic acid, a lipid, a lipid derivative, a sterol, a steroid, a metabolite, a metabolite derivative, or a small molecule. In some embodiments, the ligand stimulus is complexed with or bound to one or more different types and / or many other molecules. In some embodiments, the ligand stimulus is a multimer of the same type of ligand. In some embodiments, the ligand stimulus multimer includes 2, 3, 4, 5, 6, or more monomers.

[0131] 11.DHFR Ligand In some embodiments, the ligands of the disclosure bind to dihydrofolate reductase, hi some embodiments, the ligands bind to and inhibit dihydrofolate reductase function, and are referred to herein as dihydrofolate inhibitors.

[0132] In some embodiments, the ligands can be selective inhibitors of human DHFR. The ligands of the present disclosure can also be selective inhibitors of dihydrofolate reductase from bacteria and parasites, such as Pneumocystis, Toxoplasma, Trypanosoma, Mycobacterium, and Streptococcus. Ligands specific for other DHFRs can be modified to improve binding to human dihydrofolate reductase.

[0133] Examples of dihydrofolate inhibitors include, but are not limited to, trimethoprim (TMP), methotrexate (MTX), pralatrexate, piritrexate, pyrimethamine, tarotrexin, chlorguanide, pentamidine, trimetrexate, aminopterin, C1 898 trihydrochloride, pemetrexed disodium, raltitrexed, sulfaguanidine, folothin, iclaprim, and diaveridine.

[0134] In some embodiments, the ligand of the present disclosure may include dihydrofolic acid or any of its derivatives that can bind to human DHFR. In some embodiments, the ligand of the present disclosure may be a 2,4, diaminoheterocyclic compound. In some embodiments, the 4-oxo group in dihydrofolic acid may be modified to provide a DHFR inhibitor. In one example, the 4-oxo group may be replaced with a 4-amino group. Various diaminoheterocycles, including pteridines, quinazolines, pyridopyrimidines, pyrimidines, and triazines, may also be used as scaffolds to develop DHFR inhibitors and may be used according to the present disclosure.

[0135] In some embodiments, the ligand comprises a ligand derived from TMP that contains a portion of the ligand known to mediate binding to DHFR. The ligand can also be modified to reduce off-target binding to other folate metabolic enzymes and increase specific binding to DHFR.

[0136] 12.ER Ligand In some embodiments, the ligand of the present disclosure binds to ER. The ligand can be an agonist or an antagonist. In some embodiments, the ligand binds to and inhibits ER function, and is referred to herein as an ER inhibitor. In some embodiments, the ligand can be a selective inhibitor of human ER. The ligand of the present disclosure can also be a selective inhibitor of the ER of other species. Ligands specific for other ER can be modified to improve binding to human ER.

[0137] The ligand may be an ER agonist, such as, but not limited to, the endogenous estrogen 17b-estradiol (E2) and the synthetic nonsteroidal estrogen diethylstilbestrol (DES). In some embodiments, the ligand may be an ER antagonist, such as ICI-164,384, RU486, tamoxifen, 4-hydroxytamoxifen (4-OHT), fulvestrant, oremifene, lasofoxifene, clomiphene, femarel, and ormeloxifene, and raloxifene (RAL).

[0138] In some embodiments, the stimulus of the present disclosure can be an ER antagonist, such as, but not limited to, bazedoxifene and / or raloxifene. In some embodiments, the ligand comprises a bazedoxifene-derived ligand that contains a portion of the ligand known to mediate binding to ER. The ligand can also be modified to reduce off-target binding to other folate metabolism enzymes and increase specific binding to the DRD from the ER.

[0139] 13. Phosphodiesterase Ligands In some embodiments, the ligands of the disclosure bind to phosphodiesterases, hi some embodiments, the ligands bind to and inhibit phosphodiesterase function, and are referred to herein as phosphodiesterase inhibitors.

[0140] In some embodiments, the ligand is a small molecule that binds to phosphodiesterase 5. In one embodiment, the small molecule is a hPDE5 inhibitor. Examples of hPDE5 inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidonafil, dasantafil, beminafil, SLx-2101, LAS 34179, UK-343,664, UK-357903, UK-371800, and BMS-341400.

[0141] In some embodiments, the ligand comprises a sildenafil-derived ligand that contains a portion of the ligand known to mediate binding to hPDE5. The ligand may also be modified to reduce off-target binding to phosphodiesterase and increase specific binding to hPDE5.

[0142] In some embodiments, the stimulus can be a ligand that binds to more than one phosphodiesterase. In one embodiment, the stimulus is a pan-phosphodiesterase inhibitor that can bind to more than one hPDE, such as aminophylline, paraxanthine, pentoxifylline, theobromine, dipyridamole, theophylline, zaprinast, icariin, CDP-840, etazolate, and glaucine.

[0143] 14.FKBP Ligand In some embodiments, the ligand of the present disclosure binds to FKBP, including human FKBP, hi some embodiments, the ligand is SLF or shield-1.

[0144] 15. Stabilizing and destabilizing ratio In some embodiments, the present disclosure provides methods for regulating protein expression, function, or levels by measuring stabilization and destabilization ratios. As used herein, a stabilization ratio may be defined as the ratio of the expression, function, or level of a protein of interest in response to a stimulus to the expression, function, or level of the protein of interest in the absence of a stimulus specific for an SRE. In some aspects, the stabilization ratio is at least 1, e.g., at least 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 30-20, 30-3 ... 100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-95, 40-100, 50-60, 50-70, 50-80, 50-90, 50-95, 50-100, 60-70, 60-80, 60-90, 60-95, 60-100, 70-80, 70-90, 70-95, 70-100, 80-90, 80-95, 80-100, 90-95, 90-100, or 95-100. As used herein, a destabilization ratio may be defined as the ratio of expression, function, or levels of a protein of interest in the absence of an effector module-specific stimulus to the expression, function, or levels of a protein of interest that is constitutively expressed in the absence of an SRE-specific stimulus. As used herein, "constitutively" refers to the expression, function, or levels of a protein of interest that is not linked to an SRE and is therefore expressed in both the presence and absence of a stimulus.In some embodiments, the destabilization ratio is at least 0, e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least 0-0.1, 0-0.2, 0-0.3, 0-0.4, 0-0.5, 0-0.6, 0-0.7, 0-0.8, 0-0.9, 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0.9, 0.2-0.3, 0.2-0.4, 0 0.2-0.5, 0.2-0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.9, 0.7-0.8, 0.7-0.9, or 0.8-0.9.

[0145] In some embodiments, the SRE of the effector module can stabilize the payload of interest by one or more stabilization ratios, which may include a ratio of expression, function, or level of the payload of interest in the presence of a stimulus to the expression, function, or level of the payload of interest in the absence of the stimulus.

[0146] In some embodiments, the SRE can destabilize the immunotherapeutic agent by a destabilization ratio between 0 and 0.09, which can comprise a ratio of expression, function, or level of the payload of interest expressed in the absence of a stimulus specific to the SRE to the expression, function, or level of the payload of interest expressed in the absence of a stimulus specific to the SRE constitutively.

[0147] 16. Additional effector module functions The effector module of the present disclosure may further comprise additional components that may be operably linked to either the DRD or the payload, or both. In some embodiments, the additional components include a signal sequence that controls the distribution of the payload of interest, a cleavage and / or processing function that facilitates cleavage of the payload from the effector module construct, a targeting and / or penetration sequence that can control the cellular localization of the effector module, a tag, and / or one or more linker sequences that link different components of the effector module, regulatory elements, polyadenylation sequences, transmembrane domains, intra-tail domains, hinges, tags, cleavage sites, and leaders. Examples of such additional effector module components are described in WO2018 / 161000; WO2018 / 231759; WO2019 / 241315; WO2018 / 160993; WO2018 / 237323; and WO2018 / 161038. In one embodiment, the transmembrane domain region of the first payload may be replaced with a transmembrane domain, variant or fragment thereof from a second parent protein.

[0148] 17. Payload As used herein, a "payload" or "protein of interest" (used interchangeably herein) is any polypeptide, protein, or portion thereof that is linked, attached, or operably linked to a DRD of the present disclosure.

[0149] The payload may comprise any polypeptide or any protein or fragment thereof. The payload may comprise a wild-type sequence, a fragment of a wild-type sequence, and / or one or more mutations. The payload may be a native protein from an organism genome, or a variant, mutant, and derivative thereof. The native protein may be from, for example, a mammal, a bacteria, and a virus. The payload may be a protein or polypeptide encoded by a recombinant nucleic acid molecule, a fusion or chimeric polypeptide, or a polypeptide that functions as part of a protein complex.

[0150] In one example, the payload can be a polypeptide encoded by a nucleic acid sequence derived from the human genome.

[0151] In some embodiments, the payload can be a variant sequence of the parent polypeptide. In some aspects, the variant sequence can have the same or similar activity as the reference sequence. Alternatively, the variant can have an altered activity (e.g., increased or decreased) compared to the reference sequence. In general, a variant of a particular polypeptide of the present disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polypeptide as determined by sequence alignment programs known to those skilled in the art.

[0152] 18. Therapeutic drugs as payloads In some embodiments, the payload of the present disclosure can be a therapeutic agent. For example, the payload can be a cancer therapeutic agent, an autoimmune disease therapeutic agent, an immunotherapeutic agent, an anti-inflammatory agent, an anti-pathogen agent, or a gene therapy agent. In some aspects, the immunotherapeutic agent can be an antibody and its fragments and variants, a TCR receptor, a chimeric antigen receptor (CAR), a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a co-stimulatory molecule, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor, and a safety switch.

[0153] In some embodiments, the payload of the present disclosure can be an immunotherapeutic agent that induces an immune response in an organism. The immunotherapeutic agent can be, but is not limited to, an antibody and its fragments and variants, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, a chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, or any agent that induces an immune response, including any agent that alters the activity, function, or response of an immune cell. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell or a subject.

[0154] 19. Cytokines, chemokines, and other soluble factors as payloads In some embodiments, the payloads of the present disclosure can be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells, and other cell types that act as chemical messengers between cells and tissues in the body. These proteins mediate a wide range of physiological functions, from influencing cell proliferation, differentiation, migration, and survival to a host of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic functions to eliminate tumor cells.

[0155] In some embodiments, the payload of the present disclosure may be a cytokine, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGF-betas, and chemokines, as well as fragments, variants, analogs, and derivatives thereof. In some embodiments, the payload of the present invention may be a cytokine that stimulates an immune response. In other embodiments, the payload of the present invention may be an antagonist of a cytokine that negatively impacts anti-cancer immune responses.

[0156] For example, the transmembrane of the first payload can be replaced with a transmembrane domain, either a variant or a fragment thereof.

[0157] In some embodiments, the payload can be a fusion protein that includes any of the immunotherapeutic agents described and ubiquitin. Within the fusion protein, ubiquitin can be located at the N-terminus and the immunotherapeutic agent can be located at the C-terminus. In one aspect, the immunotherapeutic agent can itself be a fusion protein, and ubiquitin can be located between the fused proteins. The payload can include a single ubiquitin protein or a chain of ubiquitin proteins. The ubiquitin protein can be linked to the immunotherapeutic agent via a single amino acid.

[0158] 20. Immunotherapy In some embodiments, the payload of the present disclosure can be an immunotherapeutic agent that induces an immune response in an organism. The immunotherapeutic agent can be, but is not limited to, an antibody and its fragments and variants, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, a chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell or a subject.

[0159] 21. CD40L In various embodiments, the payload of the present disclosure comprises an immunotherapeutic. In various embodiments, the immunotherapeutic is CD40 Ligand (CD40L), also known as CD154 or TNFRF5, or a variant containing one or more amino acid substitutions, deletions, or additions to the wild-type sequence of human CD40L. CD40L belongs to the TNF superfamily and is expressed primarily on T cells. CD40L binds to CD40, which is expressed on many immune cells, and initiates a cascade of cellular responses depending on the cell type. CD40L can also bind to α5β1 integrin and αIIbβ3 integrin. CD40L is a type II membrane polypeptide with a cytoplasmic domain at the N-terminus, a transmembrane region, and then an extracellular domain at the C-terminus. In some embodiments, the CD40L of the present disclosure can be engineered to bind only one of its binding partners, e.g., CD40. In some aspects, the CD40L described herein can be capable of binding to all of its cognate binding partners.

[0160] Unless otherwise indicated, full length CD40L is referred to herein as "CD40L". The nucleotide and amino acid sequences of CD40L from mouse and human are well known in the art and can be found, for example, in U.S. Patent No. 5,962,406 (Armitage et al.). The meaning of CD40 ligand also includes variations in the sequence, including conservative amino acid changes, etc., that do not alter the ability of the ligand to elicit an immune response against mucins.

[0161] CD40L can bind to CD40 expressed on antigen-presenting cells (APCs), B cells, monocytes, macrophages, platelets, neutrophils, dendritic cells, endothelial cells, and αSMCs (smooth muscle cells). Binding of CD40L to CD40 expressed on dendritic cells can promote dendritic cell (DC) licensing. DCs can be converted to a functional state by antigen-specific T helper cells to activate cytotoxic CD8+ T cells, a process called DC licensing. Binding of CD40 on DCs results in DC stimulation as evidenced by surface expression of costimulatory molecules and MHC molecules, production of inflammatory cytokines (e.g., IL12 and TNF), and epitope spreading.

[0162] In some embodiments, CD40L controlled by the regulatable protein expression system described herein may be utilized in the treatment of solid immunogenic tumors. CD40L may improve the efficacy of solid tumor-targeted T cells in immunogenic tumors by activating adaptive innate immune responses in situ. The regulatable CD40L-based biological circuit system described herein may be desirable because endogenous CD40L expression in T cells is transient. Furthermore, the tumor microenvironment is rich in sheddases that can cleave endogenous CD40L expressed by T cells. Exogenously expressed constitutive CD40L expression can cause hepatotoxicity and excessive B cell proliferation, leading to lymphoma (Schmitz et al., 2003; Sch ... (2006) Hepatology 44(2):430-9, Vonderheide et al (2007) J Clin Oncol. 1;25(7):876-83, Sacco et al (2000) Cancer Gene Ther.;7(10):1299-306). Constitutive (unregulated) expression can lead to CRS, thromboembolic syndromes, autoimmune responses, hyperimmune stimulation and AICD due to tumor angiogenesis, thus creating the need for the disclosed biological circuits.

[0163] In some embodiments, the immunotherapeutic agent may be a multimer of CD40L molecules, such as, but not limited to, a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer. In one embodiment, CD40L may form a trimer. Multimerization of CD40L may enhance signaling through the CD40L / CD40 axis. Binding of trimeric CD40L to CD40 may initiate CD40 clustering and activation of TRAF, ultimately resulting in activation of NF-κB.

[0164] CD40L as described herein may be resistant to proteinases and sheddases, such as those found in the tumor microenvironment, such as ADAM10 or ADAM17. Increased activity of ADAM17 in the tumor microenvironment is associated with decreased signaling through the CD40 / CD40L axis (see Lowe and Corvaia (2016), Int J Cancer Clin Res, 3:058, the contents of which are incorporated herein by reference in their entirety).

[0165] In some aspects, CD40L can be co-expressed with a chimeric antigen receptor. CD40L expressed on CAR T cells can enhance the function of CAR T cells and bystander effector cells through activation of CD40+ immune cells, such as, but not limited to, dendritic cells, macrophages, myeloid cells, B cells, platelets, endothelial cells, epithelial cells, and fibroblasts in the tumor microenvironment, as well as the tumor cells themselves. In one embodiment, the payload can be a bicistronic construct comprising CD40L and CD19CAR with CD28 and CD3ζ costimulatory domains (see Curran et al. (2015) Mol Ther. 23: 4; 769-778, the contents of which are incorporated by reference in their entirety).

[0166] In some embodiments, the cells of the present disclosure may also be engineered to express a chimeric antigen receptor as described herein in conjunction with CD40L. CD40L may be constitutively expressed or may be used as a payload in the effector module of the present disclosure. CD40L is involved in antigen presentation of dendritic cells, production of IL12, and generation of CD8+ T cell immunity. Any of the methods described by Curren et al. for enhancing the anti-tumor effect of CARs using CD40L may be useful in the present disclosure (Curren et al. Mol Ther. 2015 Apr;23(4): 769-778, the contents of which are incorporated by reference in their entirety). In one embodiment, agonistic CD40 antibodies may be useful in the present disclosure. CD40 monoclonal antibodies have shown clinical activity without abrogating toxicity.

[0167] The combination of T regulatory cells, myeloid-derived suppressor cells (MDSCs), and extensive stromal networks within the tumor microenvironment (TME) can attenuate antitumor immune responses by preventing T cell infiltration and / or activation with current immunotherapies (Ma et al. A CD40 agonist and PD-1 antagonist antibody reprogram the microenvironment of non-immunogenic tumors to allow T cell-mediated anticancer activity. Cancer Immunol Res Jan. 2013, the contents of which are incorporated by reference in their entirety). 14, 2019;doi: 10.1158 / 2326-606.CIR-18-0061). Current CAR T therapies are ineffective due to immune suppression, tumor antigen escape, insufficient CAR T expansion, and healthy tissue toxicity.

