WO2018161038A1 - Il12 compositions and methods for immunotherapy - Google Patents

Abstract

The present invention provides biocircuit systems, effector modules and compositions for cancer immunotherapy. Methods for inducing anti-cancer immune responses in a subject are also provided.

Description

IL12 COMPOSITIONS AND METHODS FOR IMMUNOTHERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Patent Application No. 62/466,601, filed on March 3, 2017 entitled Compositions and Methods for Immunotherapy; the US Provisional Patent Application No. 62/484,060, filed on April 11, 2017, entitled IL12

Compositions and Methods for Immunotherapy and the US Provisional Patent Application No. 62/555,328, filed on September 7, 2017, entitled IL12 Compositions and Methods for

Immunotherapy, the contents of each of which are herein incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2095_1206PCT_SL.txt, created on March 2, 2018, which is 671,080 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for immunotherapy.

Provided in the present invention include polypeptides of biocircuit systems, effector modules, stimulus response elements (SREs) and immunotherapeutic agents, polynucleotides encoding the same, vectors and cells containing the polypeptides and/or polynucleotides for use in cancer immunotherapy. In one embodiment, the compositions comprise destabilizing domains (DDs) which tune protein stability.

BACKGROUND OF THE INVENTION

[0004] Cancer immunotherapy aims to eradicate cancer cells by rejuvenating the tumoricidal functions of tumor-reactive immune cells, predominantly T cells. Strategies of cancer immunotherapy including the recent development of checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines which can increase the anti-tumor immune effector cells have produced remarkable results in several tumors.

[0005] The impact of host anti-tumor immunity and cancer immunotherapy is impeded by three major hurdles: 1) low number of tumor antigen-specific T cells due to clonal deletion; 2) poor activation of innate immune cells and accumulation of tolerogenic antigen-presenting cells in the tumor microenvironment; and 3) formation of an immunosuppressive tumor

microenvironment. Particularly, in solid tumors the therapeutic efficacy of immunotherapeutic regimens remains unsatisfactory due to lack of an effective an anti-tumor response in the immunosuppressive tumor microenvironment. Tumor cells often induce immune tolerance or suppression and such tolerance is acquired because even truly foreign tumor antigens will become tolerated. Such tolerance is also active and dominant because cancer vaccines and adoptive transfer of pre-activated immune effector cells (e.g., T cells), are subject to suppression by inhibitory factors in the tumor microenvironment (TME).

[0006] In addition, administration of engineered T cells could result in on/off target toxicities as well as a cytokine release syndrome (reviewed by Tey Clin. Transl. Immunol., 2014, 3: el7 10.1038).

[0007] Development of a tunable switch that can turn on or off the transgenic

immunotherapeutic agent expression is needed in case of adverse events. For example, adoptive cell therapies may have a very long and an indefinite half-life. Since toxicity can be progressive, a safety switch is desired to eliminate the infused cells. Systems and methods that can tune the transgenic protein level and expression window with high flexibility can enhance therapeutic benefit, and reduce potential side effects.

[0008] To develop regulatable therapeutic agents for disease therapy, in particular cancer immunotherapy, the present invention provides biocircuit systems to control the expression of immunotherapeutic agents. The biocircuit system comprises a stimulus and at least one effector module that responds to the stimulus. The effector module may include a stimulus response element (SRE) that binds and is responsive to a stimulus and an immunotherapeutic agent operably linked to the SRE. In one example, a SRE is a destabilizing domain (DD) which is destabilized in the absence of its specific ligand and can be stabilized by binding to its specific ligand.

SUMMARY OF THE INVENTION

[0009] The present invention provides compositions and methods for immunotherapy. The compositions relate to tunable systems and agents that induce anti-cancer immune responses in a cell or in a subject. The tunable system and agent may be a biocircuit system comprising at least one effector module that is responsive to at least one stimulus. The biocircuit system may be, but is not limited to, a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. These systems are further taught in co- owned U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016, 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).

[0010] In some embodiments, the composition for inducing an immune response may comprise an effector module. In some embodiments, the effector module may comprise a stimulus response element (SRE) operably linked to at least one payload. In one aspect, the payload may be an immunotherapeutic agent.

[0011] In some embodiments, the immunotherapeutic agent may be selected from, but not limited to a cytokine, a cytokine receptor, a cytokine-cytokine receptor fusion, and any combinations thereof.

[0012] In one aspect, the SRE of the composition may be responsive to or interact with at least one stimulus.

[0013] In some embodiments, the SRE may comprise a destabilizing domain (DD). The DD may be derived from a parent protein or from a mutant protein having one, two, there, 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, human protein FKBP, comprising the amino acid sequence of SEQ ID NO. 3; human DHFR (hDHFR), comprising the amino acid sequence of SEQ ID NO. 2; E. Coli DHFR, comprising the amino acid sequence of SEQ ID NO. 1; PDE5, comprising the amino acid sequence of SEQ ID NO. 4; PPAR, gamma comprising the amino acid sequence of SEQ ID NO. 5; CA2, comprising the amino acid sequence of SEQ ID NO. 6; or NQ02, comprising the amino acid sequence of SEQ ID NO. 7.

[0014] In one aspect, the parent protein is hDHFR and the DD comprises a mutant protein. The mutant protein may comprise a single mutation and may be selected from, but not limited to hDHFR (I17V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81R), hDHFR (A107V), hDHFR (Y122I), hDHFR (N127Y), hDHFR (M140I), hDHFR (K185E), hDHFR (N186D), and hDHFR (M140I), hDHFR (Amino acid 2-187 of WT; N127Y), hDHFR (Amino acid 2-187 of WT; I17V), hDHFR (Amino acid 2-187 of WT; Y1221), and hDHFR (Amino acid 2-187 of WT; K185E). In some embodiments, the mutant protein may comprise two mutations and may be selected from, but not limited to, hDHFR (C7R, Y163C), hDHFR (Al 0V, H88Y), hDHFR (Q36K, Y122I), hDHFR (M53T, R138I), hDHFR (T57A, I72A), hDHFR (E63G, I176F), hDHFR (G21T, Y122I), hDHFR (L74N, Y122I), hDHFR (V75F, Y122I), hDHFR (L94A, T147A), DHFR (V121A, Y22I) , hDHFR (Y122I, A125F), hDHFR (H131R, E144G), hDHFR (T137R, F143L), hDHFR (Y178H, E18IG), and hDHFR (Y183H, K185E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; I17V, Y122I), hDHFR (Amino acid 2-187 of WT; Y122I, M140I), hDHFR (Amino acid 2-187 of WT; N127Y, Y122I), hDHFR (Amino acid 2-187 of WT; E162G, I176F), and hDHFR (Amino acid 2-187 of WT; H131R, E144G), and hDHFR (Amino acid 2-187 of WT; Y122I, A125F). In some embodiments, the mutant may comprise three mutations and the mutant may be selected from hDHFR (V9A, S93R, P150L), hDHFR (I8V, K133E, Y163C), hDHFR (L23S, V121A, Y157C), hDHFR (K19E, F89L, E181G), hDHFR (Q36F, N65F, Y122I), hDHFR (G54R, M140V, S168C), hDHFR (VI 10A, V136M, K177R), hDHFR (Q36F, Y122I, A125F), hDHFR (N49D, F59S, D153G), and hDHFR (G21E, I72V, I176T), hDHFR (Amino acid 2-187 of WT; Q36F, Y122I, A125F), hDHFR (Amino acid 2-187 of WT; Y122I, H131R, E144G), hDHFR (Amino acid 2-187 of WT; E31D, F32M, VI 161), and hDHFR (Amino acid 2-187 of WT; Q36F, N65F, Y122I). In some embodiments, the mutant may comprise four or more mutations and the mutant may be selected from hDHFR (V2A, R33G, Q36R, L100P, K185R), hDHFR (Amino acid 2-187 of WT; D22S, F32M, R33S, Q36S, N65S), hDHFR (I17N, L98S, K99R, Ml 12T, E151G, E162G, E172G), hDHFR (G16S, I17V, F89L, D96G, K123E, M140V, D146G, K156R), hDHFR (K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, K177E, Y178C, K185E, N186S), hDHFR (N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L), hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, I139V, M140I, D142G, D146G, E173G, D187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N, L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, VI 10A, II 15V, Y122D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, V113A, Wl 14R, Y122C, M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S), and hDHFR (A 10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, Ml 12V, W114R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N).