[0168] The present disclosure addresses these issues regarding the use of effector modules with CD40L as immunotherapeutics, fused directly or indirectly to SREs containing the DRDs described herein. CD40L may not be the only immunotherapeutic in the effector module. The effector module may also include a CAR construct. The combination of CD40L and CAR as immunotherapeutics with SREs may cause any of the following, alone or in combination: (1) repolarization of CD40+ macrophages in the tumor microenvironment to a proinflammatory state, (2) activation of CD40+ dendritic cells promoting epitope spreading that can reduce tumor antigen escape (e.g., reducing loss of CAR target antigens), (3) reverse signaling and cytokine production that enhances antigen-dependent T cell expansion, and (4) controllable protein production from the SRE that reduces toxicity of the therapeutic to healthy tissues. As a non-limiting example, an effector module comprising an SRE and a CAR immunotherapeutic fused to CD40L can be used to overcome loss of CAR target antigen (e.g., antigen escape) by inducing dendritic cells to recruit tumor infiltrating lymphocytes (TILs) to expand the population of anti-tumor specific T cells. As a non-limiting example, an effector module comprising an SRE and a CAR immunotherapeutic fused to CD40L can be used to alleviate the constraints of the tumor microenvironment (TME) of solid tumors by repolarizing tumor associated macrophages (TAMs) from an inhibitory to an inflammatory phenotype. As a non-limiting example, an effector module comprising an SRE and a CAR immunotherapeutic fused to CD40L can be used to enhance CAR cell expansion by triggering antigen-dependent T cell expansion.

[0169] In some embodiments, the effector module comprises at least one immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is CD40L. In one embodiment, the effector module comprises two or more immunotherapeutic agents, which can be of the same type, such as two antibodies, or of different types, such as CD40L and a CAR construct.

[0170] Provided herein is a composition for inducing an immune response in a cell or subject. The composition may include an effector module. The effector module may include a stimulatory response element (SRE) operably linked to a first payload. The first payload may include, in whole or in part, human CD40L (SEQ ID NO: 3820) or a variant thereof. In one embodiment, the first payload is the entire CD40L (SEQ ID NO: 3820).

[0171] In one aspect, the first payload may be a region of CD40L (SEQ ID NO: 3820). In one aspect, the region of CD40L may include amino acids 113-269 of SEQ ID NO: 3820 (SEQ ID NO: 3822). In one embodiment, the region of CD40L may include amino acids 12-261 of SEQ ID NO: 3820 (SEQ ID NO: 3824). In one embodiment, the region of CD40L may include amino acids 14-261 of SEQ ID NO: 3820 with a deletion at amino acids 110-116 of SEQ ID NO: 3820 (SEQ ID NO: 3826).

[0172] The SRE of the effector module can be derived from all or part of at least one parent protein, the parent protein being selected from the group consisting of ER, ecDHFR, FKBP, PDE5, and hDHFR.

[0173] In one embodiment, the SRE may contain one or more mutations compared to the parent protein.

[0174] In one embodiment, the SRE can be derived from the ER and the SRE can include, but is not limited to, an amino acid sequence listed in Table 6.

[0175] In one embodiment, the SRE can be derived from ecDHFR and the SRE can include, but is not limited to, an amino acid sequence listed in Table 3.

[0176] In one embodiment, the SRE can be derived from an FKBP and the SRE can include, but is not limited to, an amino acid sequence listed in Table 4.

[0177] In one embodiment, the SRE can be derived from PDE5 and the SRE can include, but is not limited to, an amino acid sequence listed in Table 5.

[0178] In some aspects, the SRE may respond to or interact with at least one stimulus. In one aspect, the effector module may include a second payload. In one embodiment, the second payload may be an immunotherapeutic. In some embodiments, the immunotherapeutic may be a chimeric antigen receptor (CAR). The CAR described herein may include (a) an extracellular targeting moiety, (b) a transmembrane domain, (c) an intracellular signaling domain, and (d) optionally, one or more costimulatory domains.

[0179] The extracellular targeting moiety of the CAR can be an scFv. In some aspects, the extracellular targeting moiety can be an scFv. In one embodiment, the scFv can be a CD19 scFv. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the CAR comprises a costimulatory domain.

[0180] Also provided herein are polynucleotides encoding the compositions described herein, vectors expressing the polynucleotides described herein, and pharmaceutical compositions comprising the compositions described herein and a pharma- ceutical acceptable excipient.

[0181] The present disclosure also provides immune cells for adoptive cell transfer expressing various therapeutic methods, e.g., cancer treatments and pharmaceutical compositions disclosed herein. The immune cells can be, for example, T cells (e.g., CD8+ T cells, CD4+ T cells), natural killer (NK) cells, NKT cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), memory T cells, regulatory T (Treg) cells, cytokine-induced killer (CIK) cells, dendritic cells, human embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, or mixtures thereof. In some embodiments, the immune cells are dendritic cells. In some embodiments, the circulating immune cells are CD8+ T cells. In one aspect, the immune cells are CD4+ T cells.

[0182] Further provided herein is a method of inducing an immune response in a subject. Such a method may include administering to a subject an effective amount of an immune cell described herein. The immune cell is an immune cell that expresses an effector module that includes a stimulus response element (SRE) operably linked to a first payload. The first payload may include, in whole or in part, human CD40L. The SRE expressed by the immune cell may respond to or interact with at least one stimulus. The method may further include exposing the subject to the stimulus and modulating CD40L expression. Modulating CD40L expression induces an immune response. In some embodiments, the method may further include administering to the subject an effective amount of a CD40 positive cell. In some embodiments, the CD40 positive cell may be a dendritic cell, a macrophage, a bone marrow cell, a B cell, a platelet, an endothelial cell, an epithelial cell, and a fibroblast.

[0183] The compositions described herein can be used to induce an immune response. Such compositions can include (a) a first immune cell capable of expressing an effector module comprising CD40L (SEQ ID NO: 3820), or a mutant CD40L as its first payload, and a second immune cell expressing CD40L. The first immune cell and the second immune cell can be independently selected from T cells (e.g., CD8+ T cells, CD4+ T cells), natural killer (NK) cells, NKT cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), memory T cells, regulatory T (Treg) cells, cytokine-induced killer (CIK) cells, dendritic cells, human embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, dendritic cells, macrophages, myeloid cells, B cells, platelets, endothelial cells, epithelial cells, and fibroblasts.

[0184] The present disclosure also provides a method for activating dendritic cells in a subject. The method may include administering a therapeutically effective amount of immune cells to the subject. The immune cells may include or express a composition comprising a stimulus response element (SRE) operably linked to a first payload. In some embodiments, the immune cells are T cells. The first payload may include, in whole or in part, human CD40L (SEQ ID NO: 3820), or a CD40L variant described herein. The immune cells may also express a pharmaceutical composition comprising a composition described herein. The method also includes administering a therapeutically effective amount of a stimulus to the subject. In one embodiment, the stimulus is a ligand. The method may further include measuring a dendritic cell activation marker, IL12, of the subject in response to the ligand to determine activation of the dendritic cells. In one embodiment, the dendritic cells may be myeloid dendritic cells, plasmacytoid dendritic cells, CD14+ dendritic cells, Langerhans cells, or microglia. In one aspect, the dendritic cells are myeloid dendritic cells.

[0185] In one embodiment, the immunotherapeutic that is CD40L or a variant thereof may be derived from UniProt ID: P29965 (also referred to herein as "WT"). The payload of the present disclosure may be a region or portion of CD40L, with or without mutations in the amino acid or nucleotide sequence encoding such a variant. Non-limiting examples of regions of CD40L include, but are not limited to, amino acids 113-269 of UniProt ID: P2996, where the cytoplasmic domain, transmembrane domain, and part of the extracellular domain have been deleted from UniProt ID: P2996, leaving part of the extracellular domain and an intact receptor binding domain. In one embodiment, the payload may be amino acids 14-261 of UniProt ID: P2996, which excludes the cytoplasmic tail of CD40L, thereby potentially reducing internalization. In one aspect, the payload can be amino acids 14-261 of UniProt ID: P2996 with the deletion of amino acids S110-G116, which renders CD40L resistant to proteolytic cleavage.

[0186] In some embodiments, mutations may be engineered in the CD40L payload such that it does not bind or binds with reduced affinity to CD40L endogenously expressed by the cells described herein. CD40L is a type II transmembrane protein that forms trimers on the cell surface. In some embodiments, trimerization occurs through the interaction of amino acid residues 47-261 of SEQ ID NO: 3820. In some embodiments, residues within 47-261 of SEQ ID NO: 3820 may be mutated in the CD40L payload to prevent trimerization (hereinafter referred to as "trimerization mutant"). In some embodiments, residues within 116-261 of SEQ ID NO: 3820 may be mutated. In some aspects, the mutations may allow selective trimerization such that the CD40L trimerization mutant can bind to another CD40L trimerization mutant protein but not to a CD40L protein lacking the mutation. The trimerization mutation site may be a site in the CD40L protein that is involved in trimerization as determined by the crystal structure of the CD40L trimer. Positions within CD40L that may be mutated include, but are not limited to, amino acids at positions 125, 170, 172, 224, 226, and / or 227 of SEQ ID NO: 3820. In some embodiments, mutations to the CD40L payload to prevent its trimerization with endogenous CD40L may include, but are not limited to, Y170G, Y172G, H224G, G226F, G226H, G226W, and / or G227F.

[0187] Sheddases present in the tumor microenvironment, such as ADAM10 / 17, can cleave CD40L, thereby preventing CD40L from achieving activation of CD40. Analysis of the sequence of CD40L reveals the ADAM10 / 17 proteolytic cleavage site. In some embodiments, deletion of amino acids 1-13 of CD40L can be engineered to reduce internalization. Deletion of amino acids 110-116 of CD40L can also be engineered to remove the ADAM10 / 17 site. Deletion or mutation of the methionine residue at amino acid position 113 of CD40L can also be utilized to reduce cleavage by the ADAM10 / 17 enzyme. In one embodiment, a region or portion of the human CD40L protein can be replaced with a mouse CD40L protein sequence to generate a CD40L protein that is resistant to cleavage by ADAM10 / 17. Any of the CD40L sequences that aim to reduce its shedding as described in US Patent Publication No. US20180085451A1 and / or US Patent No. 7,495,090B2 can be used in the effector modules and biological circuits described herein (the contents of each are incorporated by reference in their entirety). CD40L may be membrane-tethered using any of the transmembrane domains. In one embodiment, CD40L may be membrane-tethered using CD8-derived domains, such as, but not limited to, the CD8 transmembrane domain, the CD8 hinge domain, and / or the CD8 cytoplasmic tail.

[0188] In some embodiments, the effector modules described herein may include one or more cleavage sites between the DD and CD40L. The inclusion of a cleavage site may allow proteolytic turnover of the DD to be uncoupled from the payload, thereby altering the expression level of the payload independent of the DD. In some embodiments, the addition of a cleavage site may increase the expression of the payload. In other aspects, the addition of a cleavage site may decrease the expression of the payload.

[0189] In some embodiments, the CD40L payload and the SRE described herein may be linked.

[0190] The CD40L construct components and the CD40L construct are shown in Tables 7 and 8, respectively. In Tables 7 and 8, CD40L "WT" refers to Uniprot ID: P29965, hPDE5 "WT" refers to Uniprot ID: O76074, and ER "WT" refers to Uniprot ID: Refers to ID:P03372.2.

[0191] Table 7: CD40L construct components [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10]

[0192] Table 8: CD40L constructs [Table 8-1] [Table 8-2]

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

[0193] B. Pharmaceutical Compositions and Formulations The present teachings further include pharmaceutical compositions comprising one or more tunable protein expression systems, nucleic acids, polynucleotides, modified cells, or payloads of the present disclosure, and optionally at least one pharma- ceutically acceptable excipient or inactive ingredient.

[0194] As used herein, the term "pharmaceutical composition" refers to a formulation containing one or more of the tunable protein expression systems, nucleic acids, polynucleotides, payloads or components described herein, or pharma- ceutically acceptable salts thereof, and, optionally, other chemical components, such as physiologically suitable carriers and excipients.

[0195] The term "excipient" or "inactive ingredient" refers to an inert or inactive substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0196] In some embodiments, the compositions are administered to a human, human patient or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to any one or more regulatable protein expression system components delivered as described herein.

[0197] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, agricultural animals such as cows, horses, chickens, and pigs, domestic animals such as cats and dogs, and laboratory animals such as mice, rats, rabbits, dogs, and non-human primates.

[0198] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient administered to a subject and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0199] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, or inactive ingredient, and / or any additional ingredients in a pharmaceutical composition of the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as depending on the route by which the composition is administered. By way of example, the composition may contain 0.1%-100%, e.g., 0.5-50%, 1-30%, 5-80%, at least 80% (w / w) active ingredient.

[0200] The efficacy of treatment or improvement of disease can be evaluated, for example, by measuring disease progression, disease remission, severity of symptoms, pain relief, quality of life, dosage of pharmaceutical required to maintain therapeutic effect, level of disease markers, or other measurable parameters appropriate for the particular disease being treated or prevented. One skilled in the art can monitor the efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In the context of administration of the compositions of the present disclosure, "efficacious" for example against cancer indicates that administration in a clinically relevant manner results in a beneficial effect in at least a statistically significant proportion of patients, such as improvement of symptoms, cure, reduction in disease burden, reduction in tumor mass or cell count, extension of life span, improvement in quality of life, or other effects generally recognized as positive by physicians familiar with the treatment of a particular type of cancer.

[0201] The therapeutic or prophylactic effect is evident by a statistically significant improvement in one or more parameters of the disease state, or by the absence of aggravation or onset of otherwise expected symptoms. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease may indicate an effective treatment. The efficacy of a given composition or formulation of the present disclosure may also be determined using an experimental animal model of a given disease known in the art. When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant change is observed.

[0202] 1. Preparation The compositions of the present disclosure, such as polypeptide, protein, polynucleotide, and vector compositions, can be formulated in any manner suitable for delivery. Formulations can be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, and combinations thereof.

[0203] In one embodiment, the polynucleotide and vector formulation is a nanoparticle that may include at least one lipid.The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipid.In another aspect, the lipid may be a cationic lipid, such as, but is not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0204] For polynucleotides of the disclosure, formulations can be selected from, for example, any of those taught in International Application PCT / US2012 / 09610.

[0205] 2.Inactive ingredients In some embodiments, a pharmaceutical or other formulation may include at least one excipient that is an inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more inactive agents included in a formulation. In some embodiments, all, none, or some of the inactive ingredients that may be used in the formulations of the present disclosure may be approved by the U.S. Food and Drug Administration (FDA). Inactive ingredients suitable for the formulations of the present disclosure may be found in applicant's PCT International Publication Nos. WO2018 / 161000; WO2018 / 231759; WO2019 / 241315; and WO2018 / 237323.

[0206] 3. Administration, Delivery, and Administration The compositions of the present disclosure can be delivered to cells or subjects via one or more routes and modalities. They can be delivered to cells and / or subjects using viral vectors that contain one or more regulatable protein expression systems, nucleic acids, polynucleotides, payloads, and other components described herein. Other modalities such as mRNA, plasmids, etc. can also be used as recombinant proteins.

[0207] 4. Naked Delivery The pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be delivered to cells, tissues, organs, and / or organisms in naked form. As used herein, the term "naked" refers to pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads that are delivered without agents or modifications that promote transfection or permeability. Naked pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads may be delivered to cells, tissues, organs, and / or organisms using routes of administration known in the art and described herein. In some embodiments, naked delivery may include formulation in a simple buffer, such as saline or PBS.

[0208] 5. Formulated Delivery In some embodiments, the pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be formulated using the methods described herein. The formulations may include pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads, which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell permeation agents, pharma- ceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and / or sustained release delivery depots. The formulations of the present disclosure may be delivered to cells using routes of administration known in the art and described herein.

[0209] The pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, or payloads may also be formulated for direct delivery to an organ or tissue in any of a number of ways in the art, including, but not limited to, direct immersion or bathing, such as via a catheter, by gels, powders, ointments, creams, gels, lotions, and / or drops, by using a substrate such as a fabric or biodegradable material coated or impregnated with the composition, or the like.

[0210] 6. Delivery to cells In another aspect of the disclosure, polynucleotides encoding the tunable protein expression system, the DRD, or the payload of interest, as well as the compositions of the disclosure and vectors comprising such polynucleotides, can be introduced into cells, such as immune effector cells.

[0211] In one aspect of the disclosure, the polynucleotides encoding the regulatable protein expression system, DRD, or payload of interest and the compositions of the disclosure can be packaged into a plasmid, viral vector, or integrated into the viral genome, allowing for transient or stable expression of the polynucleotide. Preferred viral vectors are retroviral vectors, including lentiviral vectors and gamma retroviral vectors. To construct a retroviral vector, a polynucleotide molecule encoding the regulatable protein expression system, DRD, or payload of interest is inserted into the viral genome in place of a specific viral sequence to generate a replication-deficient virus. The recombinant viral vector is then introduced into a packaging cell line that contains the gag, pol, and env genes, but does not contain the LTR and packaging components. The recombinant retroviral particles are secreted into the medium, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred, as they can infect both dividing and non-dividing cells.

[0212] Vectors can also be introduced into cells by non-viral methods, such as physical methods, such as needles, electroporation, sonoporation, hydroporation, chemical carriers, such as inorganic particles (e.g., calcium phosphate, silica, gold), and / or chemical methods. In some embodiments, synthetic or natural biodegradable agents can be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors. In some embodiments, vectors can be introduced into cells by transient membrane disruption, e.g., by rapid cell deformation.

[0213] In some embodiments, the polypeptides of the present disclosure can be delivered directly to cells. In one embodiment, the polypeptides of the present disclosure can be delivered using a synthetic peptide that includes an endosomal leakage domain (ELD) fused to a cell permeability domain (CLD). The polypeptides of the present disclosure are co-introduced into cells with an ELD-CLD-synthetic peptide. ELDs facilitate the escape of endosome-trapped proteins into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the art. CPDs allow transport of proteins across the plasma membrane and have also been described in the art. ELD-CLD fusion proteins synergistically increase transduction efficiency when compared to co-transduction with either domain alone. In some embodiments, a histidine-rich domain can be optionally added to the shuttle construct as an additional method to allow escape of cargo from endosomes into the cytosol. The shuttle can also include a cysteine ​​residue at the N- or C-terminus to generate multimers of the fusion peptide. Multimers of ELD-CLD fusion peptides, generated by the addition of cysteine ​​residues to the termini of the peptides, show even higher transduction efficiency when compared to single fusion peptide constructs. The polypeptides of the present disclosure can also be appended to appropriate localization signals to direct the cargo to the appropriate subcellular location, for example, the nucleus. In some embodiments, any of the ELD, CLD, or fusion ELD-CLD synthetic peptides taught in International Patent Publications WO2016161516 and WO2017175072 (the contents of each of which are incorporated herein by reference in their entirety) can be useful in the present disclosure.

[0214] 7. Delivery Modalities and / or Vectors The tunable protein expression system, DRD, or payload of interest of the present disclosure can be delivered using one or more modalities. The present disclosure also provides vectors that package the polynucleotides of the present disclosure that code for the tunable protein expression system, DRD, or payload constructs, and combinations thereof. The vectors of the present disclosure can also be used to deliver the packaged polynucleotides to cells, local tissue sites, or subjects. These vectors can be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors, and particles. Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses useful as vectors include, but are not limited to, adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses. In some embodiments, the virus is selected from a lentiviral vector, a gammaretroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, and a herpes viral vector.

[0215] Generally, a vector contains an origin of replication functional in at least one organism, a promoter sequence, and convenient restriction endonuclease sites, as well as one or more selectable markers, such as drug resistance genes.

[0216] In some embodiments, the recombinant expression vector may include regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific for the type of host cell into which the vector will be introduced.

[0217] In some embodiments, a vector of the present disclosure can include one or more payloads as taught herein, and two or more payloads can be included in one ligand response, where the two or more payloads are simultaneously regulated by the same ligand or response agent.

[0218] 8. Lentiviral Vehicles / Particles In some embodiments, lentiviral vehicles / particles can be used as a delivery modality. Lentiviruses are a subgroup of the Retroviridae family of viruses, named because reverse transcription of the viral RNA genome into DNA is required before integration into the host genome. Thus, the most important feature of lentiviral vehicles / particles is that they integrate their genetic material into the genome of the target / host cell. Some examples of lentiviruses include human immunodeficiency viruses: HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi, and caprine arthritis-encephalitis virus (CAEV).

[0219] Typically, lentiviral particles that constitute gene delivery vehicles are replication defective by themselves (also called "self-inactivating"). Lentiviruses can infect both dividing and non-dividing cells by an entry mechanism via the intact host envelope. Recombinant lentiviral vehicles / particles have been generated by multiple attenuation of HIV pathogenicity genes. For example, genes Env, Vif, Vpr, Vpu, Nef, and Tat have been deleted, making the vector biologically safe. Correspondingly, lentiviral vehicles derived from, for example, HIV-1 / HIV-2, can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells. As used herein, the term "recombinant" refers to vectors or other nucleic acids that contain both lentiviral and non-lentiviral retroviral sequences.

[0220] Lentiviral particles can be produced by co-expressing viral packaging elements and the vector genome itself in producer cells such as human HEK293T cells. These elements are usually provided in three or four separate plasmids. Producer cells are co-transfected with plasmids encoding lentiviral components including the viral core (i.e., structural proteins) and enzymatic components, and the envelope proteins (called the packaging system), as well as a plasmid encoding the genome containing the foreign transgene to be introduced into the target cell, i.e., the vehicle itself (also called the transfer vector). Generally, the plasmid or vector is contained in the producer cell line. The plasmid / vector is introduced into the producer cell line via transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. As a non-limiting example, the packaging and transfer constructs can be introduced into the producer cell line by calcium phosphate transfection, lipofection, or electroporation, generally together with a key selection marker such as neo, DHFR, Gln synthetase, or ADA, followed by the presence of the appropriate drug and clonal isolation.

[0221] The producer cells produce recombinant viral particles that contain the foreign gene, e.g., the tunable protein expression system of the present disclosure, the DRD, and the payload. The recombinant viral particles are recovered from the culture medium and titered by standard methods used by those skilled in the art. The recombinant lentiviral vehicle can be used to infect target cells.

[0222] Cells that can be used to produce high titer lentiviral particles include HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11: 452-459), the FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T-based producer cell lines (see, e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Ther. 2012, 23(3): 357-369, the contents of each of which are incorporated by reference in their entirety). Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al., Blood. 2009, 113(21): 5104-5110).

[0223] In some embodiments, the envelope protein can be a heterologous envelope protein from another virus, such as the G protein of vesicular stomatitis virus (VSV G) or the baculovirus gp64 envelope protein. The VSV-G glycoprotein is specifically expressed in the following species classified in the genus Vesicular stomatitis virus: Karahas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), Vesicular stomatitis New Jersey virus (VSNJV), and / or species provisionally classified in the genus Vesiculovirus, Grasscar prabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Grey Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Quatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Ergonomic Bat virus (MEBV), Perinet virus (PERV), Pike fly rhabdo virus (PFRV), Porton virus (PORV), Radi virus (RADIV), Carp spring viremia virus (SVCV), Tree shrew virus (TUPV), Ulcerative disease rhabdo virus (UDRV), and Yugbogdanova virus (YBV).The gp64 or other baculovirus env protein can be derived from Autographa californica nuclear polyhedrosis virus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nuclear polyhedrosis virus, Orgyia pseudotsugata monocapsid nuclear polyhedrosis virus, Epiphyas postvittana nuclear polyhedrosis virus, Hyphantria cunea nuclear polyhedrosis virus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nuclear polyhedrosis virus, or Batken virus.

[0224] Other elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or an active portion thereof.

[0225] Methods for producing recombinant lentiviral particles are discussed in the art, e.g., U.S. Patent Nos. 8,846,385; 7,745,179; 7,629,153; 7,575,924; 7,179,903; and 6,808,905.

[0226] The lentiviral vector used may be selected from, but is not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII. 9. Adeno-associated virus particles

[0227] Delivery of any of the polynucleotides of the tunable protein expression system, DRD, or payload constructs of the present disclosure can be accomplished using recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles can be designed to utilize any of the known serotype capsids or a combination of serotype capsids.

[0228] AAV vectors include not only single-stranded vectors, but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA that anneals together to form a double-stranded vector genome. By skipping the synthesis of the second strand, scAAV allows for rapid expression in cells.

[0229] rAAV vectors can be produced by standard methods in the art, such as triple transfection in suspension cell cultures of sf9 insect cells, or human cells such as HEK293 cells.

[0230] The tunable protein expression system, DRD, or payload construct can be encoded in one or more viral genomes that are packaged into the AAV capsids taught herein.

[0231] Such vectors or viral genomes may also contain, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements required for expression from the vector or viral genome. Such regulatory elements are well known in the art and include, for example, promoters, introns, spacers, stuffer sequences, etc.

[0232] The tunable protein expression system, DRD, or payload constructs of the present disclosure can be administered in one or more or separate AAV particles.

[0233] In some embodiments, the tunable protein expression system may be administered in one or more AAV particles. In some embodiments, more than one tunable protein expression system, DRD, or payload may be encoded in the viral genome.

[0234] 10. Retroviral vehicles / particles (γ-retroviral vectors) In some embodiments, retroviral vehicles / particles can be used to deliver the tunable protein expression system, DRD, or payload constructs of the present disclosure. Retroviral vectors (RVs) allow for the permanent integration of transgenes into target cells. In addition to complex HIV-1 / 2-based lentiviral vectors, simple gammaretrovirus-based retroviral vectors have been widely used to deliver therapeutic genes and have been clinically proven as one of the most efficient and powerful gene delivery systems capable of transducing a wide range of cell types. Exemplary species of gammaretroviruses include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0235] In some embodiments, gammaretroviral vectors derived from mammalian gammaretroviruses, such as murine leukemia viruses (MLV), are recombinant. The MLV family of gammaretroviruses includes ecotropic, amphotropic, xenotropic, and polytropic subfamilies. Ecotropic viruses can only infect mouse cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect mice, humans, and other species via the Pit-2 receptor. An example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor but have different species tropism. Xenotropic viruses, such as NZB-9-1, infect humans and other species but not mouse species, whereas polytropic viruses, such as focus forming virus (MCF), infect mice, humans, and other species.

[0236] Gammaretroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including a plasmid encoding the retroviral structural and enzymatic (gag-pol) polyproteins, a plasmid encoding the envelope (env) proteins, and a plasmid encoding a vector mRNA that includes a polynucleotide encoding a composition of the present disclosure that is packaged into newly formed viral particles.

[0237] In some embodiments, recombinant gammaretroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated into the outer lipid layer of the viral particle and can increase / alter cellular tropism.

[0238] In some embodiments, the recombinant gammaretroviral vector is a self-inactivating (SIN) gammaretroviral vector. The vector is replication incompetent. SIN vectors may carry a deletion in the 3' U3 region, which initially contains enhancer / promoter activity. In addition, the 5' U3 region can be replaced with a strong promoter from cytomegalovirus or RSV (required for packaging cell lines), or an internal promoter and / or enhancer element of choice. The choice of internal promoter can be made according to the specific requirements of gene expression required for the specific purpose of the present disclosure.

[0239] In some embodiments, a polynucleotide encoding a tunable protein expression system, a DRD, or a payload construct is inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant gammaretroviral vector may be modified by inserting or removing naturally occurring sequences (e.g., inserting an IRES, inserting a heterologous polynucleotide encoding a polypeptide of interest or an inhibitory nucleic acid, shuffling a more effective promoter from a different retrovirus or virus in place of the wild-type promoter, etc.). In some examples, the recombinant gammaretroviral vector may contain a modified packaging signal, and / or a primer binding site (PBS), and / or a 5' enhancer / promoter element in the U3 region of the 5' long terminal repeat (LTR), and / or a modified 3'-SIN element in the U3 region of the 3'-LTR. These modifications may improve titer and infectivity.

[0240] 11. Oncolytic viral vectors In some embodiments, the polynucleotides of the present disclosure can be packaged into oncolytic viruses. As used herein, the term "oncolytic viruses" refers to viruses that preferentially infect or kill cancer cells, such as vaccine viruses. Oncolytic viruses can be naturally occurring or genetically engineered viruses, such as oncolytic adenoviruses and oncolytic herpes viruses.

[0241] In some embodiments, the oncolytic vaccine virus may comprise a thymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector virus particle, sufficient to induce cell lysis in tumors.See, e.g., U.S. Patent No. 9,226,977.

[0242] 12. Messenger RNA (mRNA) In some embodiments, the tunable protein expression system, DRD, or payload of the present disclosure may be designed as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Such mRNA molecules may have any of the structural components or features taught in International Application No. PCT / US2013 / 030062.

[0243] In some embodiments, the effector module may be designed as a self-amplifying RNA. As used herein, "self-amplifying RNA" refers to an RNA molecule that can replicate in a host, thereby increasing the amount of RNA and the protein encoded by the RNA. Such a self-amplifying RNA may have any of the structural features or components taught in International Patent Application Publication No. WO2011005799.

[0244] 13. Administration The present disclosure provides methods that include administering any one or more components or compositions of the regulatable protein expression system to a subject in need thereof. These can be administered to the subject in any amount and using any route of administration effective to prevent or treat or image a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with cancer or an autoimmune disease). The exact amount required may vary depending on the race, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, mode of activity, and the like.

[0245] The composition according to the present disclosure is typically formulated in unit dosage form for ease of administration and uniformity of dosage.However, it will be understood that the total daily use amount of the composition of the present disclosure can be determined by the attending physician within the scope of sound medical judgment.The therapeutically effective, prophylactically effective or suitable imaging dose level for any particular patient depends on various factors including the disorder being treated, the severity of the disorder, the activity of the compound used, the specific composition used, the age, weight, general condition, sex and diet of the patient, the time of administration, route of administration and excretion rate of the specific compound used, duration of treatment, the drug used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0246] In some embodiments, the compositions of the present disclosure may be used in cancer immunotherapy at various doses to avoid T cell exhaustion, prevent cytokine release syndrome, and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present disclosure may be used to initially treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of the compositions of the present disclosure to ensure recognition of minimal tumor antigen load. In another example, the compositions of the present disclosure may be delivered in a pulsatile manner to reduce persistent T cell signaling and enhance persistence in vivo. In some aspects, toxicity may be minimized by initially using low doses of the compositions of the present disclosure before administering high doses. Dosages may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-γ, and TNF-α are elevated.

[0247] In some embodiments, neurotoxicity may be associated with CAR or TIL therapy. Such neurotoxicity may be associated with CD19-CAR. Toxicity may be due to excessive T cell infiltration into the brain. In some embodiments, neurotoxicity may be reduced by preventing T cell passage through the blood-brain barrier. This can be achieved by targeted gene deletion of endogenous alpha-4 integrin inhibitors such as tisaburi / natalizumab, which may also be useful in the present disclosure.

[0248] Also provided herein is a method of administering a ligand or DRD ligand according to the present disclosure to a subject in need thereof. The ligand can be administered to a subject or cell using any amount and any route of administration effective to modulate the tunable protein expression system, DRD, or payload of the present disclosure. The exact amount required may vary depending on the subject's race, age, and general condition, the severity of the disease, the particular composition, its mode of administration, mode of activity, and the like. The subject may be a human, a mammal, or an animal. The compositions according to the present disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure may be determined by the attending physician within the scope of sound medical judgment. In certain embodiments, the ligands of the present disclosure are administered at a dose of about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 5 mg / kg of the subject's body weight per day, one or more times per day, to achieve the desired effect. The compound may be administered at a dosage level sufficient to deliver about 0 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg, about 10 mg / kg to about 100 mg / kg, about 50 mg / kg to about 500 mg / kg, or about 100 mg / kg to about 1000 mg / kg. In some embodiments, dosage levels can be 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg or mg / kg of subject body weight per day, one or more times per day to obtain the desired effect.

[0249] The present disclosure provides methods for delivering any of the ligands described herein to a cell or tissue, comprising contacting the cell or tissue with the ligand, which may be accomplished in vitro, ex vivo, or in vivo. In certain embodiments, the ligands according to the present disclosure may be administered to a cell at a dosage level sufficient to deliver about 1 nM to about 10 nM, about 5 nM to about 50 nM, about 10 nM to about 100 nM, about 50 nM to about 500 nM, about 100 nM to about 1000 nM, about 1 μM to about 10 μM, about 5 μM to about 50 μM, about 10 μM to about 100 μM, about 25 μM to about 250 μM, about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to cells at a dose selected from, but not limited to, 0.00064 μM, 0.0032 μM, 0.016 μM, 0.08 μM, 0.4 μM, 1 μM, 2 μM, 10 μM, 50 μM, 75 μM, 100 μM, 150 μM, 175 μM, 200 μM, 250 μM.

[0250] The desired dosage of the ligand of the present disclosure can be delivered only once, three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are employed, a split-dosing regimen as described herein can be used. As used herein, a "split dose" is the division of a "single unit dose" or total daily dose into two or more doses, e.g., two or more administrations of a "single unit dose". As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose / at one time / single route / single contact point, i.e., a single administration event. The desired dosage of the ligand of the present disclosure can be administered as a "pulse dose" or a "continuous flow". As used herein, a "pulse dose" is a series of single unit doses of any therapeutic agent administered at a set frequency over a period of time. As used herein, a "continuous flow" is a dose of a therapeutic agent administered continuously over a period of time in a single route / single contact point, i.e., continuous administration events. The total daily dose, which is the amount administered or prescribed in a 24-hour period, may be administered by any of these methods, or as a combination of these methods, or by any other method suitable for pharmaceutical administration.

[0251] 14. Administration In some embodiments, the composition for cancer immunotherapy or treatment of autoimmune disease can be administered to cells ex vivo and then administered to a subject. Immune cells can be isolated and expanded ex vivo using various methods known in the art. For example, the method of isolating cytotoxic T cells is described in U.S. Patent No. 6,805,861 and U.S. Patent No. 6,531,451. The isolation of NK cells is described in U.S. Patent No. 7,435,596.