[0015] In one aspect, the stimulus of the SRE may be Trimethoprim or Methotrexate.

[0016] In some embodiments, the immunotherapeutic agent of the composition may be a cytokine. The cytokine may be an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor B, a CC chemokine, a CXC chemokine, a CX3C chemokine or a growth factor.

[0017] In one aspect, the interleukin may be a whole or a portion of a IL12 and may comprise a p40 subunit (the amino acid sequence of SEQ ID NO. 58) or portion thereof and/or a p35 subunit (the amino acid sequence of SEQ ID NO. 59). In one aspect, the IL12 may be modified. In some embodiments, the modifications may comprise fusing SEQ ID NO. 58 and/or SEQ ID NO. 59 to the whole or a portion of, a transmembrane domain. The IL12 may optionally be modified by incorporating a hinge domain. [0018] In one aspect, the composition may include a first effector module (e.g., an effector module comprising IL12 or a portion thereof) and a second effector module. The second effector module may be a second SRE linked to an immunotherapeutic agent. As a non-limiting example, the immunotherapeutic agent is IL 15 or an IL15/IL15Ra fusion polypeptide.

[0019] In one aspect, the SRE of the composition may stabilize the immunotherapeutic agent by a stabilization ratio of 1 or more. The stabilization ratio may comprise the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the expression, function or level of the immunotherapeutic agent in the absence of the stimulus.

[0020] In one aspect, the SRE of the composition may destabilize the immunotherapeutic agent by a destabilization ratio between 0, and 0.09. The destabilization ratio may comprise the ratio of expression, function or level of an immunotherapeutic agent in the absence of the stimulus specific to the SRE to the expression, function or level of the immunotherapeutic agent that is expressed constitutively, and in the absence of the stimulus specific to the SRE.

[0021] The present invention also provides polynucleotides comprising the compositions of the invention.

[0022] In one aspect, the polynucleotides may be a DNA or RNA molecule. In one aspect, the polynucleotides may comprise spatiotemporally selected codons. In one aspect, the

polynucleotides of the invention may be a DNA molecule. In some embodiments, the polynucleotides may be an RNA molecule. In one aspect, the RNA molecule may be a messenger molecule. In some embodiments, the RNA molecule may be chemically modified.

[0023] In some embodiments, the polynucleotides may further comprise, at least one additional feature selected from, but not limited to, a promoter, a linker, a signal peptide, a tag, a cleavage site and a targeting peptide.

[0024] The present invention also provides vectors comprising polynucleotides described herein. In one aspect, the vector may be a viral vector. In some embodiments, the viral vector may be a retroviral vector, a lenti viral vector, a gamma retroviral vector, a recombinant AAV vector, an adeno viral vector, and an oncolytic viral vector.

[0025] The present invention also provides immune cells for adoptive cell transfer (ACT) which may express the compositions of the invention, the polynucleotides described herein. In one aspect, the immune cells may be infected or transfected with the vectors described herein. The immune cells for ACT may be selected from, but not limited to a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine- induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

[0026] In some embodiments, the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

[0027] The present invention provides methods for reducing a tumor volume or burden in a subject comprising contacting the subject with the immune cells of the invention. Also provided herein, is a method for inducing an anti-tumor immune response in a subject, comprising administering the immune cells of the system to the subject.

[0028] The present invention also provides methods for enhancing the expansion and/or survival of immune cells, comprising contacting the immune cells with the compositions of the invention, the polynucleotides of the invention, and/or the vectors of the invention.

[0029] Also provided herein, is a method for inducing an immune response in a subject, administering the compositions of the invention, the polynucleotides of the invention, and/or the immune cells of the invention to the subject.

[0030] In some embodiments, the effector module comprises a stimulus response element

(SRE) and at least one pay load comprising a protein of interest (POI).

[0031] In some embodiments, the SRE may be a destabilizing domain (DD). In some examples, the DD is a mutant domain derived from a protein such as FKBP (FK506 binding protein), E. coli DHFR (Dihydrofolate reductase) (ecDHFR), human DHFR (hDHFR), or any protein of interest. In this context, the biocircuit system is a DD biocircuit system.

[0032] The payload may be any immune-therapeutic agent used for cancer immunotherapy such as a cytokine. In one embodiment, the cytokine may be IL12. The SRE and payload may be operably linked through one or more linkers and the positions of components may vary within the effector module.

[0033] In some embodiments, the effector module may further comprise one or more additional features such as linker sequences (with specific sequences and lengths), cleavage sites, regulatory elements (that regulate expression of the protein of interest such as microRNA targeting sites), signal sequences that lead the effector module to a specific cellular or subcellular location, penetrating sequences, or tags and biomarkers for tracking the effector module.

[0034] In some embodiments, the DD may stabilize the immune-therapeutic agent with a stabilization ratio of at least one in the presence of the stimulus. According to the present invention, the DD may destabilize the immunotherapeutic agent in the absence of ligand with a destabilization ratio between 0, and 0.99. [0035] The invention provides isolated biocircuit polypeptides, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing; vectors comprising polynucleotides of the invention; and cells expressing

polypeptides, polynucleotides and vectors of the invention. The polypeptides, polynucleotides, viral vectors and cells are useful for inducing anti-tumor immune responses in a subject.

[0036] In some embodiments, the vector of the invention is a viral vector. The viral vector may include, but is not limited to a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.

[0037] In some embodiments, the vector of the invention may be a non-viral vector, such as a nanoparticles and liposomes.

[0038] The present invention also provides immune cells engineered to include one or more polypeptides, polynucleotides, or vectors of the present invention. The cells may be immune effector cells, including T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), and tumor infiltrating lymphocytes (TTLs). The engineered cell may be used for adoptive cell transfer for treating a disease (e.g., a cancer).

[0039] Also provided herein are compositions and vectors containing a second effector module comprising second a stimulus response element (SRE) and at least second immunotherapeutic agent. In some embodiments, the immunotherapeutic agent may IL15 or IL15/IL15Ra fusion polypeptide.

[0040] The present invention also provides methods for inducing immune responses in a subject using the compositions of the invention. Also provided are methods for reducing a tumor burden in a subject using the compositions of the invention.

[0041] Provided herein are methods for tuning the expression and function of

immunotherapeutic agent in cells or subjects. Such method may involve the administering effector modules containing an SRE operably linked to an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is IL12. In some embodiments, the SRE is derived from FKBP, DHFR, PDE5, PPAR gamma, CA2 and NQ02. Methods for pulsatile regulation of an immunotherapeutic agent using compositions described herein are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows an overview diagram of a biocircuit system of the invention. The biocircuit comprises a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces a signal or outcome. The effector module comprises at least one stimulus response element (SRE) and one payload. [0043] Figure 2 shows representative effector modules carrying one payload. The signal sequence (SS), SRE and payload may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.

[0044] Figure 3 shows representative effector modules carrying two payloads without a cleavage site. The two payloads may be either directly linked to each other or separated.

[0045] Figure 4 shows representative effector modules carrying two payloads with a cleavage site. In one embodiment, an SS is positioned at the N-terminus of the construct, while other components: SRE, two payloads and the cleavage site may be located at different positions (A to L). In another embodiment, the cleavage site is positioned at the N-terminus of the construct (M to X). An optional linker may be inserted between each component of the effector module.

[0046] Figure 5 shows effector modules of the invention carrying two payloads, where an SRE is positioned at the N-terminus of the construct (A to L), while SS, two payloads and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.

[0047] Figure 6 shows effector modules of the invention carrying two payloads, where either the two payloads (A to F) or one of the two payloads (G to X) is positioned at the N-terminus of the construct (A to L), while SS, SRE and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.