[0252] In some embodiments, depending on the nature of the cells, the cells can be introduced into a host organism, e.g., a mammal, in a variety of ways, including injection, transfusion, infusion, local instillation, or transplantation. In some aspects, the cells of the present disclosure can be introduced into the site of a tumor. The number of cells used will depend on several circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., the number of administrations, the ability of the cells to grow, etc. The cells can be present in a physiologically acceptable medium.

[0253] In some embodiments, the cells of the present disclosure may be administered in multiple doses to a subject having a disease or condition, generally resulting in amelioration of one or more symptoms of the cancer or clinical condition and / or treating or preventing the cancer or clinical condition or symptoms thereof.

[0254] 15. Delivery Route The pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the present disclosure can be administered by any route to achieve a therapeutically effective result, including enteral (into the gut), gastrointestinal, epidural (into the dura), oral (through the mouth), transdermal, epidural, intracerebral (into the brain), intraventricular (into the ventricles of the brain), epicutaneous (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), intranasal (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (injected or injected into the abdominal cavity), intravesical, intravitreal (through the eye), intracavernous injection (into a pathological cavity), intracavitary (into the penis), intravenous (into the bladder ... to the base), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (nose), sublingual, sublabial, edema, ophthalmic (on the conjunctiva), ear, auricular (in or through the ear), buccal (towards the cheek), conjunctival, dermal, dental (into one or more teeth), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-articular, intrabiliary, intra-bronchial, intracapsular, intrachondral (in cartilage), caudal (in the cauda equina), intracapsular (in the cisterna magna cerebellum medulla), intracorneal (in the cornea) , intradental, intracoronary (inside the coronary arteries), intracavernosal (in the expandable space of the corpus cavernosum of the penis), intradiscal (inside the intervertebral disc), intraductal (inside the glandular ducts), intraduodenal (inside the duodenum), intradural (in the dura or subdural), intraepidermal (up to the epidermis), intraesophageal (up to the esophagus), intragastric (inside the stomach), intragingival (inside the gums), intraileal (in the distal part of the small intestine), intralesional (introduced directly into a localized lesion or into a localized lesion), intraluminal (in the lumen of a duct), intralymphatic (inside the lymph), intramedullary (in the bone marrow cavity of a bone), intrameningeal (inside the meninges), intramyocardial (inside the myocardium), intraocular (inside the eye), intraovarian ( in the ovary), intrapericardial (in the pericardium), intrapleural (in the pleura), intraprostatic (in the prostate), intrapulmonary (in the lungs or their bronchi), intranasal (in the nose or periorbital sinuses), intraspinal (in the spinal column), intrasynovial (in the synovial cavity of a joint), intratendinous (in the tendons), intratesticular (in the testicles), intrathecal (in the cerebrospinal fluid at any level of the neuraxis), intrathoracic (in the chest), intratubular (in the tubules of an organ), intratumoral (in a tumor), intratympanic (in the middle membrane of the ear), intravascular (in one or more blood vessels), intraventricular (inside the brain), iontophoresis (by electric current that moves ions of soluble salts to the tissues of the body),irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly to the larynx), nasogastric (through the nose to the stomach), occlusive dressing technique (topical route of administration, then covered with a bandage that occludes the area), ophthalmic (outside the eye), oropharyngeal (directly to the mouth and pharynx), parenteral, transdermal, periarticular, peridural, perineural, periodontal, rectal, respiratory (into the airways by inhalation orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (into the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the laryngeal cavity), ureteral (up to the ureter), urethral (to the urethra), vaginal, sacral anesthesia, diagnostic, nerve block, biliary irrigation, cardiac perfusion, phototherapy, or spinal cord.

[0255] 16. Parenteral and Injectable Administration In some embodiments, the pharmaceutical compositions, tunable protein expression systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the present disclosure may be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as CREMOPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof. In other embodiments, a surfactant such as hydroxypropylcellulose is included.

[0256] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, as 1,3-butanediol solutions. Acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and physiological saline solution. Sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, can also be used in the preparation of injectables.

[0257] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0258] 17. Detectable Agents and Labels The regulatable protein expression systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the present disclosure may be associated with or linked to one or more radioactive or detectable agents.

[0259] These agents include various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent substances, luminescent substances (e.g., luminol), bioluminescent substances (e.g., luciferase, luciferin, and aequorin), chemiluminescent substances, radioactive substances (e.g., 18F, 67Ga, 81mKr, 82Rb, 111In, 123I, 133Xe, 201Tl, 125I, 35S, 14C, 3H, or 99mTc (e.g., pertechnetate (technetium salt)). tungsten phosphate (TcO4-), as well as contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxide (e.g., superparamagnetic iron oxide (SPIO), single crystal iron oxide nanoparticles (MION), ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast agents (iohexol), microbubbles, or perfluorocarbons).

[0260] In some embodiments, the detectable agent can be an undetectable precursor that becomes detectable upon activation (e.g., a fluorogenic tetrazine-fluorophore construct (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or an enzyme-activatable fluorogenic agent (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme-labeled composition can be used include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIAs), radioimmunoassays (RIAs), and Western blot analysis.

[0261] 18. Application and Use The regulatable protein expression systems, constructs, ligands, or compositions of the present disclosure may be utilized in a wide variety of applications, including, but not limited to, therapeutics, diagnostics and prognostics, bioengineering, bioprocessing, biomanufacturing, research reagents, metabolomics, gene expression, enzyme supplementation, and the like.

[0262] The disclosure provides methods that include administering a composition, eg, a pharmaceutical composition, that includes one or more components of a regulatable protein expression system to a subject in need thereof.

[0263] While there may be some uses not involving medical treatment, for example to generate cell lines and reagents for scientific research, one use involves administration of the disclosed compositions for in vivo gene therapy or adoptive cell therapy, e.g., to generate modified cells for the treatment of cancer, autoimmune diseases and other diseases. An exemplary method for medical treatment or prevention of a disease, condition, or disorder in a subject in need thereof may include: (a) providing a population of cells (including human, animal, either primary or cell culture, autologous, allogeneic, or syngeneic); (b) introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule comprises (i) a first nucleic acid sequence encoding a protein of interest for treating the disease, and a second nucleic acid sequence encoding a drug responsive domain (DRD), wherein the payload nucleic acid sequence comprises a first polynucleotide operably linked to the DRD nucleic acid sequence encoding the protein of interest for treating the disease; (c) delivering the cells to a subject; and (d) administering to the subject a ligand that stabilizes the DRD sufficiently to permit expression of the protein of interest in the cells, wherein expression of the protein of interest is controlled by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest to treat the disease.

[0264] In the above-described methods, the protein of interest can be used to ameliorate, cure, prevent, or alleviate one or more symptoms of a disease, condition, or disorder.

[0265] The compositions of the present disclosure may be administered to a subject in any amount and using any route of administration effective to prevent or treat or image a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with working memory impairment). The exact amount required may vary depending on the subject's race, age, and general condition, the severity of the disease, the particular composition, its mode of administration, mode of activity, and the like.

[0266] The composition according to the present disclosure is typically formulated in unit dosage form for ease of administration and uniformity of dosage.However, it will be understood that the total daily usage of the composition of the present disclosure can be determined by the attending physician within the scope of sound medical judgment.The specific therapeutically effective or suitable imaging dose level for any particular subject depends on various factors including the disorder being treated, the severity of the disorder, the activity of the compound used, the specific composition used, the age, weight, general condition, sex and diet of the patient, the time of administration, route of administration and excretion rate of the specific compound used, duration of treatment, the drug used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0267] Also provided herein is a method of administering one or more stabilizing ligands (as used herein, a ligand that stabilizes a DRD may be referred to as a stabilizing ligand or simply a ligand, with the understanding that the ligand is effective to stabilize a DRD used in a regulatable protein expression system according to the present disclosure) to a subject in need thereof. The ligand may be administered to a subject or cell using any amount and any route of administration effective to regulate the amount of a protein of interest of the present disclosure in a cell transformed with a regulatable protein expression system. The exact amount of stabilizing ligand required may vary depending on the subject's race, age, and general condition, the severity of the disease, the particular composition, its mode of administration, mode of activity, etc. The subject may be a human, a mammal, or an animal.

[0268] C. Therapeutic use 1. Cancer immunotherapy Cancer immunotherapy aims to induce or restore immune system responsiveness against cancer. Significant advances in immunotherapy research have led to the development of various strategies that can be broadly classified as active and passive immunotherapy. In general, these strategies can be utilized to directly kill cancer cells or to counteract the immunosuppressive tumor microenvironment. Active immunotherapy aims to induce an endogenous, long-lasting tumor antigen-specific immune response. The response can be further enhanced by nonspecific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy involves the administration of effector immune molecules, such as tumor antigen-specific cytotoxic T cells or antibodies, to the host. This approach is short-lived and requires multiple applications.

[0269] Despite significant advances, the efficacy of current immunotherapeutic strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy and emerge in part from the need to push therapeutic doses to the edge of potentially lethal toxicity to obtain clinically meaningful therapeutic effects. Furthermore, doses are expanded in vivo and are often unpredictable, as adoptively transferred immune cells continue to expand within the patient.

[0270] The main risk associated with immunotherapy is on-target but off-tumor side effects resulting from the activation of T cells in response to normal tissue expression of tumor-associated antigens (TAA). Skin rash, colitis, and hearing loss have been reported in response to immunotherapy in clinical trials utilizing T cells expressing T cell receptors against specific TAAs.

[0271] Immunotherapy may also produce on-target on-tumor toxicity that appears when tumor cells are killed in response to immunotherapy. Adverse effects include tumor lysis syndrome, cytokine release syndrome, and related macrophage activation syndrome. Importantly, these adverse effects may occur during tumor destruction, and thus may result in toxicity even if on-tumor immunotherapy is successful. Therefore, an approach to control immunotherapy through the regulation of immunotherapy drugs is highly desirable to reduce toxicity and maximize efficacy.

[0272] The present disclosure provides systems, compositions, immunotherapeutic agents, and methods for cancer immunotherapy. These compositions provide adjustable regulation of gene expression and function in immunotherapy. In one aspect, the systems, compositions, immunotherapeutic agents, and other components of the present disclosure can be controlled by separately added stabilizing ligands, which provides significant flexibility for controlling cancer immunotherapy. Furthermore, the systems, compositions, and methods of the present disclosure can also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies.

[0273] The tunable nature of the disclosed systems and compositions has the potential to improve the efficacy and duration of immunotherapy. The disclosed compositions can be used to reversibly silence the biological activity of adoptively transferred cells, maximizing the potential of cell therapy without irreparably killing or terminating treatment.

[0274] The present disclosure provides methods for the fine-tuning of immunotherapies after administration to patients, thereby improving their safety and efficacy and expanding the subject population that may benefit from immunotherapy.

[0275] In some embodiments, the immune cells of the present disclosure can be T cells, NK cells, antigen-presenting cells, such as dendritic cells, or tumor-infiltrating tumor cells, and the immune cells are engineered to express CD40L in addition to a second payload, such as an antigen-specific T cell receptor (TCR) as taught herein, or an antigen-specific chimeric antigen receptor (CAR) (known as a CAR T cell). Thus, at least one polynucleotide encoding both CD40L and a CAR system (or TCR), or a first polynucleotide encoding a CD40L payload operably linked to a DRD, and a second polynucleotide encoding a different payload, such as an antigen-specific T cell or an antigen-specific chimeric antigen receptor (CAR) as described herein, can be linked to the same or different DRD as the DRD linked to CD40L. In some embodiments, the tunable protein expression system of the present disclosure may include a first polynucleotide encoding CD40L linked to a first DRD and a second payload, e.g., an antigen-specific T cell receptor (TCR) or an antigen-specific chimeric antigen receptor (CAR), operably linked to the first DRD or optionally to a second, different DRD. The second payload may not be linked to a DRD and may be expressed in a transformed or transfected cell. In various embodiments, the first and second polynucleotides may be present in a single vector, or the two polynucleotides may each be present separately in two different vectors. In some embodiments, when CD40L and the second payload are encoded by the same polynucleotide, the second payload can be operably linked to a DRD or it can be expressed independently of CD40L, not linked to any DRD, and separated from CD40L and / or the DRD by an IRES or some other transcription termination signal, such that translation and expression of the second payload is independent of translation and expression of the CD40L payload operably linked to a DRD. One or more vectors can then be introduced into immune cells, such as T cells, NK cells, dendritic cells, or tumor-infiltrating tumor cells.

[0276] In related embodiments, T cells expressing a CAR or TCR bind to a specific antigen via the extracellular targeting portion of the CAR or TCR, which transmits a signal to the T cell via the intracellular signaling domain(s), resulting in activation of the T cell. Activated CAR T cells change their behavior, including releasing cytotoxic cytokines (e.g., tumor necrosis factor, lymphotoxin, etc.), improving cell proliferation rates, changing cell surface molecules, etc. Such changes cause the destruction of target cells expressing the antigen recognized by the CAR or TCR. In addition, the release of cytokines or changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0277] In related exemplary embodiments, the CAR introduced into the T cell can be a first generation CAR that contains only the intracellular signaling domain from the TCR CD3ζ, or a second generation CAR that contains the intracellular signaling domain from the TCR CD3ζ and a costimulatory signaling domain, or a third generation CAR that contains the intracellular signaling domain from the TCR CD3ζ and two or more costimulatory signaling domains, or a split CAR system, or an on / off switch CAR system. In one example, the expression of CD40L, CAR, or TCR is controlled by stabilization of the operably linked DRD, and in the absence of a stabilizing ligand, there is little or no accumulation of the payload, i.e., CAR or TCR. In these examples, two or more payloads can be linked to the same or different DRDs, or one of the two payloads can be one that is not controlled by the DRD.

[0278] Since the payload of interest is operably linked to the DRD, little or no protein of interest is produced in the absence of the stabilizing ligand. Administration of the stabilizing ligand to cells transformed with the tunable protein expression system stabilizes the payload linked to the DRD, allowing the protein of interest to accumulate in the cell. In some exemplary embodiments, the presence or absence of the DRD stabilizing ligand is used to regulate CAR or TCR expression in transduced T cells or NK cells. In various embodiments, the payload can be optionally linked to a signal sequence, a leader sequence, a cleavage site, or some other peptide or polypeptide sequence or sequences that allow the protein of interest to be separated from the DRD in the cell after its accumulation.

[0279] In some embodiments, the CAR T cells of the present disclosure may be further modified to express one, two, three, or more additional immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific for a different target molecule; a cytokine such as IL2, IL12, IL15, and IL18, or a cytokine receptor such as IL15Ra; a chimeric switch receptor that converts an inhibitory signal into a stimulatory signal; a homing receptor that guides the adoptively transferred cells to a target such as a tumor tissue; a drug that optimizes the metabolism of the immune cells; or a safety switch gene (e.g., a suicide gene) that kills the activated T cells if a severe event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed. These molecules may be included in the same effector module or in separate effector modules.

[0280] In one embodiment, the CAR T cells (including TCR T cells) of the present disclosure can be "armed" CAR T cells transformed with one or more of the tunable protein expression systems, including the CAR payload and either the same or different polynucleotide sequence encoding CD40L operably linked to the same or the same or different DRD. Inducible or constitutively secreted active cytokines further arm the CAR T cells to improve efficacy and persistence. In this context, such CAR T cells are also referred to as "armored CAR T cells." The "armor" molecules can be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecules enhance the CAR T cell's ability to respond to the hostile tumor microenvironment by various mechanisms. These may be stimulatory factors such as IL2, IL12, IL15, IL18, type I IFN, CD40L, and 4-1BBL, which have been shown to further enhance T cell potency and persistence.

[0281] In one embodiment, the tunable protein expression system, which adjusts the expression level and activity of any of the described payloads or proteins of interest (used interchangeably), and its components, can be used in immunotherapy. As non-limiting examples, immunotherapeutic agents can be antibodies and their fragments and variants, cancer-specific T-cell receptors (TCRs) and their variants, anti-tumor specific chimeric antigen receptors (CARs), chimeric switch receptors, inhibitors of co-inhibitory receptors or ligands, agonists of co-stimulatory receptors and ligands, cytokines, chemokines, cytokine receptors, chemokine receptors, soluble growth factors, metabolic factors, suicide genes, homing receptors, or any agent that induces an immune response in cells and subjects.

[0282] In some embodiments, a composition for inducing or suppressing an immune response may comprise one or more components of a tunable protein expression system or one or more polypeptides encoded by a tunable protein expression system. In some embodiments, the tunable protein expression system may comprise a first polynucleotide comprising a first nucleic acid sequence encoding a payload and a second nucleic acid sequence encoding a drug responsive domain (DRD).

[0283] In some embodiments, the tunable protein expression system and compositions of the present disclosure relate to tunable protein expression (protein of interest or payload) function, including, for example, immunotherapeutic anti-tumor immune responses. In some embodiments, immunotherapeutic agents may include cytokines, chemokines, antibodies, integrins, intracellular proteins, membrane proteins, extracellular proteins, such as CD40L, which may be used to upregulate or improve the function of one or more immune cell types or downregulate the activity of one or more immune cell types. In various embodiments, immunotherapeutic agents useful for treating a disease, condition, or disorder may include CD40L, alone or in combination with other cytokines, chemokines, antibodies, integrins, intracellular proteins, membrane proteins, extracellular proteins. In various embodiments, the tunable protein expression system provides a protein of interest or payload that includes CD40L to promote or upregulate the lifespan and activity of one or more immune cell types useful for treating a disease, condition, or disorder, or symptoms associated with any of these.

[0284] 2. Adoptive cell transplantation (adoptive immunotherapy) In some embodiments, the controlled expression of cells genetically modified to encode and express at least one payload, e.g., CD40L operably linked to a DRD, can be used for adoptive cell therapy (ACT). As used herein, adoptive cell transfer refers to the administration of immune cells (autologous, allogeneic, or from a genetically modified host) with direct anti-cancer activity. ACT has shown promise in clinical applications against malignancies and infectious diseases.

[0285] According to the present disclosure, one or more components of the tunable protein expression system may be used in the development and implementation of cell therapies, such as adoptive cell therapy. In some embodiments, one or more components of the tunable protein expression system may be used for cell therapy to perform CAR therapy, to manipulate or control TILs, allogeneic cell therapy, combination of T cell therapy with other lines of treatment (e.g., radiation, cytokines), to encode engineered or modified TCRs, or to enhance T cells other than TCRs (e.g., by introducing cytokine genes, genes for checkpoint inhibitors PD1, CTLA4).

[0286] Provided herein is a method for use in adoptive cell therapy, comprising pre-treating a subject in need thereof, modulating immune cells with one or more components of a tunable protein expression system and / or a composition of the disclosure, administering engineered immune cells expressing the composition of the disclosure to the subject, and successful engraftment of the engineered cells in the subject.

[0287] In some embodiments, the controllable protein expression system and compositions of the present disclosure may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein, "preconditioning" refers to any therapeutic regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to, total body irradiation and / or lymphodepleting chemotherapy. Clinical trials of adoptive therapy without preconditioning have failed to demonstrate clinical benefit, indicating its importance in ACT. Nevertheless, preconditioning is associated with significant toxicity, limiting the subject cohort suitable for ACT. In some cases, to reduce the need for preconditioning, immune cells for ACT express CD40L as a payload, either alone or with cytokines such as IL-2, IL-6, IL12, and IL15, using the tunable protein expression constructs described herein that allow for selective expression of the protein of interest, which can be regulated using the stabilized ligands of the present disclosure.

[0288] In some embodiments, immune cells for ACT can be dendritic cells, T cells such as CD8+ T cells and CD4+ T cells, natural killer (NK) cells, NK T cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, immune stimulatory cells for ACT can be generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, autologous or allogeneic immune cells are used for ACT.

[0289] In some embodiments, the cells used for ACT can be antigen-presenting cells, such as dendritic cells and T cells engineered to express CD40L alone or in combination with a CAR that includes an antigen-binding domain specific for an antigen on the tumor cells of interest. In other embodiments, the cells used for ACT can be NK cells engineered to express CD40L alone or in combination with a cytokine or CAR that can be used for adoptive immunotherapy. In one example, a mixture of dendritic cells, T cells and / or NK cells can be used for ACT. According to the present disclosure, the expression level of CD40L in antigen-presenting cells, T cells and / or NK cells is regulated and controlled by a payload, such as a small molecule that binds to the DRD(s) operably linked to CD40L. This allows for selective expression of CD40L in transformed antigen-presenting cells, T cells and NK cells, alone or in combination with other payloads, such as a CAR as a payload or cytokines, such as IL-2, IL-6, IL12 and IL15.

[0290] In some embodiments, NK cells engineered to express one or more components of a tunable protein expression system can be used for ACT. Activation of NK cells induces perforin / granzyme-dependent apoptosis in target cells. Activation of NK cells also induces cytokine secretion, such as IFNγ, TNF-α, and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity, and enhance adaptive immune responses via upregulation of antigen presentation by antigen-presenting cells, such as dendritic cells (DCs).

[0291] Other examples of genetic modifications may include the introduction of chimeric antigen receptors (CARs) and downregulation of inhibitory NK cell receptors such as NKG2A.

[0292] NK cells can also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, genetic modification of NK cells using CRISPR, ZFN, or TALEN to silence their inhibitory receptors can enhance the antitumor potential of NK cells.

[0293] Immune cells can be isolated and expanded ex vivo using various methods known in the art. For example, methods for isolating and expanding cytotoxic T cells are described in U.S. Patent Publication Nos. 6,805,861 and 6,531,451; U.S. Patent Publication No. US20160348072A1 and International Patent Publication No. WO2016168595A1, the contents of each of which are incorporated herein by reference in their entirety. Isolation and expansion of NK cells are described in U.S. Patent Publication No. US20150152387A1, U.S. Patent No. 7,435,596, and Oyer, JL (2016). Cytotherapy 18(5):653-63, the contents of each of which are incorporated herein by reference in their entirety. Specifically, human primary NK cells can be expanded in the presence of feeder cells, such as myeloid cell lines genetically modified to express membrane-bound IL15, IL21, IL12, and 4-1BBL.

[0294] In some cases, subpopulations of immune cells may be enriched in ACT. Methods for enriching immune cells are described in International Patent Publication No. WO2015039100A1. In another example, T cells positive for B and T lymphocyte attenuation marker BTLA can be used to enrich anti-cancer reactive T cells, as described in U.S. Patent No. 9,512,401, the contents of which are incorporated herein by reference in their entirety.

[0295] In some embodiments, immune cells for ACT can be depleted of selected subpopulations to enhance T cell expansion. For example, immune cells can be depleted of Foxp3+ T lymphocytes to minimize anti-tumor immune responses using the methods taught in U.S. Patent Publication US20160298081A1, the contents of which are incorporated herein by reference in their entirety.

[0296] In some embodiments, activation and expansion of T cells for ACT is achieved by antigen stimulation of a chimeric antigen receptor (CAR) transiently expressed on the cell surface. Such activation methods are disclosed in International Patent WO2017015427, the contents of which are incorporated herein by reference in their entirety.

[0297] In some embodiments, immune cells can be activated by antigen binding to antigen-presenting cells (APCs). In some embodiments, APCs can be dendritic cells, macrophages, or B cells that are antigen-specific or non-specific. APCs can be autologous or allogeneic within an organ. In some embodiments, APCs can be artificial antigen-presenting cells (aAPCs), such as cell-based aAPCs or acellular aAPCs. Cell-based aAPCs can be selected from either genetically modified allogeneic cells, such as human erythroleukemia cells, or xenogeneic cells, such as mouse fibroblasts and Drosophila cells. Alternatively, APCs can be acellular, and antigens or costimulatory domains are presented on synthetic surfaces, such as latex beads, polystyrene beads, lipid vesicles, or exosomes.

[0298] In some embodiments, the cells of the present disclosure, specifically T cells, can be expanded using an artificial cell platform. In one embodiment, mature T cells are expanded using Seet CS et al. Artificial thymic organoids (ATOs) can be generated using the method described in (2017) Nat Methods 14, 521-530, the contents of which are incorporated herein by reference in their entirety. ATOs are based on a stromal cell line expressing delta like canonical Notch ligand (DLL1). In this method, stromal cells are aggregated with hematopoietic stem and progenitor cells by centrifugation and placed on cell culture inserts at an air-liquid interface to generate organoid cultures. ATO-derived T cells display a naive phenotype, a diverse T cell receptor (TCR) repertoire, and TCR-dependent functions.

[0299] In some embodiments, adoptive cell therapy is performed by autologous transplantation, where cells are derived from a subject in need of treatment, and the cells, after isolation and processing, are administered to the same subject. In other examples, ACT may involve allogeneic transfer, where cells are isolated and / or prepared from a donor subject other than the recipient subject who will ultimately receive the cell therapy. The donor and recipient subjects may be genetically identical or similar, or express the same HLA class or subtype.

[0300] In some embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., dendritic cells, T cells, and NK cells) for ACT can be controlled by the same or different tunable protein expression systems. In one example, two payloads, e.g., a CAR construct, such as a CD40L payload and a CD19 CAR payload, are each controlled by one or more DRDs on the same or different tunable protein expression systems. In some related embodiments, the payloads are linked to the same or different DRDs. In some embodiments, CD40L is operably linked to a DRD, and a CAR construct, such as a CD19 CAR, is located upstream or downstream of CD40L and is not linked to any DRD, or a CAR construct, such as a CD19 CAR, is introduced into a cell and encoded by a nucleotide sequence separate from the nucleotide sequence encoding the first payload, and the second payload can be linked to the same or different DRD as the DRD linked to CD40L, or may not be linked to any DRD. The payload is transcribed, translated, and expressed once the DRD(s) are stabilized with a stabilizing ligand specific for the DRD(s). Expression of CD40L and optionally a second payload, e.g., IL12 and / or CD19 CAR, can be regulated using one or more stabilizing ligands. In other embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., T cells and NK cells) for ACT can be controlled by different tunable protein expression systems. In one example, CAR constructs such as CD40L and CD19 CARs can each be operably linked to a different DRD, and thereby can be regulated separately using different stimuli.

[0301] Following gene regulation using one or more components of the regulatable protein expression system and compositions of the present disclosure, the cells are administered to a subject in need thereof. Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.

[0302] In some embodiments, immune cells for ACT can be engineered to express one or more immunotherapeutic agents (proteins of interest) that promote immune cell activation, infiltration, expansion, survival, and antitumor function. The immunotherapeutic agent can be a second CAR or TCR specific for a different target molecule; a cytokine or cytokine receptor; a chimeric switch receptor that converts an inhibitory signal into a stimulatory signal; a homing receptor that guides the adoptively transferred cells to a target such as a tumor tissue; a drug that optimizes the metabolism of the immune cells; or a safety switch gene (e.g., a suicide gene) that kills the activated T cells if a severe event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed.

[0303] In some embodiments, the immune cells used for adoptive cell transfer can be genetically engineered to improve their persistence, cytotoxicity, tumor targeting ability, and ability to home to disease sites in vivo, with the goal of further improving their ability to kill tumors in cancer patients as a whole. One example is the introduction of one or more components of the regulatable protein expression system of the present disclosure encoding cytokines, such as gamma cytokines (e.g., IL2 and IL15), into immune cells to promote immune cell proliferation and survival. Transduction of immune cells with cytokine genes encoded by the regulatable protein expression system (e.g., gamma cytokines IL2 and IL15) allows the immune cells, e.g., NK cells, to proliferate without the addition of exogenous cytokines, such that the cytokine-expressing NK cells have enhanced tumor cytotoxicity.

[0304] In some embodiments, one or more components of the tunable protein expression system can be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the gradual and progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the effectiveness of antiviral and antitumor immunotherapy. Exhausted T cells have high rates of apoptosis and high surface expression of multiple inhibitory receptors, as well as reduced proliferation and cytokine production. T cell activation leading to exhaustion can occur either in the presence or absence of antigen.

[0305] In some embodiments, the tunable protein expression system and its components can be utilized to prevent T cell exhaustion in the context of chimeric antigen receptor-T cell therapy (CAR-T). In this context, in some cases, exhaustion can be caused by oligomerization of the scFv of the CAR on the cell surface, which leads to continuous activation of the intracellular domain of the CAR. As a non-limiting example, the CAR of the present disclosure can include an scFv that is unable to oligomerize. As another non-limiting example, CARs that are rapidly internalized and re-expressed after antigen exposure can also be selected to prevent chronic scFv oligomerization at the cell surface. In one embodiment, the framework region of the scFv can be modified to prevent constitutive CAR signaling (Long et al. 2014. Cancer Research. 74(19) S1; incorporated herein by reference in its entirety). One or more components of the tunable protein expression system of the present disclosure can also be used to control the surface expression of the CAR on the T cell surface to prevent chronic T cell activation. The CAR of the present disclosure can also be designed to minimize exhaustion. As a non-limiting example, a 41-BB signaling domain can be incorporated into the CAR design to ameliorate T cell exhaustion. In some embodiments, exhaustion can be prevented using any of the strategies disclosed by Long HA et al. (Long AH et al. (2015) Nature Medicine 21, 581-590; the contents of which are incorporated herein by reference in their entirety).

[0306] In some embodiments, the adjustable nature of the adjustable protein expression system of the present disclosure can be utilized to reverse the exhaustion of human T cells observed with persistent CAR signaling.Reversible silencing of the biological activity of adoptively transferred cells using the composition of the present disclosure can be used to reverse persistent signaling, which can then reactivate T cells.Reversal of exhaustion can be measured by downregulation of multiple inhibitory receptors associated with exhaustion.

[0307] In some embodiments, T cell metabolic pathways may be modified to reduce the susceptibility of T cells to exhaustion. Metabolic pathways may include, but are not limited to, glycolysis, urea cycle, citric acid cycle, beta-oxidation, fatty acid biosynthesis, pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolic pathways. As a non-limiting example, payloads that reduce the rate of glycolysis can be utilized to limit or prevent T cell exhaustion (Long et al., 2003). al. Journal for Immunotherapy of Cancer 2013, 1(Suppl 1): P21; the contents of which are incorporated by reference in their entirety. In one embodiment, the T cells of the present disclosure can be used in combination with 2-deoxyglucose and a glycolysis inhibitor, such as rapamycin.

[0308] In some embodiments, the payload or protein of interest of the present disclosure may be used in combination with an antibody or fragment that targets a T cell surface marker associated with T cell exhaustion. T cell surface markers associated with T cell exhaustion that may be used may include, but are not limited to, CTLA-1, PD-1, TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96. In some embodiments, one or more components of a tunable protein expression system may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the gradual and progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the efficacy of antiviral and antitumor immunotherapy. Exhausted T cells have a high rate of apoptosis and high surface expression of multiple inhibitory receptors, as well as a reduced ability to proliferate and produce cytokines. T cell activation resulting in exhaustion may occur either in the presence or absence of antigen.

[0309] In some embodiments, one or more components of the tunable protein expression system and those components may be utilized to prevent T cell exhaustion in the context of chimeric antigen receptor-T cell therapy (CAR-T). In this context, in some cases, exhaustion may be caused by oligomerization of the scFv of the CAR on the cell surface, which results in continuous activation of the intracellular domain of the CAR. As a non-limiting example, the CAR of the present disclosure may include an scFv that is unable to oligomerize. As another non-limiting example, a CAR that is rapidly internalized and re-expressed after antigen exposure may also be selected to prevent chronic scFv oligomerization at the cell surface. In one embodiment, the framework region of the scFv may be modified to prevent constitutive CAR signaling (Long et al. 2014. Cancer Research. 74(19) S1; incorporated herein by reference in its entirety). One or more components of the tunable protein expression system of the present disclosure may also be used to control the surface expression of the CAR on the T cell surface to prevent chronic T cell activation. The CAR of the present disclosure may also be designed to minimize exhaustion. As a non-limiting example, the 41-BB signaling domain can be incorporated into the CAR design to ameliorate T cell exhaustion.

[0310] In some embodiments, the compositions of the present disclosure can be utilized to modify TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein can be utilized to alter the ratio of CD4 positive cells to CD8 positive populations. In some embodiments, TILs can be ex vivo selected and engineered to express any of the cytokines described herein. The payloads of the present disclosure can be used to expand the CD4 and / or CD8 populations of TILs and enhance TIL-mediated immune responses.

[0311] Parameters for improving CAR-T therapy outcomes are described in Finney et al. JCI. 2019;129(5):2123-2132, the contents of which are incorporated herein by reference in their entirety. Levels of the biomarkers LAG3(high) / TNF-α(low) in peripheral blood CD8+ T cells at the time of apheresis can also predict subsequent dysfunctional responses in subjects with high antigen loads who do not achieve a durable complete response beyond a few weeks. T cell intrinsic characteristics that are a result of the starting T cell repertoire and an effect of the manufacturing process may also play a role in the outcome of CAR-T therapy, converging with CD19 antigen-induced activation after adoptive transfer. The starting T cell repertoire may be partially influenced by the timing of apheresis. In one embodiment, apheresis may be performed prior to chemotherapy. The cumulative burden of CD19-expressing leukemic and normal B cells, assessed in bone marrow prior to lymphodepleting chemotherapy, may be important for determining the outcome of CAR-T therapy. According to Finney et al., increasing antigen load improves the outcome of CAR-T therapy. To increase the CD19 antigen load in vivo, subjects can also be infused with expanded subject-derived T cells (also called T-APCs) that have been genetically modified to express CD19.

[0312] 3. Cancer vaccines In some embodiments, the tunable protein expression system constructs, payloads of interest (e.g., immunotherapeutics), vectors, cells, and compositions of the present disclosure may be used in combination with a cancer vaccine.

[0313] In some embodiments, cancer vaccines may include peptides and / or proteins derived from tumor-associated antigens (TAA). Such strategies may be utilized to induce immune responses in subjects, which may be cytotoxic T lymphocyte (CTL) responses in some cases. The peptides used in cancer vaccines may also be modified to match the mutation profile of the subject. For example, EGFR-derived peptides with mutations that match those found in subjects in need of treatment have been successfully used in patients with lung cancer.