[0048] Figure 7 depicts representative configurations of the stimulus and effector module within a biocircuit system. A trans-membrane effector module is activated either by a free stimulus (Figure 7A) or a membrane bound stimulus (Figure 7B) which binds to SRE. The response to the stimulus causes the cleavage of the intracellular signal/payload, which activates down-stream effector/payload.

[0049] Figure 8 depicts a dual stimulus-dual presenter biocircuit system, where two bound stimuli (A and B) from two different presenters (e.g., different cells) bind to two different effector modules in a single receiver (e.g., another single cell) simultaneously and create a dual- signal to downstream payloads.

[0050] Figure 9 depicts a dual stimulus-single presenter biocircuit system, where two bound stimuli (A and B) from the same presenter (e.g., a single cell) bind to two different effector modules in another single cell simultaneously and create a dual-signal.

[0051] Figure 10 depicts a single-stimulus-bridged receiver biocircuit system. In this configuration, a bound stimulus (A) binds to an effector module in the bridge cell and creates a signal to activate a payload which is a stimulus (B) for another effector module in the final receiver (e.g., another cell).

[0052] Figure 11 depicts a single stimulus-single receiver biocircuit system, wherein the single receiver contains the two effector modules which are sequentially activated by a single stimulus.

[0053] Figure 12 depicts a biocircuit system which requires a dual activation. In this embodiment, one stimulus must bind the transmembrane effector module first to prime the receiver cell being activated by the other stimulus. The receiver only activates when it senses both stimuli (B).

[0054] Figure 13 A is a bar graph depicting IL12 levels in the various dilutions of media derived from cells expressing DD-IL12. Figure 13B is a bar graph depicting the Shield- 1 dose responsive induction of DD- IL12. Figure 13C depicts plasma IL12 levels in mice implanted with SKOV3 cells. Figure 13D depicts plasma IL12 levels in mice in response to different Shield- 1 dosing regimens.

[0055] Figure 14A is a western blot of IL15 protein levels in 293 cells. Figure 14B and Figure 14C are histograms depicting surface expression of IL15 and lL15Ra. Figure 14D is a western blot of IL15 and hDHFR in HCT116 ceUs.

[0056] Figure 15 denotes the frequency of IFNgamma positive T cells.

[0057] Figure 16A depicts IFN gamma production in T cells. Figure 16B depicts T cell expansion with IL15/IL15Ra treatment. Figure 16C is a dot plot depicting percentage human cells after in vivo cell transfer. Figure 16D is scatter plot depicting CD4+/CD8+ T cells.

[0058] Figure 17 depicts plasma IL12 levels in mice in response to different Shield- 1 dosing regimens.

[0059] Figure 18A and Figure 18B depict the effect of promoter on IL12 levels. Figure 18C depicts the effect of ligand concentration and promoter on IL12 levels. Figure 18D shows the effect of promoter on IL12 levels in HCT116 cells. Figure 18E depicts IL12 levels in Raji cells.

[0060] Figure 19A depicts the expansion of T cells in response to cytokine treatment. Figure 19B, Figure 19C and Figure 19D depict the frequency of IFN gamma positive cells with IL12 treatment.

[0061] Figure 20A is a bar graph depicting IL15Ra positive cells with 24 hour TMP treatment. Figure 20B is a bar graph depicting IL15Ra positive cells with 48 hour TMP treatment. Figure 20C is a bar graph depicting IL15Ra positive cells in response to varying concentrations of TMP.

[0062] Figure 21 is a western blot of IL15Ra protein levels in HCT116 cells.

[0063] Figure 22A represents the percentage of human T cells blood with respect to mouse T cells. Figure 22B represents the number of T cells in blood. Figure 22C represents ratio of CD8 to CD4 cells in the blood. Figure 22D represents the percentage of IL15Ra positive CD4 and CD8 T cells in the blood.

[0064] Figure 23 is a bar graph representing the effect of promoters on transgene expression.

[0065] Figure 24A provides the final IL12 concentration for each of the four groups tested. Figure 24B shows that IL12 is detectable in kidney and Figure 24C shows that IL12 is detectable in tumor.

[0066] Figure 25A shows the regulation of IL12 over 24 hours. Figure 25B shows the regulation in the plasma and Figure 25C shows the detection of flexi-IL12 in the kidneys.

[0067] Figure 26A shows that restimulation increased the expression of IL12. Figure 26B and Figure 26C show that ligand increased production of IL12.

[0068] Figure 27A shows the concentration-dependent induction of 1L12 secretion of IL12 secretion from primary human T cells. Figure 27B shows the time course induction of IL12 secretion from primary human T cells.

[0069] Figure 28A shows the dose response of Aquashield-Induced DD-IL12 regulation in vivo. Figure 28B shows that plasma levels of IL12 remain high in animals transplanted with constitutive IL12 transduced T cells.

[0070] Figure 29A and 29B show the expression of IL12 in vivo over 7 days. Figure 29C and 29D show the expression of 1L12 in vivo over 11 days. Figure 29E shows the Geometric MFI (GeoMFI) of Granzyme B (GrB) after 7 days in CD8+ T cells. Figure 29F shows the GeoMFI of Perforin at day 7 in CD8+ T cells.

[0071] Figure 30A shows the regulation of IL12 with PGK and EFla promoters and FKBP domains. Figure 30B shows the relative expression of 1L12.

[0072] Figure 31 depicts the kinetics of IL 15Ra surface expression on CD4 T cells after TMP treatment.

[0073] Figure 32 represents a western blot of !L15-lL15Ra protein in HCT116 tumors from mice treated with TMP for 17 days in xenograft assays.

[0074] Figure 33 is a graph of the results of the MSD assay of IL15 protein levels in HEK293 cells.

[0075] Figure 34A provides FACS plots showing the expression of membrane bound XL 15 after a dose response study of TMP. Figure 34B is two graphs showing the dose and time of exposure of TMP in vitro influences membrane bound IL15 expression.

[0076] Figures 35A- 35C show the regulation of membrane bound IL15 using IL15 (Figure 35A), IL15Ra (Figure 35B), or lL15/IL15Ra double ++ staining (Figure 35C). Figure 35D shows FACS plots of the expression of IL15. Figure 35E is a graph of the regulation of IL15 in blood and Figure 35F is a graph of the plasma TMP levels.

[0077] Figure 36 represents the regulation of membrane bound IL15 with PO or IP dosing of TMP.

DETAILED DESCRIPTION OF THE INVENTION

[0078] The details of one or more embodiments of the invention are set forth 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 invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, 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 the case of conflict, the present description will control.

I. INTRODUCTION

[0079] Cancer immunotherapy aims at the induction or restoration of the reactivity of the immune system towards cancer. Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment. Active immunotherapy aims at induction of an endogenous, long-lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are

administered to the host. This approach is short lived and requires multiple applications.

[0080] Despite significant advances, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, which in part, emerges from the need to push therapy dose to the edge of potentially fatal toxicity to get a clinically meaningful treatment effect. Further, dose expands in vivo since adoptively transferred immune cells continue to proliferate within the patient, often unpredictably.

[0081] A major risk involved in immunotherapy is the on-target but off tumor side effects resulting from T-cell activation in response to normal tissue expression of the tumor associated antigen (TAA). Clinical trials utilizing T cells expressing T-cell receptor against specific TAA reported skin rash, colitis and hearing loss in response to immunotherapy.

[0082] Immunotherapy may also produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy. The adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.

Importantly, these adverse effects may occur during the destruction of tumors, and thus even a successful on-tumor immunotherapy might result in toxicity. Approaches to regulatably control immunotherapy are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.

[0083] The present invention provides systems, compositions, immunotherapeutic agents and methods for cancer immunotherapy. These compositions provide tunable regulation of gene expression and function in immunotherapy. The present invention also provides biocircuit systems, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing. In one aspect, the systems, compositions, immunotherapeutic agents and other components of the invention can be controlled by a separately added stimulus, which provides a significant flexibility to regulate cancer immunotherapy. Further, the systems, compositions and the methods of the present invention may also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies.