[0314] In one embodiment, the cancer vaccine of the present disclosure may comprise superagonist modified peptide ligands (APLs) derived from TAAs. These are mutant peptide ligands that deviate from the native peptide sequence by one or more amino acids, and activate specific CTL clones more effectively than native epitopes. These modifications allow the peptides to better bind to restricted class I MHC molecules or to interact more favorably with the TCR of certain tumor-specific CTL subsets. APLs can be selected using methods known in the art.

[0315] In some embodiments, effector immune cells genetically modified to encode components of a tunable protein expression system and payloads of the present disclosure can be combined with biological adjuvants as described herein. Dual control of CARs with cytokines and ligands separates kinetic control of target-mediated activation from endogenous T cell expansion. Such dual control also minimizes the need for patient preconditioning regimens. As a non-limiting example, a DRD-controlled payload, e.g., CD40L, in combination with a CAR, e.g., a CD19 CAR, can be combined with a cytokine, e.g., IL12, to enhance the antitumor effect of the CAR. As another non-limiting example, dendritic cell-based vaccination in combination with recombinant human IL7 to improve outcomes for high-risk pediatric sarcoma patients can be used in the methods described herein.

[0316] In some embodiments, effector immune cells engineered to express one or more antigen-specific TCRs or CARs can be combined with compositions of the present disclosure that include immunotherapeutic agents that transform the immunosuppressant tumor microenvironment, such as CD40L.

[0317] In one embodiment, effector immune cells modified to express CARs specific to different target molecules on the same cell can be combined. In another aspect, different immune cells modified to express the same CAR construct, such as NK cells and T cells, can be used in combination for tumor treatment. For example, T cells modified to express CD40L in combination with CD19 CAR can be combined with NK cells modified to express the same CD19CAR to treat B cell malignancies.

[0318] In other embodiments, immune cells engineered to express a CAR can be combined with checkpoint blockade.

[0319] In some embodiments, one or more components of the tunable protein expression system, e.g., effector immune cells genetically engineered to express the payloads of the present disclosure, can be combined with the cancer vaccines and other immunotherapies and adjuvant therapies of the present disclosure.

[0320] In some embodiments, the methods of the present disclosure may include a combination of the compositions of the present disclosure with other agents, such as anti-cancer agents, that are effective in treating cancer, infectious diseases, and other immune deficiency disorders. As used herein, the term "anti-cancer" agent refers to any agent that negatively affects cancer in a subject, for example, by killing cancer cells, including apoptosis in cancer cells, reducing the rate of proliferation of cancer cells, reducing the occurrence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the growth of cancer, or increasing the life span of a subject with cancer.

[0321] In some embodiments, the anti-cancer agent or therapy can be a chemotherapeutic agent, or radiation therapy, immunotherapy, surgery, or any other therapeutic agent that improves the therapeutic effect of the treatment in combination with the present disclosure.

[0322] In one embodiment, one or more components of the tunable protein expression system, including the CD19 CAR, can be used in combination with an aminopyrimidine derivative, such as a Burkitt tyrosine receptor kinase (BTK) inhibitor.

[0323] In some embodiments, the compositions of the present disclosure may be used in combination with immunotherapies other than the inventive treatments described herein, such as antibodies specific for some target molecule on the surface of tumor cells.

[0324] Exemplary chemotherapeutics include acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperin, sulindac, curcumin, alkylating agents including: nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; carmustine (BCU), lomustine (CCNU), semustine (methyl-CC Nitrosoureas such as triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine), etc.; alkylsulfonates such as busulfan; triazines such as dacarbazine (DTIC); folic acid analogs such as methotrexate and trimetrexate, 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), pyrrolidine analogs such as 5-azathioprine, 2,2'-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), antimetabolites, including purine analogs such as erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); mitotic inhibitors such as paclitaxel, vinca alkaloids such as vinblastine (VLB), vincristine, and vinorelbine, natural products such as taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin;Enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta, and GM-CSF, anti-angiogenic factors such as angiogenesis factor and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenesis factors including soluble VGF / VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methyl ureas such as N-methylhydrazine (MIFf) and procarbazine, adrenal cortical suppressants such as hydrazine derivatives, mitotane (ο,ρ'-DDD) and aminoglutethimide; hormones and antagonists including adrenergic corticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; testosterone propionate and fluoxymethol. androgens, including steroids / equivalents; antiandrogens, such as flutamide, gonadotropin releasing hormone analogs, and leuprolide; nonsteroidal antiandrogens, such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors, and receptor tyrosine kinase inhibitors, such as imatinib mesylate (sold as Gleevec or Gleevac), and estrogen, currently sold as Tarbeca. Rulotinib (EGF receptor inhibitor); antivirals such as oseltamivir phosphate, amphotericin B, and palivizumab; Sdi1 mimetics; semustine; senescence-derived inhibitory factor 1; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide agonists; veraresol; veramine; verdine; verteporfin; vinorelbine; vinxartin; vitaxin; vorozole; zanoterone; zeniplatin; ziralscob;and zinostatin stimulators; PI3Kβ small molecule inhibitors, GSK2636771; pan-PI3K inhibitors (BKM120); BRAF inhibitors, vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or analogs or derivatives and variations of the foregoing.

[0325] Radiotherapy agents and factors include radiation and waves that induce DNA damage, such as gamma irradiation, X-rays, UV irradiation, microwaves, electronic emissions, radioisotopes, etc. Treatment can be accomplished by irradiating the localized tumor site with the above-mentioned forms of radiation. All of these factors most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0326] In some embodiments, the chemotherapeutic agent can be an immunostimulant such as lenalidomide (LEN). Recent studies have shown that lenalidomide can enhance the anti-tumor function of CAR-modified T cells. Some examples of anti-tumor antibodies include tocilizumab, siltuximab.

[0327] Other agents that may be used in combination with the compositions of the present disclosure may also include, but are not limited to, agents that affect upregulation of cell surface receptors and their ligands, such as Fas / Fas ligand, DR4 or DR5 / TRAIL, as well as agents that enhance the sensitivity of hyperproliferative cells to inhibitors of GAP junctions, cytostatic and differentiating agents, cell adhesion such as focal adhesion kinase (FAK) inhibitors and lovastatin, or apoptosis inducers such as the antibody C225.

[0328] The combination may include administering the composition of the present disclosure and the other agent simultaneously or separately. Alternatively, the present immunotherapy may precede or follow the other agent / therapy by intervals ranging from minutes, days, weeks to months. 4. Disease

[0329] Provided in the present disclosure is a method of reducing tumor volume or tumor burden in a subject in need thereof, the method comprising introducing to the subject a composition of the present disclosure.

[0330] The present disclosure also provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of effector immune cells genetically modified to include a regulatable protein expression system of the present disclosure.

[0331] 5. Cancer Various cancers may be treated with the pharmaceutical compositions, tunable protein expression system components, and constructs comprising their DRDs and payloads of the present disclosure. As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to disease states characterized by such malignant neoplastic proliferation. The cancer may be a tumor or hematological malignancy, including, but not limited to, all types of lymphoma / leukemia, carcinoma and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid, and uterus.

[0332] Types of carcinomas that can be treated with the compositions of the present disclosure include, but are not limited to, papilloma / carcinoma, choroidal carcinoma, intradermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyosarcoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and undifferentiated carcinoma of the nasal and paranasal sinuses.

[0333] Types of sarcomas that can be treated with the compositions of the present disclosure include, but are not limited to, soft tissue sarcomas such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0334] 6. Infectious disease In some embodiments, the tunable protein expression system of the present disclosure may be used for the treatment of infectious diseases. The tunable protein expression system of the present disclosure may be introduced into cells suitable for adoptive cell transfer, such as macrophages, dendritic cells, natural killer cells, and / or T cells. Infectious diseases treated by the tunable protein expression system of the present disclosure may include diseases caused by viruses, bacteria, fungi, and / or parasites. The IL15-IL15Ra payload of the present disclosure may be used to increase immune cell proliferation and / or immune cell persistence useful for the treatment of infectious diseases.

[0335] "Infectious disease" herein refers to any pathogen or disease caused by a pathogen that infects mammalian cells, preferably human cells, and causes a disease state. Examples include bacteria, yeast, fungi, protozoa, mycoplasma, viruses, prions, and parasites.Examples include (a) a virus that is an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus, such as Variola or Vaccinia, or Mollusca contagiosum), a picornavirus (e.g., a rhinovirus or an enterovirus), an orthomyxovirus (e.g., influenza virus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., a papillomavirus, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., hepatitis C virus or dengue virus), or a retrovirus (e.g., (b) viral diseases, such as Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, and the like; (c) bacterial diseases, such as diseases resulting from infection with bacteria of the genus Bacillus, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella; (d) fungal diseases, including but not limited to chlamydia, candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomal infections, and other infectious diseases, including but not limited to prions that cause human diseases such as Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru.

[0336] 7. Immuno-oncology and cell therapy Recent advances in the field of cancer immunology have enabled the development of several approaches to enable the immune system to keep cancer at bay. Such immunotherapeutic approaches include targeting cancer antigens with monoclonal antibodies or adoptive transfer of ex vivo engineered T cells (e.g., containing chimeric antigen receptors or engineered T cell receptors).

[0337] In some embodiments, the pharmaceutical composition, adjustable protein expression system of the present disclosure can be used in regulating or modifying or utilizing the immune system to target one or more cancers.This approach can also be considered together with other such biological approaches, such as immune response modifier therapy, such as administration of interferon, interleukin, colony stimulating factor, other monoclonal antibodies, vaccines, gene therapy, and non-specific immunomodulators are also envisioned as anti-cancer treatments in combination with the pharmaceutical composition, adjustable protein expression system of the present disclosure, including their payloads.

[0338] Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce a patient's own immune system to fight cancer. In some embodiments, the pharmaceutical compositions, pharmaceutical compositions, and tunable protein expression systems comprising those payloads of the present disclosure are designed as immuno-oncology therapeutics.

[0339] 8. Cell therapy Several types of cellular immunotherapy exist, including tumor-infiltrating lymphocyte (TIL) therapy, genetically engineered T cells bearing chimeric antigen receptors (CARs), and recombinant TCR technology.

[0340] According to the present disclosure, the tunable protein expression system may be used in the development and implementation of cell therapies such as adoptive cell therapy. The tunable protein expression system and their payloads can be used in cell therapy to perform TCR ablation-TCR gene disruption, in TCR engineering, to control epitope-tagged receptors, in APC platforms for stimulating T cells, as a tool to enhance ex vivo APC stimulation, to improve methods of T cell expansion, in ex vivo stimulation with antigen, in TCR / CAR combinations, in manipulating or controlling TILs, in allogeneic cell therapy, in combination of T cell therapy with other lines of therapy (e.g., radiation, cytokines), to encode engineered or modified TCRs, or to enhance T cells other than TCRs (e.g., by introducing cytokine genes, genes for checkpoint inhibitors PD1, CTLA4).

[0341] In some embodiments, improved response rates are obtained in support of cell therapy.

[0342] Expansion and persistence of cell populations can be achieved through control or fine-tuning of payloads, such as receptors or pathway components in T cells, NK cells, or other immune-related cells. In some embodiments, the tunable protein expression system of the present disclosure is designed to spatially and / or temporally control expression of proteins that enhance T cell or NK cell responses. In some embodiments, the tunable protein expression system is designed to spatially and / or temporally control expression of proteins that inhibit T cell or NK cell responses.

[0343] The immune system can also be used to treat diseases other than cancer. Regulatable protein expression systems, their components, can be used in immunotherapy for the treatment of diseases, including but not limited to autoimmune diseases, allergies, graft-versus-host disease, and diseases and disorders that may result in immune deficiency, such as acquired immune deficiency syndrome (AIDS).

[0344] The present disclosure provides compositions for immunotherapy. Such compositions may include an effector module comprising CD40L as a payload. The composition may further include a chimeric antigen receptor as an additional payload. The composition may be expressed in immune cells suitable for adoptive cell therapy. The cells expressing the effector module may be delivered directly to a subject. In some embodiments, the cells expressing the effector module may be co-administered with immune cells expressing CD40. Expression of CD40 in these cells may be ectopic or endogenous. Expression of CD40 may also be induced by co-culture with other immune cells expressing the effector module of the present disclosure. Some non-limiting examples of CD40 positive cells include cells such as dendritic cells, macrophages, bone marrow cells, B cells, platelets, endothelial cells, epithelial cells, and fibroblasts.

[0345] In some embodiments, the payloads described herein can be used to activate dendritic cells. In some embodiments, the dendritic cells can be myeloid dendritic cells, plasmacytoid dendritic cells, CD14+ dendritic cells, Langerhans cells, or microglia. In some embodiments, myeloid DCs (mDCs) express the typical myeloid antigens CD11c, CD13, CD33, and CD11b, which correspond to mouse CD11c+ "classical" or "conventional" DCs. In humans, both monocytes and mDCs express CD11c, but DCs lack CD14 or CD16 and can be divided into CD1c+ and CD141+ fractions. These two fractions share homology with mouse classical DCs, which express either CD11b (CD1c+DCs) or CD8 / CD103 (CD141+DCs). In some embodiments, the dendritic cells can be plasmacytoid dendritic cells (pDCs). Plasmacytoid dendritic cells (pDCs) typically lack myeloid antigens and can be distinguished by the expression of CD123, CD303, and CD304. In one embodiment, dendritic cells can be CD14+. Such cells are found in tissues and lymph nodes and are a third subset of CD11c+ myeloid cells originally called "interstitial DCs". They are more monocyte- or macrophage-like than CD1c+ and CD141+ mDCs and can arise from classical monocytes. Comparable cells were recently discovered in mice as a new monocyte-derived subset of CD11b classical DCs that express ESAM. In one embodiment, dendritic cells can be Langerhans cells or microglia. Langerhans cells (LCs) and microglia are two specialized self-renewing DC populations found in stratified squamous epithelium and brain parenchyma, respectively. LCs can be capable of differentiating into migratory DCs, while microglia are considered a type of macrophage.

[0346] In some embodiments, the payload of the present disclosure can be a chimeric antigen receptor (CAR) that, when transduced into immune cells (e.g., T cells and NK cells), can redirect the immune cells to targets (e.g., tumor cells) that express a molecule recognized by the extracellular targeting portion of the CAR.

[0347] In some embodiments, pharmaceutical compositions comprising the tunable protein expression systems containing their payload or protein of interest may be used in modulating or modifying or harnessing the immune system to target one or more autoreactive immune components, such as autoantibodies and autoreactive immune cells, to alleviate autoimmune disease.

[0348] In some embodiments, the tunable protein expression system can be utilized in immunotherapy-based treatments to attenuate or alleviate graft-versus-host disease (GVHD). GVHD refers to a condition in which immune cells of an allogeneic donor react against host tissues after stem cell or bone marrow transplantation. In some embodiments, the tunable protein expression system can be designed to code for cytokines or immunological agents designed to regulate Tregs for the treatment of GVHD.

[0349] In some embodiments, the tunable protein expression system may be significantly less immunogenic than other biological circuits or switches in the art due to the expression of a native human protein of interest.

[0350] A variety of autoimmune and autoimmune-related diseases can be treated with pharmaceutical compositions comprising the regulatable protein expression system of the present disclosure. As used herein, the term "autoimmune disease" refers to diseases in which the body produces antibodies that attack its own tissues.

[0351] A variety of hematological disorders can be treated with pharmaceutical compositions containing one or more components of the regulatable protein expression system of the present disclosure.

[0352] 9. Central Nervous System (CNS) In some embodiments, pharmaceutical compositions comprising one or more components of the regulatable protein expression system of the present disclosure may be used in the regulation or modification or utilization of proteins in the central nervous system, including cerebrospinal fluid (CSF) proteins.

[0353] 10. Stem Cell Applications The tunable protein expression systems of the present disclosure and / or components thereof may be utilized in regulated reprogramming of cells, stem cell engraftment, or other applications in which controlled or tunable expression of such reprogramming factors is useful.

[0354] The tunable protein expression system constructs of the present disclosure can be used for reprogramming cells, including stem cells or induced stem cells. The derivation of induced pluripotent stem cells (iPSCs) was first achieved by Takahashi and Yamanaka (Cell, 2006. 126(4):663-76; incorporated herein by reference in its entirety) by using viral vectors to express KLF4, c-MYC, OCT4, and SOX2, collectively known as KMOS.

[0355] Excisable lentiviral and transposon vectors, repeated application of transient plasmids, episomal and adenoviral vectors have also been used to attempt to derive iPSCs.

[0356] DNA-free methods for generating human iPSCs have also been derived using sequential protein transduction with recombinant proteins incorporating cell-penetrating peptide moieties, and infectious transgene delivery using Sendai virus.

[0357] The tunable protein expression system of the present disclosure may include a payload that includes any of the genes, including but not limited to OCT, such as OCT4, SOX, such as SOX1, SOX2, SOX3, SOX15, and SOX18, KLF, such as NANOG, KLF1, KLF2, KLF4, and KLF5, MYC, such as c-MYC and n-MYC, REM2, TERT, and LIN28, and variants thereof that support cellular reprogramming. Sequences of such reprogramming factors are taught, for example, in International Application PCT / US2013 / 074560, the contents of which are incorporated herein by reference in their entirety.