[0084] The tunable nature of the systems and compositions of the invention has the potential to improve the potency and duration of the efficacy of immunotherapies. Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present invention allows maximizing the potential of cell therapy without irretrievably killing and terminating the therapy.

[0085] The present invention provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.

II. COMPOSITIONS OF THE INVENTION

[0086] According to the present invention, biocircuit systems are provided which comprise, at their core, at least one effector module system. Such effector module systems comprise at least one effector module having associated, or integral therewith, one or more stimulus response elements (SREs). The overall architecture of a biocircuit system of the invention is illustrated in Figure 1. In general, a stimulus response element (SRE) may be operably linked to a payload construct which could be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form an effector module. The SRE, when activated by a particular stimulus, e.g., a small molecule, can produce a signal or outcome, to regulate transcription and/or protein levels of the linked payload either up or down by perpetuating a stabilizing signal or destabilizing signal, or any other types of regulation. A much-detailed description of a biocircuit system are taught in co-owned U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016, 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587 (the contents each of which are herein incorporated by reference in their entirety). In accordance with the present invention, biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided.

[0087] As used herein, a ^c¾iocircuit" or '¾iocircuit system" is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system. Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral. It is also understood that biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present invention and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits. The artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts.

[0088] In accordance with the present invention, a biocircuit system may be a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems.

Effector modules and SREs for immunotherapy

[0089] In accordance with the present invention, biocircuit systems, effector modules, SREs, and components that tune expression levels and activities of any agents used for immunotherapy are provided. As non-limiting examples, an immune-therapeutic agent may be an antibody and fragments and variants thereof, a cancer specific T cell receptor (TCR) and variants thereof, an anti-tumor specific chimeric antigen receptor (CAR), a chimeric switch receptor, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a suicide gene, a homing receptor, or any agent that induces an immune response in a cell and a subject. [0090] As stated, the biocircuits of the invention include at least one effector module as a component of an effector module system. As used herein, an "effector module" is a single or multi-component construct or complex comprising at least (a) one or more stimulus response elements (i.e. proteins of interest (POIs). As used herein a "stimulus response element (SRE)" is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the "responsive" nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, the response of any SRE to a stimulus may be a matter of degree or kind. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1% and 100% or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.

[0091] In some embodiments, the present invention provides methods for modulating protein expression, function or level. In some aspects, the modulation of protein expression, function or level refers to modulation of expression, function or level by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 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%, 40-50%, 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%.

[0092] In some embodiments, the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabilization ratio. As used herein, the stabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in response to the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus specific to the SRE. In some aspects, the stabilization ratio is at least 1, such as by 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, 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, the destabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the effector module to the expression, function or level of the protein of interest, that is expressed constitutively and in the absence of the stimulus specific to the SRE. As used herein "constitutively" refers to the expression, function or level of a protein of interest that is not linked to an SRE, and is therefore expressed both in the presence and absence of the stimulus. In some aspects, the destabilization ratio is at least 0, such as by 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, 0.2-0.5, 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.

[0093] In some embodiments, the stimulus of the present invention maybe ultrasound stimulation. In some embodiments, the SREs of the present invention may derived from mechanosensitive proteins. In one embodiment, the SRE of the present invention may be the mechanically sensitive ion channel, Piezol.

[0094] Expression of the payload of interest in such instances is tuned by providing focused ultrasound stimulation. In other embodiments, the SREs of the present invention may be derived from calcium biosensors, and the stimulus of the present invention may calcium. The calcium may be generated by the ultrasound induced mechanical stimulation of mechanosensitive ion channels. The ultrasound activation of the ion channel causes a calcium influx thereby generating the stimulus. In one embodiment, the mechanosensitive ion channel is Piezo 1.

Mechanosensors may be advantageous to use since they provide spatial control to a specific location in the body.

[0095] The SRE of the effector module may be selected from, but is not limited to, a peptide, peptide complex, peptide-protein complex, protein, fusion protein, protein complex, protein- protein complex. The SRE may comprise one or more regions derived from any natural or mutated protein, or antibody. In this aspect, the SRE is an element, when responding to a stimulus, can tune intracellular localization, intramolecular activation, and/or degradation of payloads.

[0096] In some embodiments, effector modules of the present invention may comprise additional features that facilitate the expression and regulation of the effector module, such as one or more signal sequences (SSs), one or more cleavage and/or processing sites, one or more targeting and/or penetrating peptides, one or more tags, and/or one or more linkers. Additionally, effector modules of the present invention may further comprise other regulatory moieties such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA targeting sites. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, an SRE may represent a destabilizing domain, while mutations in the protein payload may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits may be engineered to contain one, two, three, four or more tuned features.

[0097] In some embodiments, effector modules of the present invention may include one or more degrons to tune expression. As used herein, a "degron" refers to a minimal sequence within a protein that is sufficient for the recognition and the degradation by the proteolytic system. An important property of degrons is that they are transferrable, that is, appending a degron to a sequence confers degradation upon the sequence. In some embodiments, the degron may be appended to the destabilizing domains, the payload or both. Incorporation of the degron within the effector module of the invention, confers additional protein instabilityto the effector module and may be used to minimize basal expression. In some embodiments, the degron may be an N- degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron. As a non-limiting example, the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J et al. (2008). Biochem J. 2008 Mar l;410(2):401-7; the contents of which are incorporated by reference in their entirety). Other examples of degrons useful in the present invention include degrons described in International patent publication Nos. WO2017004022, WO2016210343, and WO2011062962; the contents of each of which are incorporated by reference in their entirety.

[0098] As shown in Figure 2, representative effector module embodiments comprising one payload, i.e. one immunotherapeutic agent are illustrated. Each components of the effector module may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.

[0099] Figures 3 to 6 illustrate representative effector module embodiments comprising two payloads, i.e. two immunotherapeutic agents. In some aspects, more than two

immunotherapeutic agents (payloads) may be included in the effector module under the regulation of the same SRE (e.g., the same DD). The two or more agents may be either directly linked to each other or separated (Figure 3). The SRE may be positioned at the N-terminus of the construct, or the C -terminus of the construct, or in the internal location. [00100] In some aspects, the two or more immunotherapeutic agents may be as represented in given in Figures 7-12.

1. Destabilizing domains (DDs)

[00101] In some embodiments, biocircuit systems, effector modules, and compositions of the present invention relate to post-translational regulation of protein (payload) function, in particular, anti-tumor immune responses of immunotherapeutic agents. In one embodiment, the SRE is a stabilizing/destabilizing domain (DD). The presence, absence or an amount of a small molecule ligand that binds to or interacts with the DD, can, upon such binding or interaction modulate the stability of the payload(s) and consequently the function of the payload. Depending on the degree of binding and or interaction the altered function of the payload may vary, hence providing a ''tuning" of the payload function.

[00102] In some embodiments, destabilizing domains described herein or known in the art may be used as SREs in the biocircuit systems of the present invention in association with any of the immunotherapeutic agents (payloads) taught herein. Destabilizing domains (DDs) are small protein domains that can be appended to a target protein of interest. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell (Stankunas, K., et al., Mol. Cell, 2003, 12: 1615-1624; Banaszynski, et al, Cell; 2006, 126(5): 995-1004; reviewed in Banaszynski, L.A., and Wandless, T.J. Chem. Biol.; 2006, 13:11-21 and Rakhit R et al, Chem Biol. 2014; 21(9): 1238-1252). However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates.

[00103] In some embodiments, the desired characteristics of the DDs may include, but are not limited to, low protein levels in the absence of a ligand of the DD (i.e. low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation. DDs that bind to a desired ligand but not endogenous molecules may be preferred.

[00104] Several protein domains with destabilizing properties and their paired small molecules have been identified and used to control protein expression, including FKBP/shield-1 system (Egeler et al., J Biol. Chem. 2011, 286(36): 32328-31336; the contents of which are incorporated herein by reference in their entirety), ecDHFR and its ligand trimethoprim (TMP); estrogen receptor domains which can be regulated by several estrogen receptor antagonists (Miyazaki et al, J Am Chem. Soc, 2012, 134(9): 3942-3945; the contents of which are incorporated by reference herein in their entirety); and fluorescent destabilizing domain (FDD) derived from bilirubin-inducible fluorescent protein, UnaG and its cognate ligand bilirubin (BR) ( Navarro et al., ACS Chem Biol., 2016, June 6; the contents of which are incorporated herein by reference in their entirety).

[00105] Known DDs also include those described in U.S. Pat. NO. 8,173,792 and U.S. Pat. NO. 8,530,636, the contents of which are each incorporated herein by reference in their entirety.

[00106] In some embodiments, the DDs of the present invention may be derived from some known sequences that have been approved to be capable of post-translational regulation of proteins. For example, Xiong et al ., have demonstrated that the non-catalytic N-terminal domain (54-residues) of ACS7 (1-aminocyclopropane-l-carboxylate synthase) in Arabidopsis, when fused to the β-glucuromdase (GUS) reporter, can significantly decrease the accumulation of the GUS fusion protein (Xiong et al., J. Exp. Bot, 2014, 65(15): 4397-4408). Xiong et al. further demonstrated that both exogenous 1 -aminocyclopropane- 1 -carboxylic acid (ACQ treatment and salt can rescue the levels of accumulation of the ACS N-terminal and GUS fusion protein. The ACS N-terminus mediates the regulation of ACS7 stability through the ubiquitin-26S proteasome pathway.

[00107] Another non-limiting example is the stability control region (SCR, residues 97-118) of Tropomyosin (Tm), which controls protein stability. A destabilizing mutation LI 10A, and a stabilizing mutation A109L dramatically affect Tropomyosin protein dynamics (Kirwan and Hodges, J. Biol. Chem., 2014, 289: 4356-4366). Such sequences can be screened for ligands that bind them and regulate their stability. The identified sequence and ligand pairs may be used as components of the present invention.

[00108] In some embodiments, the DDs of the present invention may be developed from known proteins. Regions or portions or domains of wild type proteins may be utilized as SREs/DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs/DDs or the starting point for the design of further SREs and/or DDs.

[00109] Ligands such as small molecules that are well known to bind candidate proteins can be tested for their regulation in protein responses. The small molecules may be clinically approved to be safe and have appropriate pharmaceutical kinetics and distribution. In some embodiments, the stimulus is a ligand of a destabilizing domain (DD), for example, a small molecule that binds a destabilizing domain and stabilizes the POI fused to the destabilizing domain. In some embodiments, ligands, DDs and SREs of the present invention, include without limitation, any of those taught in Tables 2-4 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety. Some examples of the proteins that may be used to develop DDs and their ligands are listed in Table 1.

[00110] In some embodiments, DDs of the invention may be FKBP DD or ecDHFR DDs such as those listed in Table 2. In some embodiments, binding ligand of FKBP DD may be

Aquashield, which has considerably improved solubility in aqueous medium compared to Shield- 1, but retains all the binding properties of Shield- 1. The position of the mutated amino acid listed in Table 2 is relative to the ecDHFR (Uniprot ID: P0ABQ4) of SEQ ID NO. 1 for ecDHFR DDs and relative to FKBP (Uniprot ID: P62942) of SEQ ID NO. 3 for FKBP DDs.

Table 2: ecDHFR DDs and FKBP DDs

[00111] Inventors of the present invention have tested and identified several candidate human proteins that may be used to develop destabilizing domains. As show in Table 2, these candidates include human DHFR (hDHFR), PDE5 (phosphodiesterase 5), PPAR gamma (peroxisome proliferator-activated receptor gamma), CA2 (Carbonic anhydrase II) and NQ02 (NRH:

Quinone oxidoreductase 2). Candidate destabilizing domain sequence identified from protein domains of these proteins (as a template) may be mutated to generate libraries of mutants based on the template candidate domain sequence. Mutagenesis strategies used to generate DD libraries may include site-directed mutagenesis e.g. by using structure guided information; or random mutagenesis e.g. using error-prone PCR, or a combination of both. In some embodiments, destabilizing domains identified using random mutagenesis may be used to identify structural properties of the candidate DDs that may be required for destabilization, which may then be used to further generate libraries of mutations using site directed mutagenesis.

[00112] In some embodiments, novel DDs derived from E.coli DHFR (ecDHFR) may comprise amino acids 2-159 of the wild type ecDHFR sequence. This may be referred to as an Mldel mutation.

[00113] In some embodiments, novel DDs derived from ecDHFR may comprise amino acids 2- 159 of the wild type ecDHFR sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, R12Y, R12H, Y 1001, and E129K.

[00114] In some embodiments, novel DDs derived from FKBP may comprise amino acids 2- 107 of the wild type FKBP sequence. This may be referred to as an Mldel mutation.

[00115] In some embodiments, novel DDs derived from FKBP may comprise amino acids 2- 107 of the wild type FBKP sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, E31G, F36V, R71G, K105E, and L106P.

[00116] In some embodiments, DD mutant libraries may be screened for mutations with altered, preferably higher binding affinity to the ligand, as compared to the wild type protein. DD libraries may also be screened using two or more ligands and DD mutations that are stabilized by some ligands but not others may be preferentially selected. DD mutations that bind preferentially to the ligand compared to a naturally occurring protein may also be selected. Such methods may be used to optimize ligand selection and ligand binding affinity of the DD. Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.

[00117] In some embodiments, suitable DDs may be identified by screening mutant libraries using barcodes. Such methods may be used to detect, identify and quantify individual mutant clones within the heterogeneous mutant library. Each DD mutant within the library may have distinct barcode sequences (with respect to each other). In other instances, the polynucleotides can also have different barcode sequences with respect to 2,3,4,5,6,7,8,9,10 or more nucleic acid bases. Each DD mutant within the library may also comprise a plurality of barcode sequences. When used in plurality may be used such that each barcode is unique to any other barcode. Alternatively, each barcode used may not be unique, but the combination of barcodes used may create a unique sequence that can be individually tracked. The barcode sequence may be placed upstream of the SRE, downstream of the SRE, or in some instances may be placed within the SRE. DD mutants may be identified by barcodes using sequencing approaches such as Sanger sequencing, and next generation sequencing, but also by polymerase chain reaction and quantitative polymerase chain reaction. In some embodiments, polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel. In other instances, each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.

[00118] In some embodiments, DDs of the invention may be derived from human dihydrofolate reductase (hDHFR). hDHFR is a small (18 kDa) enzyme that catalyzes the reduction of dihydrofolate and plays a vital role in variety of anabolic pathway. Dihydrofolate reductase (DHFR) is an essential enzyme that converts 7,8-dihydrofolate (DHF) to 5,6,7,8, tetrahydrofolate (THLF) in the presence of nicotinamide adenine dihydrogen phosphate (NADPH). Anti-fblate drugs such as methotrexate (MTX), a structural analogue of folic acid, which bind to DHFR more strongly than the natural substrate DHF, interferes with folate metabolism, mainly by inhibition of dihydrofolate reductase, resulting in the suppression of purine and pyrimidine precursor synthesis. Other inhibitors of hDHFR such as folate, TQD, Trimethoprim (TMP), epigallocatechin gallate (EGCG) and ECG (epicatechin gallate) can also bind to hDHFR mutants and regulates its stability.In one aspect of the invention, the DDs of the invention may be hDHFR mutants including the single mutation hDHFR (Y122I), hDHFR (K81R), hDHFR (F59S), hDHFR (117V), hDHFR (N65D), hDHFR (A107V), hDHFR (N127Y), hDHFR

(K185E), hDHFR (N186D), and hDHFR (M140I); double mutations: hDHFR (M53T, R138I), hDHFR (V75F, Y122I), hDHFR (A125F, Y122I), hDHFR (L74N, Y122I), hDHFR (L94A, T147A), hDHFR (G21T, Y122I), hDHFR (V121A, Y1221), hDHFR (Q36K, Y122I), hDHFR (C7R, Y163C)-hDHFR (Y178H, E18IG), hDHFR (A10V, H88Y), hDHFR (T137R, F143L), hDHFR (E63G, I176F), hDHFR (T57A, I72A), hDHFR (H131R, E144G), and hDHFR (Y183H, K185E); and triple mutations: hDHFR (Q36F, N65F, Y122I), hDHFR (G21E, I72V, I176T), hDHFR (I8V, K133E, Y163C), hDHFR (V9A, S93R, P150L), hDHFR (K19E, F89L, E181G), hDHFR (G54R, M140V, S168C), hDHFR (L23S, V121A, Y157C), hDHFR (VI 10A, V136M, K177R), and hDHFR (N49D, F59S, D153G).