[0358] The tunable protein expression system of the present disclosure may include a payload that includes any of the factors that contribute to stem cell mobilization. In autologous stem cell therapy, sources of stem cells for transplantation may include bone marrow, peripheral blood mononuclear cells, and umbilical cord blood. Stem cells are stimulated from these sources (e.g., bone marrow) to enter the bloodstream. Thus, sufficient stem cells are available to be collected for future reinfusion. Stem cells can be mobilized using one or a combination of cytokine strategies, including but not limited to G-CSF (filgrastim), GM-CSF, and chemotherapy preceding cytokines (chemomobilization).

[0359] 11. Tools and drugs for creating medicines Provided in the present disclosure are tools and agents that can be used in generating therapeutics, such as, but not limited to, immunotherapies, for reducing tumor volume or tumor burden in a subject in need. There are a significant number of variables involved in the production of therapeutics, including payload structure, cell type, method of gene transfer, method and time of ex vivo expansion, preconditioning, amount and type of tumor burden in the subject, etc. Such parameters can be optimized using the tools and agents described herein.

[0360] 12. Cell lines The present disclosure provides mammalian cells genetically modified with the compositions of the present disclosure. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include, but are not limited to, human embryonic kidney cell line 293, fibroblast cell line NIH3T3, human colorectal cancer cell line HCT116, ovarian cancer cell line SKOV-3, immortalized T cell lines (e.g., Jurkat cells and SupT1 cells), lymphoma cell lines Raji cells, NALM-6 cells, K562 cells, HeLa cells, PC12 cells, HL-60 cells, NK cell lines (e.g., NKL, NK92, NK962, and YTS), and the like. In some cases, the cells are not immortalized cell lines, but are instead cells obtained from an individual, referred to herein as primary cells. For example, the cells are T lymphocytes obtained from an individual. Other examples include, but are not limited to, cytotoxic cells, stem cells, peripheral blood mononuclear cells, or progenitor cells obtained from an individual.

[0361] 13. Cell Assay In some embodiments, the effectiveness of the compositions of the present disclosure as immunotherapeutic agents can be evaluated using cell assays. The expression and / or identity levels of the compositions of the present disclosure can be determined according to any method known in the art for identifying proteins and / or quantifying protein levels. In some embodiments, such methods can include Western blotting, flow cytometry, and immunoassays.

[0362] Provided herein are methods for functionally characterizing cells transformed or transduced with the regulatable protein expression system constructs and compositions of the present disclosure. In some embodiments, functional characterization can be performed on primary immune cells or immortalized immune cell lines and determined by expression of cell surface markers. Examples of cell surface markers for T cells include, but are not limited to, CD3, CD4, CD8, CD14, CD20, CD11b, CD16, CD45 and HLA-DR, CD69, CD28, CD44, IFN gamma. Markers of T cell exhaustion include PD1, TIM3, BTLA, CD160, 2B4, CD39, and LAG3. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC class II, CD40, CD45, B7-1, B7-2, IFN gamma receptor and IL2 receptor, ICAM-1 and / or Fc gamma receptor. Exemplary cell surface markers for dendritic cells include, but are not limited to, MHC class I, MHC class II, B7-2, CD18, CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR and / or Dectin-1, and in some cases, CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56, and / or CD57 may be absent. Examples of cell surface markers for NK cells include, but are not limited to, CCL3, CCL4, CCL5, CCR4, CXCR4, CXCR3, NKG2D, CD71, CD69, CCR5, phospho-JAK / STAT, phospho-ERK, phospho-p38 / MAPK, phospho-AKT, phospho-STAT3, granulysin, granzyme B, granzyme K, IL10, IL22, IFNg, LAP, perforin, and TNFa.

[0363] In some embodiments, T cell metabolic pathways can be modified to reduce the susceptibility of T cells to exhaustion. Metabolic pathways can include, but are not limited to, glycolysis, urea cycle, citric acid cycle, beta-oxidation, fatty acid biosynthesis, pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolic pathways. As a non-limiting example, payloads that reduce the rate of glycolysis can be utilized to limit or prevent T cell exhaustion. In one embodiment, the T cells of the present disclosure can be used in combination with glycolysis inhibitors such as 2-deoxyglucose and rapamycin.

[0364] In some embodiments, the tunable protein expression system constructs of the present disclosure useful for immunotherapy can be placed under the transcriptional control of the T cell receptor alpha locus (TRAC) locus in T cells. Eyquem et al. show that expression of a CAR from the TRAC locus prevents T cell exhaustion and accelerated T cell differentiation caused by excessive T cell activation.

[0365] In some embodiments, the payload of the present disclosure may comprise an antibody or fragment that targets a T cell surface marker associated with T cell exhaustion. T cell surface markers associated with T cell exhaustion that can be used as a payload may include, but are not limited to, CTLA-1, PD-1, TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96.

[0366] In one embodiment, the payload of the present disclosure may be a CD276 CAR (having CD28, 4-IBB, and CD3ζ intracellular domains), which does not show upregulation of markers associated with early T cell exhaustion.

[0367] 14.Cells According to the present disclosure, a cell is provided that is genetically modified to express at least one protein or payload of interest under the control of an encoded DRD ligand of the present disclosure. The cell of the present disclosure may non-specifically include immune cells, stem cells, and tumor cells. In some embodiments, the immune cell is an effector immune cell, including but not limited to T cells, such as CD8+T cells and CD4+T cells (e.g., Th1, Th2, Th17, Foxp3+ cells), memory T cells, such as T memory stem cells, central T memory cells, and effector memory T cells, terminally differentiated effector T cells, natural killer (NK) cells, NK T cells, tumor infiltrating lymphocytes (TIL), cytotoxic T lymphocytes (CTL), regulatory T cells (Treg), and dendritic cells (DCs, e.g., myeloid dendritic cells, plasmacytoid dendritic cells, CD14+ dendritic cells, Langerhans cells, or microglia), other immune cells that can induce effector functions, or mixtures thereof. The T cells can be Tαβ cells and Tγδ cells. In some embodiments, the stem cells can be derived from human embryonic stem cells, mesenchymal stem cells, and neural stem cells. In some embodiments, the T cells can be endogenous T cell receptor depleted.

[0368] In some embodiments, the cells of the present disclosure may be autologous, allogeneic, syngeneic, or xenogeneic with respect to a particular individual subject.

[0369] In some embodiments, the cells of the disclosure can be mammalian cells, particularly human cells. The cells of the disclosure can be primary cells or immortalized cell lines.

[0370] An engineered immune cell can be achieved by a method comprising introducing into a cell a nucleic acid molecule comprising a first nucleic acid sequence encoding at least one payload operably linked to a second nucleic acid sequence encoding a drug responsive domain (DRD).

[0371] The vector can be a viral vector, such as a lentiviral vector, a gamma retroviral vector, a recombinant AAV, an adenoviral vector, and an oncolytic viral vector. In other aspects, non-viral vectors, such as nanoparticles and liposomes, can also be used. In some embodiments, the immune cells of the present disclosure are genetically modified to express at least one immunotherapeutic agent of the present disclosure that can be regulated using a stabilizing ligand. In some examples, two, three, or more immunotherapeutic agents constructed with the same regulatable protein expression system construct are introduced into the cell. In other examples, two, three, or more regulatable protein expression system constructs can be introduced into the cell.

[0372] In some embodiments, the immune cells of the present disclosure may be T cells and / or NK cells that are engineered to express CD40L, and optionally, in combination with an antigen-specific T cell receptor (TCR) or an antigen-specific chimeric antigen receptor (CAR) as taught herein.

[0373] 15. Polynucleotides The components of the tunable protein expression system, including the effector modules, their SREs and payloads, can be nucleic acid based. The term "nucleic acid" in its broadest sense includes any compound and / or substance that includes a polymer of nucleotides, e.g., linked nucleosides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribo configuration, α-LNA (diastereomer of LNA) with α-L-ribo configuration, 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization), or hybrids thereof.

[0374] In some embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to generate the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. The polynucleotide of the present disclosure can be an mRNA or any nucleic acid molecule, and may or may not be chemically modified.

[0375] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5'cap, and a polyA tail. Based on this wild-type modular structure, the present disclosure expands the scope of functionality of conventional mRNA molecules by providing payload constructs that maintain the modular organization but contain one or more structural and / or chemical modifications or alterations that confer useful properties, such as tunability of function, to the polynucleotide. As used herein, a "structural" feature or modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification to the nucleosides themselves. Since chemical bonds are necessarily broken and reformed to effect the structural modification, the structural modification is of a chemical nature and is therefore a chemical modification. However, the structural modification results in a different sequence of nucleotides. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" is inserted, resulting in a structural modification of the polynucleotide.

[0376] In some embodiments, the polynucleotides of the present disclosure may include 5'UTR sequences that play a role in translation initiation. The 5'UTR sequence may include features such as the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate the translation of genes. The Kozak sequence has the consensus XCCR(A / G)CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering features that are typically found in abundantly expressed genes in target cells or tissues, the stability and protein production of the polynucleotides of the present disclosure can be enhanced.

[0377] Also provided are polynucleotides that may contain an internal ribosome entry site (IRES), which plays an important role in initiating protein synthesis when a polynucleotide lacks a 5' cap structure. An IRES may function as the only ribosome binding site or as one of multiple binding sites. Polynucleotides of the present disclosure that contain more than one functional ribosome binding site may encode several peptides or polypeptides that are independently translated by the ribosome giving rise to bicistronic and / or multicistronic nucleic acid molecules.

[0378] In one embodiment, the polynucleotide of the present disclosure can encode a variant polypeptide that has a certain identity with a reference polypeptide sequence. As used herein, "reference polypeptide sequence" refers to the starting polypeptide sequence. The reference sequence can be a wild type sequence, or any sequence that is referenced in the design of another sequence.

[0379] The term "identity" as known in the art refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between sequences, as determined by the number of matches between strings of two or more residues (amino acids or nucleic acids). Identity measures the percent of identity matches between two or more sequences, with gap alignment (if present), as addressed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0380] In some embodiments, the variant sequence may have the same or similar activity as the reference sequence. Alternatively, the variant may have an altered activity (e.g., increased or decreased) compared to the reference sequence. In general, a variant of a particular polynucleotide or polypeptide of the present disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Such tools for alignment include the BLAST suite of tools (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402).

[0381] 16. Codon Selection In some embodiments, one or more codons of the polynucleotides of the present disclosure may be replaced with other codons that encode a native amino acid sequence to modulate expression of the SRE through a process called codon selection. Because mRNA codons and tRNA anticodon pools tend to change across organisms, cell types, subcellular locations, and over time, the codon selection described herein is spatiotemporal (ST) codon selection.

[0382] In some embodiments of the present disclosure, certain polynucleotide features can be codon-optimized. Codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of a native sequence with the codons most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage can be measured using the codon adaptation index (CAI), which measures the deviation of a coding polynucleotide sequence from a reference gene set. Codon usage tables are available at the codon usage database (http: / / www.kazusa.or.jp / codon / ), and CAI can be calculated by the EMBOSS CAI program (http: / / emboss.sourceforge.net / ). Codon optimization methods are known in the art and can be useful in efforts to achieve one or more of several goals.

[0383] The stop codons of the polynucleotides of the present disclosure can be modified to include sequences and motifs to alter the expression levels of the SREs, payloads, and effector modules of the present disclosure. Such sequences can be incorporated to induce stop codon read-through, where the stop codon can specify an amino acid, for example, selenocysteine ​​or pyrrolysine. In other examples, the stop codon can be skipped entirely to resume translation via an alternative open reading frame. Stop codon read-through can be utilized to adjust the expression of components of the effector module in a particular ratio (e.g., as determined by the context of the stop codon). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is either C or U.

[0384] Suppression of termination occurs during the translation of many viral mRNAs as a means of generating a second protein with an extended carboxy terminus. In retroviruses, the gag and pol genes are encoded by a single mRNA and are separated by the amber stop codon UAG. Translational suppression of the amber codon allows synthesis of the gag pol precursor. Translational suppression is mediated by suppressor tRNAs that can recognize the stop codon and insert specific amino acids. In some embodiments, the effector modules described herein may incorporate an amber stop codon. Such codons can be used instead of or in addition to IRES and p2A sequences in bicistronic constructs. Stop codon read-through can be combined with P2A to obtain low levels of expression of downstream genes (e.g., IL12). In some embodiments, the amber stop codon can be combined with tRNA expression or amino-acyl tRNA synthetases for further control. In one aspect, the payload can be a regulated tRNA synthetase.

[0385] 17.Target area In some embodiments, the stimulus is a site of interest. The site of interest may be a location within a subject, such as, but not limited to, an organ selected from blood, plasma, liver, kidney, brain, heart, lung, bone, and bone marrow.

[0386] 18. Promoter In some embodiments, a composition of the present disclosure comprises a promoter.

[0387] As used herein, a promoter is defined as a DNA sequence recognized by the transcriptional machinery of a cell, required to initiate specific transcription of a polynucleotide sequence of the present disclosure. A vector can include a native or non-native promoter operably linked to a polynucleotide of the present disclosure. The promoter selected can be strong, weak, constitutive, inducible, tissue-specific, developmental stage-specific, and / or organism-specific. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter, such as, but not limited to, SEQ ID NOs: 6384-6386. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of a polynucleotide sequence operably linked to it. Another example of a promoter is elongation growth factor-1 alpha (EF-1 alpha), such as, but not limited to, SEQ ID NOs: 6387-6391. Other constitutive promoters may also be used, including, but not limited to, Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, Avian Leukemia Virus promoter, Epstein-Barr Virus immediate early promoter, Rous Sarcoma Virus promoter, and human gene promoters, including, but not limited to, the phosphoglycerate kinase (PGK) promoter (non-limiting examples include SEQ ID NOs: 6392-6399), actin promoter, myosin promoter, hemoglobin promoter, ubiquitin C (Ubc) promoter, human U6 small nucleoprotein promoter, and creatine kinase promoter. In some cases, inducible promoters may be used, such as, but not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0388] In some embodiments, an optimal promoter can be selected based on its ability to achieve minimal expression of the SRE and payload of the present disclosure in the absence of ligand, and detectable expression in the presence of ligand.

[0389] Additional promoter elements, such as enhancers, can be used to control the frequency of transcription initiation. Such regions can be located 10-100 base pairs upstream or downstream of the start site. In some cases, two or more promoter elements can be used to activate transcription, either coordinately or independently.

[0390] 19.Other control functions In some embodiments, the compositions of the present disclosure may include any proteasome adaptor. As used herein, the term "proteasome adaptor" refers to any nucleotide / amino acid sequence that targets an added payload for degradation. In some aspects, the adaptor directly targets the payload for degradation, thereby avoiding the need for a ubiquitination reaction. Proteasome adaptors can be used in combination with drug-responsive domains to reduce basal expression of the payload. Exemplary proteasome adaptors include the UbL domain of Rad23 or hHR23b, HPV E7, which binds with high affinity to both the target protein Rb and the S4 subunit of the proteasome, bypassing the ubiquitination machinery to allow direct proteasome targeting, and gankyrin, a protein that binds to Rb and the proteasome subunit S6.

[0391] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference and understanding, and the inclusion of such definitions herein should not necessarily be interpreted as meaning a substantial difference from what is commonly understood in the art. Commonly understood definitions of molecular biology terms and / or methods and / or protocols are described in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;Lewin, Genes V, Oxford University Press: New York, 1991; York, 1994; Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001), and Ausubel et al., Current Protocols in Molecular Biology (1994), Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717;Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929.

[0392] Where appropriate, procedures involving the use of commercially available kits and / or reagents are generally performed according to manufacturer's guidance and / or protocols and / or parameters unless otherwise noted.

[0393] "Affinity" refers to the strength of binding, with increased binding affinity correlated with a decreased Kd.

[0394] Adoptive Cell Therapy (ACT): As used herein, the term "adoptive cell therapy" or "adoptive cell transfer" refers to cell therapy involving the transplantation of cells into a patient, where the cells may originate from the patient or are from another individual and are manipulated (modified) before being returned to the patient. Therapeutic cells may be derived from the immune system, such as effector immune cells: CD4+ T cells; CD8+ T cells, natural killer cells (NK cells); B cells and tumor infiltrating lymphocytes (TILs) derived from resected tumors. The most commonly transplanted cells are autologous antitumor T cells after ex vivo expansion or manipulation. For example, autologous peripheral blood lymphocytes can be genetically engineered to recognize specific tumor antigens by expressing T cell receptors (TCRs) or chimeric antigen receptors (CARs).

[0395] Agent: As used herein, the term "agent" refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include simple or complex, organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, receptors, and soluble factors.

[0396] Agonist: As used herein, the term "agonist" refers to a compound that can combine with a receptor to produce a cellular response. An agonist can be a ligand that directly binds to the receptor. Alternatively, an agonist can indirectly bind to the receptor, for example, (a) by forming a complex with another molecule that directly binds to the receptor, or (b) by otherwise effecting the modification of another compound so that the other compound directly binds to the receptor. An agonist can be referred to as an agonist of a particular receptor or family of receptors, for example, an agonist of a costimulatory receptor.

[0397] Antagonist: As used herein, the term "antagonist" refers to any agent that inhibits or reduces the biological activity of a target or targets to which it binds.

[0398] Approximately: As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the reference value mentioned. In some embodiments, the term "approximately" or "about" refers to a range of values ​​within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less in either direction (greater or smaller) of the reference value mentioned, unless otherwise specified or clear from the context (except when such number exceeds 100 possible values).