[00119] In one embodiment, the stimulus is a small molecule that binds to a SRE to post- translationally regulate protein levels. In one aspect, DHFR ligands: trimethoprim (TMP) and methotrexate (MTX) are used to stabilize hDHFR mutants. The hDHFR based destabilizing domains are listed in Table 3. The position of the mutated amino acid listed in Table 3 is relative to the human DHFR (Uniprot ID: P00374) of SEQ ID NO. 2 for human DHFR. In Table 3,

[00120] In some embodiments, DD mutations that do not inhibit ligand binding may be preferentially selected. In some embodiments, ligand binding may be improved by mutation of residues in DHFR. Amino acid positions selected for mutation include aspartic acid at position 22 of SEQ ID NO. 2, glutamic acid at position 31 of SEQ ID NO. 2; phenyl alanine at position 32 of SEQ ID NO. 2; arginine at position 33 of SEQ ID NO. 2; glutamine at position 36 of SEQ ID NO. 2; asparagine at position 65 of SEQ ID NO. 2; and valine at position 115 of SEQ ID NO. 2. In some embodiments, one or more of the following mutations may be utilized in the DDs of the present invention to improve TMP binding, including but not limited to, D22S, E3 ID, F32M, R33S, Q36S, N65S, and VI 161. The position of the mutated amino acids is relative to the wildtype human DHFR (Uniprot ID: P00374) of SEQ ID NO. 2.

[00121] In some embodiments, novel DDs derived from human DHFR may include one, two, three, four, five or more mutations including, but not limited to, Mldel, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, NHS, G16S, I17N, I17V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, I61T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R, II 15V, I115L, VI 161, Gl 17D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

[00122] In some embodiments, novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence. This may be referred to as an Mldel mutation.

[00123] In some embodiments, novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, 16 IT, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, VI 10A, Dl 1 IN, Ml 12T, Ml 12V, VI 13A, Wl 14R, II 15V, I115L, VI 161, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138I, I139T, 1139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

2, PaylQads; Immunofterapeutic agents

[00124] In some embodiments, payloads of the present invention may be immunotherapeutic agents that induce immune responses in an organism. The immunotherapeutic agent may be a cytokine, chemokine, a cytokine receptor, a chemokine receptor, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell, or in a subject.

[00125] In some embodiments, ligands that do not affect the activity of the immune cell, and/or the chimeric antigen receptor, in the absence of the SREs may be preferably selected.

[00126] In some embodiments, the IL12 levels secreted by the immune cells of the invention may approximately be comparable to the IL12 levels secreted by human myeloid dendritic cells (mDCl), when activated with TLR agonists. In one embodiment, the TLR agonist may be the combination of lipopolysaccharide administered with R848.

[00127] In some embodiments, the IFN gamma secreted by IL12 induced activation of the immune cells is at least 5 fold greater in the presence of ligand, compared to the levels in the absence of ligand.

[00128] In some embodiments, the IFN gamma secreted by IL15 induced activation of the immune cells is at least 10-fold greater in the presence of ligand, compared to the levels in the absence of ligand.

[00129] In some embodiments, regulation of IL12 provides the necessary safety switch. In some embodiments, IL12 secretion recruit and/or activates effector cells in the tumor

microenvironment. In some embodiments, the IL 12 regulation provides a benefit to CAR T function without causing toxicity.

[00130] In some embodiments, regulation of IL15-IL15Ra fusion proteins provides a safety switch as compared to constitutively expressed IL15-IL15Ra. In some embodiments, IL15- IL15Ra leads to better expansion, and/or persistence of CAR T cells.

Cytokines, chemokines and other soluble factors

[00131] In accordance with the present invention, payloads of the present invention may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical communicators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

[00132] In some embodiments, payloads of the present invention may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGF beta and chemokines. It is understood in the art that certain gene and/or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash "-" or symbolic such as Greek letters.

Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL2, IL2 and IL 2 refer to the same interleukin. Likewise, TNFalpha, TNFot, TNF-alpha, TNF-a, TNF alpha and TNF a all refer to the same protein. In some embodiments, payloads of the present invention may be cytokines that stimulate immune responses. In other embodiments, payloads of the invention may be antagonists of cytokines that negatively impact anti-cancer immune responses.

[00133] In one embodiment, the payloads of the present invention may be cytokines fused to TNF alpha ectodomain. Such payloads are produced as membrane associated cytokines fused to the TNF ectodomain. In one embodiment, the cytokine may be shed from the cell surface by the action of membrane associated proteases, and/or proteases in the extracellular space e.g. MMP9. Any of the cytokines described herein may be useful in the present invention. Such cytokine- TNF scaffold constructs may be used to preserve the native sequence of the processed cytokine while preserving regulation.

[00134] In some embodiments, payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines.

[00135] In some embodiments, cytokines of the present invention may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+TEM, natural killer cells and tumor infiltrating lymphocytes (TIL) cells used for immunotherapy. In other embodiments, T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, the present invention provides biocircuits and compositions to minimize toxicity related to cytokine therapy. Despite its success in mitigating tumor burden, systemic cytokine therapy often results in the development of severe dose limiting side effects. Two factors contribute to the observed toxicity (a) Pleiotropism, wherein cytokines affect different cells types and sometimes produce opposing effects on the same cells depending on the context (b) Cytokines have short serum half-life and thus need to be administered at high doses to achieve therapeutic effects, which exacerbates the pleiotropic effects. In one aspect, cytokines of the present invention may be utilized to modulate cytokine expression in the event of adverse effects. In some embodiments, cytokines of the present invention may be designed to have prolonged life span or enhanced specificity to minimize toxicity.

[00136] In some embodiments, the payload of the present invention may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein. In some aspects, the inteiieukin payload is selected from IL1, IL1 alpha (also called hematopoietin-1), ILlbeta (catabolin), IL1 delta, ILlepsilon, ILleta, IL1 zeta, interleukin-1 family member 1 to 11 (IL1F1 to IL1F11), interleukin-1 homolog 1 to 4 (IL1H1 to IL1H4), IL1 related protein 1 to 3 (IL1RP1 to IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL11, ILlla, ILllb, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, Π17Β, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20 like (IL20L), 1121, IL22, IL23, IL23A, IL23-pl9, IL23-p40, IL24, Π25, IL26, IL27, IL28A, IL28B, IL29, IL30, JL31, IL32, IL33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma, IL36RN, IL37, IL37a, IL37b, IL37c, IL37d, IL37e and IL38. In other aspects, the payload of the present invention may be an interleukin receptor selected from CD121a, CDwl21b, IL2Ro/CD25, IL2RpVCD122, IL2Ry/CD132, CDwl31, CD124, CD131, CDwl25, CD126, CD130, CD127, CDw210, IL8RA, ILllRo, CD212, CD213al, CD213a2, IL14R, IL15Ra, CDw217, IL18Ra, IL18Rp\ IL20Ra, and IL20Rp\

[00137] In one embodiment, the payload of the invention may comprise IL12. IL12 is a heterodimeric protein of two subunits (p3S, p40) that is secreted by antigen presenting cells, such as macrophages and dendritic cells. Expression of IL12 requires the simultaneous expression of the two subunits to produce a biologically active heterodimer. In some embodiments, payloads of the invention may be p35 subunit or the p40 subunit. IL12 is type 1 cytokine that acts on natural killer (NK) cells, macrophages, CD8⁺ Cytotoxic T cells, and CD4⁺ T helper cells through STAT4 pathway to induce IFN-γ production in these effector immune cells (reviewed by Trinchieri G, Nat Rev Immunol. 2003; 3(2): 133-146). IL12 can promote the cytotoxic activity of NK cells and CD8⁺ T cells, therefore has anti-tumor function as well as promote T cell persistence in vivo. Intravenous injection of recombinant IL12 exhibited modest clinical efficacy in a handful of patients with advanced melanoma and renal cell carcinoma (Gollob et al., Clin. Cancer Res. 2000; 6(5): 1678-1692). IL12 has been used as an adjuvant to enhance cytotoxic immunity using a melanoma antigen vaccine, or using peptide pulsed peripheral blood mononuclear cells; and to promote NK cell activity in breast cancer with trastuzumab treatment. Local delivery of IL12 to the tumor microenvironment promotes tumor regression in several tumor models. These studies all indicate that locally increased IL12 level can promote antitumor immunity. One major obstacle of systemic or local administration of recombinant IL12 protein, or through oncolytic viral vectors is the severe side effects when IL12 is presented at high level. Developing a system that tightly controls IL12 level may provide a safe use of IL12 in cancer treatment. A regulatable IL12 composition may also prevent negative feedback loops, thereby enhancing T cell effector functions. [00138] In one aspect, the effector module of the invention may be a DD-IL12 fusion polypeptide. This regulatable DD-IL12 fusion polypeptide may be directly used as an immunotherapeutic agent or be transduced into an immune effector cell (T cells and TIL cells) to generate modified T cells with greater in vivo expansion and survival capabilities for adoptive cell transfer. The need for harsh preconditioning regimens in current adoptive cell therapies may be minimized using regulated IL12 DD-IL12 may be utilized to modify tumor microenvironment and increase persistence in solid tumors that are currently refractory to tumor antigen targeted therapy. In some embodiments, CAR expressing T cells may be armored with DD regulated IL12 to relieve immunosuppression without systemic toxicity. In some embodiments, the payloads of the present invention may be used to enhance cell therapies with performance optimized for challenging tumor microenvironments.

[00139] In some embodiments, the IL12 may be a Flexi IL12, wherein both p35 and p40 subunits, are encoded by a single cDNA that produces a single chain polypeptide. The single chain polypeptide may be generated by placing p35 subunit at the N terminus or the c terminus of the single chain polypeptide. Similarly, the p40 subunit may be at the N terminus or C terminus of the single chain polypeptide. In some embodiments, the IL12 constructs of the invention may be placed under the transcriptional control of the CMV promoter (SEQ ID NO. 49), an EFla promoter (SEQ ID NO. 50, and SEQ ID NO. 312) or a PGK promoter (SEQ ID NO. 51). Any portion of IL12 that retains one or more functions of full length or mature IL12 may be useful in the present invention. In some aspects, the DD-IL12 comprises the amino acid sequences listed in Table 4A. The components signal sequences, linker, cleavage sites, payload and destabilizing domains may be assembled in any order to design constructs with optimal features. In some embodiments, such optimal features may include low to virtually no basal expression in the absence of the ligand and increased expression in the presence of ligand. In Table 4A, the amino acid sequences may comprise a stop codon at the end which is denoted in the table with a "*".

Table 4A: DD-IL12 constructs

[00140] In some embodiments, DD regulated IL12 compositions of the invention may be utilized to minimize the cytotoxicities associated with systemic IL12 administration. Treatment with IL12 has been associated with systemic flu-like symptoms (fever, chills, fatigue, arthromyalgia, headache), toxic effects on the bone marrow, and liver. Hematologic toxicity observed most commonly included neutropenia and thrombocytopenia; hepatic dysfunction manifested in transient (dose dependent) increase in transaminases, hyperbilirubinemia and hypoalbuminemia. In some instances, toxicity is also associated with inflammation of the mucus membranes (oral mucositis, stomatitis or colitis). These toxic effects of IL12 were related to the secondary production of IFNgamma, TNFalpha, and chemokines such as IP 10, and MIG. In certain aspects of the invention, DD regulated IL12 may be utilized to prevent the toxic effects associated with elevated production of secondary messengers.

[00141] The format of the IL12 constructs utilized as payload of the present invention may be optimized. In one embodiment, the payload of the invention may be a bicistronic IL12 containing p40 and p35 subunits separated by an internal ribosome entry site or a cleavage site such as P2A or Furin to allow independent expression of both subunits from a single vector. This results in a configuration of secreted IL12 that is more akin to the naturally occurring 1L12 than the flexi IL12 construct, the payload of the invention may be the p40 subunit of the IL12. DD regulated p40 may be co-expressed with constitutive p35 construct to generate "regulatable IL12" expression. Alternatively, the DD regulated p40 may heterodimerize with the endogenous p35. p40 has been shown to stabilize p35 expression and stimulate the export of p35 (Jalah R, et al. (2013). Journal of Biol. Chem. 288, 6763-6776 (the contents of which are incorporated by reference in its entirety).

[00142] In some embodiments, modified forms of IL12 may be utilized as the payload. These modified forms of IL12 may be engineered to have shortened half-life in vivo compared to the non-modified form of especially when used in combination with tunable systems described herein.

[00143] Human flexi IL12 has a reported half-life of 5-19 hours which, when administered as a therapeutic compound, can result in systemic cytotoxicity (Car et al. (1999) The Toxicology of Interleukin-12: A Review" Toxicologic Path.27 A, 58-63; Robertson et al. (1999)

"Immunological Effects of Interleukin 12 Administered by Bolus Intravenous Injection to Patients with Cancer" Clin. Cancer Res. 5:9-16; Atkins et al. (1997)"Phase I Evaluation of Intravenous Recombinant Human Interleukin 12 in Patients with Advance Malignancies" Clin. Cancer Res. 3:409-417). The ligand inducible control of IL12 can regulate production in a dose dependent fashion, the time from cessation of ligand dosing to cessation of protein synthesis and IL12 clearance may be insufficient to prevent toxic accumulation of IL 12 in plasma.

[00144] In one embodiment, the modified form of IL12 utilized as the payload may be a Topo- sc IL12 which have the configuration as follows from N to C terminus (i) a first IL12 p40 domain (p40N), (ii) an optional first peptide linker, (iii) an IL12 p35 domain, (iv) an optional second peptide linker, and (v) a second IL12 p40 domain (p40C). In one embodiment, modified topo sc IL12 polypeptides exhibit increased susceptibility to proteolysis. Topo-sc IL12 is described in International Patent Publication No. WO2016048903; the contents of which are incorporated herein by reference in its entirety.

[00145] 1L12 polypeptide may also be modified (e.g. genetically, synthetically, or recombinantly engineered) to increase susceptibility to proteinases to reduce the biologically active half-life of the IL12 complex, compared to a corresponding IL12 lacking proteinases susceptibility. Proteinase susceptible forms of IL12 are described in International Patent Publication No. WO20170629S3; the contents of which are incorporated by reference in its entirety.

[00146] IL12 systemic toxicity may also be limited or tightly controlled via mechanisms involving tethering IL12 to the cell surface to limit its therapeutic efficacy to the tumor site. Membrane tethered IL12 forms have been described previously using Glycosyl

phosphatidylinositol (GPI) signal peptide or using CD80 transmembrane domain (Nagarajan S, et al. (2011) JBiomedMater Res A. 99(3):410-7; Bozeman EN, et al. (2013) Vaccine.

7;31(20):2449-56; Wen-Yu Pan et al. (2012), Mol. Ther.20:5, 927-937; the contents of each of which are incorporated by reference in their entirety). In some embodiments, transmembrane domains may be selected from any of those described in Table 4B.