[0399] Attached: As used herein, the terms "attached," "conjugated," "linked," "bonded," and "tethered," when used in reference to two or more moieties, mean that the moieties are physically attached or connected to one another, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable such that the moieties remain physically attached under the conditions in which the structure is used, e.g., physiological conditions. "Attached" need not be strictly by direct covalent chemical bonds. It may also imply that ionic or hydrogen bonds, or hybridization-based connections are sufficiently stable such that the "attached" entities remain physically attached.

[0400] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0401] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10-6 M-1 or less.

[0402] A "binding protein" is a protein that can bind non-covalently to another molecule. A binding protein can bind, for example, to a DNA molecule (DNA-binding protein), an RNA molecule (RNA-binding protein), and / or a protein molecule (protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form a homodimer, homotrimer, etc.) and / or it can bind to one or more molecules of a different protein or multiple proteins. A binding protein can have more than one binding activity. For example, a zinc finger protein has DNA-binding, RNA-binding, and protein-binding activity.

[0403] "Cleavage" refers to the cleavage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible, and double-strand cleavage can result from two distinct single-strand cleavage events. DNA cleavage can result in the generation of either blunt ends or staggered ends. In an embodiment, fusion polypeptides are used for targeted double-strand DNA cleavage.

[0404] A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then subjected to trans-RNA splicing (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0405] A "construct" is generally understood as any recombinant nucleic acid molecule, such as a linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source capable of genomic integration or autonomous replication, including a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or a nucleic acid molecule to which one or more nucleic acid molecules are operably linked. The construct may include additional regulatory nucleic acid molecules, such as, but not limited to, from the 3' untranslated region (3'UTR). The construct may include, but is not limited to, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule, which may play an important role in translation initiation and may also be a genetic component of the expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from sources that are natural or heterologous with respect to other elements present on the promoter construct.

[0406] Cytokine: As used herein, the term "cytokine" refers to a family of small soluble factors with pleiotropic functions produced by many cell types that can influence and control the functioning of the immune system.

[0407] Delivery: As used herein, the term "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload. "Delivery agent" refers to any agent that at least partially facilitates the delivery of one or more substances (including, but not limited to, compounds and / or compositions of the present disclosure) to a cell, a subject, or cells of other biological systems in vivo.

[0408] As used herein, the phrase "derived from" in reference to a DRD or payload means that the DRD or payload is at least partially derived from the parent molecule or sequence described. For example, the DRD may be derived from an epitope or region of a naturally occurring protein and modified in any of the ways taught herein to optimize DRD function.

[0409] Destabilization: As used herein, the terms "destabilization," "destabilization," "destabilization region" or "destabilization domain" refer to a region or molecule that is less stable than a starting, reference, wild-type, or native form of the same region or molecule.

[0410] A "coding sequence" or "coding region" of DNA refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences in a cell, ex vivo, in vitro, or in vivo. An "appropriate regulatory sequence" refers to a nucleotide sequence that is upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influences the transcription, RNA processing, or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0411] The term "downstream" refers to a nucleotide sequence located 3' of a reference nucleotide sequence. In particular, a downstream nucleotide sequence generally relates to a sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0412] The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. In particular, an upstream nucleotide sequence is generally associated with a sequence located 5' of a coding sequence or at the start of transcription. For example, most promoters are located upstream of the transcription start site.

[0413] Engineered: As used herein, an embodiment of the present disclosure is "engineered" if it is designed to have a feature or characteristic, whether structural or chemical, that varies from a starting, wild-type, or naturally occurring molecule.

[0414] An "exogenous" molecule is one that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical, or other methods. "Normally present in a cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is only present during embryonic development of muscle is an exogenous molecule to an adult muscle cell. Similarly, a molecule that is induced by heat shock is an exogenous molecule to a non-heat-shocked cell. Exogenous molecules can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0415] Exogenous molecules can be small molecules, such as those produced by combinatorial chemical processes, among others, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above-mentioned molecules, or any complexes containing one or more of the above-mentioned molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, can be linear, branched, or circular, and can be of any length. Nucleic acids include nucleic acids capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0416] An exogenous molecule can be the same type of molecule as an endogenous molecule, for example, an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can include an infectious viral genome, a plasmid, or an episome that is introduced into a cell, or a chromosome that is not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art, and include, but are not limited to, lipid-mediated introduction (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, microprojectile bombardment, calcium phosphate co-precipitation, DEAE-dextran mediated introduction, and viral vector mediated introduction. An exogenous molecule can also be the same type of molecule as an endogenous molecule, but can be derived from a different species than the cell is derived from. For example, a human nucleic acid sequence can be introduced into a cell line that is originally derived from a mouse or hamster.

[0417] In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, endogenous nucleic acids can include chromosomes, the genome of mitochondria, chloroplasts, or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules can include proteins, such as transcription factors and enzymes.

[0418] An "episome" is a replicating nucleic acid, nucleoprotein complex, or other structure that contains nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

[0419] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells).

[0420] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; (4) folding of the polypeptide or protein; and (5) post-translational modification of the polypeptide or protein.

[0421] An expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid produced by human intervention, including recombinant means or direct chemical synthesis, with a set of specific nucleic acid elements that allow for transcription or translation of a particular nucleic acid, for example, in a host cell. An expression vector can be part of a plasmid, a virus, or a nucleic acid fragment. Typically, an expression vector can include a nucleic acid that is operably linked to a promoter and is to be transcribed.

[0422] The term "fragment" as applied to a polynucleotide sequence refers to a nucleotide sequence that is shorter in length than a reference nucleic acid and contains a nucleotide sequence identical to the reference nucleic acid in the common portion. Such a nucleic acid fragment according to the present invention may be included in a larger polynucleotide of which it is a component, if appropriate. Such a fragment may comprise or alternatively consist of an oligonucleotide ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more consecutive nucleotides of a nucleic acid according to the present invention.

[0423] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to the full-length protein, polypeptide, or nucleic acid, but which retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment may have more, fewer, or the same number of residues as the corresponding naturally occurring molecule, and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of a protein are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assessed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays, or complementation (both genetic and biochemical). See, e.g., Fields et al. (1989) Nature 340:245-246, U.S. Patent No. 5,585,245 and PCT WO 98 / 44350.

[0424] As used herein, a "functional" biomolecule is a biological entity whose structure and form exhibit a property and / or activity by which it is characterized.

[0425] A "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be of the same chemical type or of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins, such as fusions between DNA-binding domains (e.g., ZFPs, TALEs and / or meganuclease DNA-binding domains) and nuclease (cleavage) domains (e.g., endonucleases, meganucleases, etc.) and fusion nucleic acids (e.g., nucleic acids encoding the aforementioned fusion proteins). Examples of the second type of fusion molecule include, but are not limited to, fusions between triplex-forming nucleic acids and polypeptides, and fusions between minor groove binders and nucleic acids.

[0426] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell, or delivery of a polynucleotide encoding the fusion protein to the cell, where the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation can also be involved in expression of the protein in the cell. Methods for delivering polynucleotides and polypeptides to cells are presented elsewhere in this disclosure.

[0427] "Gene" refers to a polynucleotide that includes nucleotides that encode a functional molecule, including functional molecules produced by transcription alone (e.g., a bioactive RNA species) or by transcription and translation (e.g., a polypeptide). The term "gene" encompasses cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific RNA, protein, or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, including regulatory and / or coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but arranged in a different way than found in nature. A chimeric gene may contain coding sequences derived from different sources and / or regulatory sequences derived from different sources. "Endogenous gene" refers to a native gene in its natural location within the genome of an organism. A "foreign" or "heterologous" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. A foreign gene can include a native gene inserted into a non-naturally occurring organism, or a chimeric gene. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. For example, the interleukin-12 (IL-12) gene encodes the IL-12 protein. IL-12 is a heterodimer of a 35 kD subunit (p35) and a 40 kD subunit (p40), which bind via disulfide bonds to form the fully functional IL-12p70. The IL-12 gene encodes both the p35 and p40 subunits.

[0428] A transcribed polynucleotide can have a sequence that codes for a polypeptide, such as a functional protein, which can be translated into an encoded polypeptide when placed under the control of appropriate regulatory regions. A gene can include several operably linked fragments, such as a promoter, a 5' leader sequence, a coding sequence, and a 3' untranslated sequence, such as a polyadenylation site, and can include all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational control sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0429] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristyrylation, and glycosylation.

[0430] A chimeric or recombinant gene is a gene that is not normally found in nature, e.g., a gene whose promoter is not naturally associated with some or all of the transcribed DNA region. "Expression of a gene" refers to the process by which a gene is transcribed into RNA and / or translated into a functional protein.

[0431] "Gene delivery" or "gene transduction" refers to a method for introducing recombinant or foreign DNA into a host cell. The introduced DNA can remain unintegrated or preferably be integrated into the genome of the host cell. Gene delivery can be performed, for example, by transduction using a viral vector or by transformation of cells using known methods such as electroporation, cell bombardment, etc.

[0432] The term "genome" includes chromosomal, as well as mitochondrial, chloroplast, and viral DNA or RNA.

[0433] The term "head-to-head" is used herein to describe the orientation of two polynucleotide sequences relative to one another. Two polynucleotides are arranged in a head-to-head orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, such that the direction of transcription of each polynucleotide proceeds away from the 5' end of the other polynucleotide. The term "head-to-head" is sometimes abbreviated as (5') to (5').

[0434] The term "tail-to-tail" is used herein to describe the orientation of two polynucleotide sequences relative to each other. Two polynucleotides are arranged in a tail-to-tail orientation when the 3' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, such that the direction of transcription of each polynucleotide proceeds toward the other polynucleotide. The term "tail-to-tail" may be abbreviated as (3') to (3').

[0435] The term "head-to-tail" is used herein to describe the orientation of two polynucleotide sequences relative to one another. Two polynucleotides are arranged in a head-to-tail orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, such that the direction of transcription of each polynucleotide proceeds in the same direction as the direction of transcription of the other polynucleotide. The term "head-to-tail" is sometimes abbreviated as (5') to (3').

[0436] As used herein, the terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid" each refer to a sequence that is derived from a source foreign to a particular host cell, or that is modified from its original form if derived from the same source. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell, but that has been modified, for example, by the use of DNA shuffling. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, these terms refer to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell, but is in a location within the host cell nucleic acid where the element is not normally found. The exogenous DNA segment is expressed to produce an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence that is naturally associated with the host cell into which it is introduced.

[0437] "Heterologous DNA" refers to DNA that is not naturally located within a cell or at a chromosomal site of the cell. Heterologous DNA can include genes foreign to the cell.

[0438] "Homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, for example, the insertion of a vector into a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector contains a sufficiently long homologous region to the sequence of the chromosome, allowing the vector to be complementary bound and integrated into the chromosome. A longer homologous region and a higher degree of sequence similarity can increase the efficiency of homologous recombination.

[0439] Immune Cell: The term "immune cell" as used herein refers to any cell of the immune system that is derived from hematopoietic stem cells in the bone marrow, which give rise to two major lineages, myeloid progenitors (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitors (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, Tγδ cells, Tαβ cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of APCs complexed with peptides interact with TCRs on the surface of the T cells.

[0440] Immunotherapy: The term "immunotherapy" as used herein refers to a type of treatment of disease by inducing or restoring the immune system's responsiveness to the disease.

[0441] Immunotherapeutic: The term "immunotherapeutic" as used herein refers to the treatment of disease by inducing or restoring immune system responsiveness to the disease with a biological, pharmaceutical, or chemical compound.

[0442] For purposes of the present invention, the term "isolated" refers to biological material (cells, nucleic acids, or proteins) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide naturally occurring in a plant or animal is not isolated, but the same polynucleotide separated from the adjacent nucleic acids in which it naturally occurs is considered "isolated."

[0443] "Modulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene suppression. Genome editing (e.g., truncation, modification, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to a decrease in gene expression compared to cells that do not contain the ZFP, TALE, or CRISPR / Cas system described herein. Thus, gene inactivation can be partial or complete.

[0444] Modified: As used herein, the term "modified" refers to an altered state or structure of a molecule or entity compared to a parent or reference molecule or entity. Molecules can be modified in many ways, including chemically, structurally, and functionally. In some embodiments, the compounds and / or compositions of the disclosure are modified by the introduction of a non-natural amino acid.

[0445] Mutation: As used herein, the term "mutation" refers to a change and / or modification. In some embodiments, a mutation may be a change and / or modification to a protein (including peptides and polypeptides) and / or a nucleic acid (including polynucleic acids). In some embodiments, a mutation includes a change and / or modification to a protein and / or a nucleic acid sequence. Such a change and / or modification may include an addition, substitution and / or deletion of one or more amino acids (in the case of proteins and / or peptides) and / or nucleotides (in the case of nucleic acids and / or polynucleic acids, e.g., polynucleotides). In some embodiments, where a mutation includes an addition and / or substitution of amino acids and / or nucleotides, such addition and / or substitution may include one or more amino acid and / or nucleotide residues and may include modified amino acids and / or nucleotides. The resulting construct, molecule, or sequence of the mutation, change, or modification may be referred to herein as a mutant.

[0446] "Nucleic acid", "nucleic acid molecule", "oligonucleotide", "nucleotide", and "polynucleotide" are used interchangeably and refer to the phosphate polymeric forms of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules"), or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, either in single-stranded form or double-stranded helices. Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in, inter alia, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.

[0447] As used herein, an "isolated nucleic acid fragment" refers to a polymer of RNA or DNA that is single- or double-stranded and optionally contains synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be composed of one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0448] Exogenous nucleic acid sequences may include, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more control elements (e.g., promoters). In addition, exogenous nucleic acid sequences may generate one or more RNA molecules (e.g., small hairpin RNA (shRNA), inhibitory RNA (RNAi), microRNA (miRNA), etc.).

[0449] Operably linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0450] "Operably linked" or "functionally linked" when referring to nucleic acid sequences and polynucleotides refers to the association of nucleic acid sequences in a single nucleic acid fragment, where the function of one is affected by the other, while the nucleic acid sequences are not necessarily contiguous or adjacent to each other, but may have intervening sequences between them. For example, a regulatory DNA sequence is said to be "operably linked" or "associated" with a DNA sequence encoding an RNA or polypeptide when the two sequences are positioned such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. A transcriptional regulatory sequence is generally operably associated with a coding sequence in cis, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence even if they are not contiguous, or a promoter is operably linked to a gene of interest if the promoter regulates or mediates the transcription of the gene of interest in a cell.

[0451] Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, an EF-1 promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent to or located in close proximity to the coding sequence whose transcription is enhanced. A polylinker provides a convenient place for inserting a coding sequence so that the gene is operably linked to the AP-1 promoter. A polylinker is a polynucleotide sequence that contains a series of three or more adjacent restriction endonuclease recognition sequences.

[0452] In the binding between two or more polypeptides or domains thereof to create a fusion polypeptide, the term "operably linked" means that the state or function of one polypeptide in the fusion protein is affected by the other polypeptide in the fusion protein. For example, for a fusion protein comprising a DRD and a payload, the DRD and the payload are operably linked if stabilization of the DRD by a ligand results in stabilization of the payload, while destabilization of the DRD in the absence of the ligand results in destabilization of the payload. The term operably linked can certainly include embodiments in which the DRD is adjacent to or directly fused to the payload, but other embodiments, such as when the DRD is separated from the payload by other nucleotide sequences or peptide or polypeptide sequences, are also "operably" linked if stabilization of the DRD by a ligand results in stabilization of the payload, while destabilization of the DRD in the absence of the ligand results in destabilization of the payload.

[0453] "Open reading frame", abbreviated as ORF, refers to a length of nucleic acid sequence, either DNA, cDNA, or RNA, that includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon, and that can potentially be translated into a polypeptide sequence.

[0454] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0455] The term "plasmid" refers to an extrachromosomal element that is not part of the central metabolism of the cell and often carries genes, usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of single- or double-stranded DNA or RNA from any source, and many nucleotide sequences are joined or recombined into unique constructs that can introduce promoter fragments and DNA sequences of selected gene products, along with appropriate 3' untranslated sequences, into cells. The starting plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well-known published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, one of skill in the art can readily construct any number of other plasmids suitable for use in the present invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill in the art from the present disclosure.

[0456] The term "polypeptide" is used interchangeably herein with the terms "polypeptide" and "protein(s)" and refers to a polymer of amino acid residues, e.g., as typically found in a protein in nature. A "mature protein" is a protein that is full-length and, optionally, includes glycosylation or other modifications typical of proteins in a given cell membrane.

[0457] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even include synthetic DNA segments. It is understood by those skilled in the art that different promoters may induce the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. As used herein, a "promoter" may include necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter may also include distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the start site of transcription, as needed.

[0458] In most cases, promoters that cause a gene to be expressed in most cell types are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a particular cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a particular stage of development or cell differentiation are commonly referred to as "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced to cause a gene to be expressed after exposure or treatment of cells with a drug, biomolecule, chemical, ligand, light, etc. that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that in m...

Claims

[Claim 1] The invention described in this specification.

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