Table 4B: Transmembrane domains

[00147] In one embodiment, the payload of the invention may comprise IL15. Interleukin 15 is a potent immune stimulatoiy cytokine and an essential survival factor for T cells, and Natural Killer cells. Preclinical studies comparing IL2 and IL15, have shown than IL15 is associated with less toxicity than IL2. In some embodiments, the effector module of the invention may be a DD-IL15 fusion polypeptide. IL1S polypeptide may also be modified to increase its binding affinity for the IL15 receptor. For example, the asparagine may be replaced by aspartic acid at position 72 of IL15 (SEQ ID NO. 2 of US patent publication US20140134128A1; the contents of which are incorporated by reference in their entirety). In some embodiments, the IL1S constructs of the invention may be placed under the transcriptional control of the CMV promoter (SEQ ID NO. 49), an EFla promoter (SEQ ID NO. 50, and SEQ ID NO. 312) or a PGK promoter (SEQ ID NO. 51). Any portion of IL15 that retains one or more functions of full length or mature IL15 may be useful in the present invention. Such functions include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells. In some aspects, the DD-IL15 comprises the amino acid sequences listed in Table 5. The amino acid sequences in Table 5 may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

Table 5: DD IL15 constructs

[00148] A unique feature of IL 15 mediated activation is the mechanism of trans-presentation in which IL15 is presented as a complex with the alpha subunit of IL15 receptor (IL15Ra) that binds to and activates membrane bound IL15 beta/gamma receptor, either on the same cell or a different cell. The IL15/IL15Ra complex is more effective in activating IL 15 signaling, than IL15 by itself. Thus, in some embodiments, the effector module of the invention may include a DD-IL15/IL15Ra fusion polypeptide. In one embodiment, the payload may be IL15/IL15Ra fusion polypeptide described in US Patent Publication NO. US20160158285A1 (the contents of which are incorporated herein by reference in their entirety). The IL15 receptor alpha comprises an extracellular domain called the sushi domain which contains most of the structural elements necessary for binding to IL15. Thus, in some embodiments, payload may be the IL15/IL15Ra sushi domain fusion polypeptide described in US Patent Publication NO. US20090238791A1 (the contents of which are incorporated herein by reference in their entirety).

[00149] Regulated IL15/IL15Ra may be used to promote expansion, survival and potency of CD8TEM cell populations without impacting regulatory T cells, NK cells and TIL cells. In one embodiment, DD-IL15/lL15Ra may be utilized to enhance CD 19 directed T cell therapies in B cell leukemia and lymphomas. In one aspect, IL15/IL15Ra may be used as payload of the invention to reduce the need for pre-conditioning regimens in current CAR-T treatment paradigms.

[00150] The effector modules containing DD-IL15, DD-IL15/IL15Ra and/or DD-IL15/IL15Ra sushi domain may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).

[00151] In some aspects, the DD-IL15/IL15Ra comprises the amino acid sequences provided in Table 6a with any combination of components in any order. ILlSRa may be fused to DD by the amino acid sequence SG. Examples of DD-IL15/IL15Ra are provided in Table 6b and Table 6c. In some aspects, the DD-IL115/IL15Ra comprises the amino acid sequences provided in Table 6a, 6b, and 6c. The amino acid sequences in Tables 6a, 6b and 6c may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

Table 6a: DD-IL 1 S/IL 1 SRa construct sequences

[00152] In one embodiment, the payload of the present invention may comprise IL18. IL18 is a proinflammatory and immune regulatory cytokine that promotes IFN-y production by T and NK cells. IL18 belongs to the IL1 family. Secreted IL18 binds to a heterodimer receptor complex, consisting of 1L18R« and ^-chains and initiates signal transduction. IL18 acts in concert with other cytokines to modulate immune system functions, including induction of IFN-y production, Thl responses, and NK cell activation in response to pathogen products. IL18 showed anti- cancer effects in several tumors. Administration of recombinant IL18 protein or IL18 transgene induces melanoma or sarcoma regression through the activation of CD4* T and/or NK cell- mediated responses (reviewed by Srivastava et al., Curr. Med. Chem., 2010, 17: 3353-3357). Hie combination of IL18 with other cytokines, such as IL12 or co-stimulatory molecules (e.g., CD80) increases IL18 anti-tumor effects. For example, IL18 and IL12A/B or CD80 genes have been integrated successfully in the genome of oncolytic viruses, with the aim to trigger synergistically T cell-mediated anti-tumor immune responses (Choi et al., Gene Ther., 2011, 18: 898-909). IL2/IL18 fusion proteins also display enhanced anti-tumor properties relative to either cytokine alone and low toxicity in preclinical models (Acres et al., Cancer Res., 2005, 65:9536- 9546).

[00153] 1L 18 alone, or in combination of IL12 and IL15, activates NK cells. Preclinical studies have demonstrated that adoptively transferred IL12, IL15 and IL18 pre-activated NK cells display enhanced effector function against established tumors in vivo (Ni et al., J Exp Med. 2012, 209: 2351-2365; and Romee et d., Blood. 2012,120:4751-4760). Human IL12/IL15/IL18 activated NK cells also display memory-like features and secrete more IFN-y in response to cytokines (e.g., low concentration of IL2). In one embodiment, the effector module of the present invention may be a DD-IL18 fusion polypeptide.

[00154] In one embodiment, the payload of the present invention may comprise IL21. IL21 is another pleiotropic type I cytokine that is produced mainly by T cells and natural killer T (NKT) cells. IL21 has diverse effects on a variety of cell types including but not limited to CD4⁺ and CD&* T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL21 is composed of IL21 receptor (IL21R) and the common cytokine receptor gamma chain, which is also a subunit of the receptors for IL2, IL4, IL7, IL9 and IL15. Studies provide compelling evidence that IL21 is a promising immunotherapeutic agent for cancer immunotherapy. IL21 promotes maturation, enhances cytotoxicity, and induces production of IFN-y and perforin by NK cells. These effector functions inhibit the growth of B 16 melanoma (Kasaian et al., Immunity. 2002, 16(4):559-569; and Brady et al., J Immunol.2004, 172(4):2048- 2058). IL21 together with IL15 expands antigen-specific CD8⁺ T-cell numbers and their effector function, resulting in tumor regression (Zeng et al., JExpMed.2005, 201(1): 139-148). IL21 may also be used to rejuvenate multiple immune effector cells in the tumor microenvironment. IL21 may also directly induce apoptosis in certain types of lymphoma such as diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia cells, via activation of STAT3 or STAT1 signal pathway. IL21, alone or in combination with anti-CD20 mAb

(rituximab) can activate NK cell-dependent cytotoxic effects. Interestingly, discovery of the immunosuppressive actions of IL21 suggests that this cytokine is a "double-edged sword"- IL21 stimulation may lead to either the induction or suppression of immune responses. Both stimulatory and suppressive effects of IL21 must be considered when using IL21 -related immunotherapeutic agents. The level of 1L21 needs to be tightly controlled by regulatory elements. In one aspect, the effector module of the present invention may be a DD-IL21 fusion polypeptide.

[00155] In some embodiments, payloads of the present invention may comprise type I interferons. Type I interferons (IFNs-I) are soluble proteins important for fighting viral infection in humans. IFNs-I include IFN-alpha subtypes (IFN- al, IFN- alb, IFN- ale), IFN-beta, IFN- delta subtypes (IFN-delta 1, IFN-delta 2, IFN-delta 8), IFN-gamma, IFN-kappa, and IFN- epsilon, lFN-lambda, IFN -omega, IFN-tau and IFN-zeta. IFN -a and IFN-β are the main IFN -I subtypes in immune responses. All subtypes of IFN -I signal through a unique heterodimeric receptor, interferon alpha receptor (IFNAR), composed of 2 subunits, IFNAR1 and IFNAR2. IFNR activation regulates the host response to viral infections and in adaptive immunity. Several signaling cascades can be activated by IFNR, including the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway, the mitogen activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase (PI3K) pathway, the v-crk sarcoma virus CT10 oncogene homolog (avian)-like (CRKL) pathway, and NF-tcB cascade. It has long been established that type I IFNs directly inhibit the proliferation of tumor cells and virus- infected cells, and increase MHC class I expression, enhancing antigen recognition. IFNs-I have also proven to be involved in immune system regulation. IFNs can either directly, through interferon receptor (IFNR), or indirectly by the induction of chemokines and cytokines, regulate the immune system. Type I IFNs enhance NK cell functions and promote survival of NK cells. Type I IFNs also affect monocytes, supporting the differentiation of monocytes into DC with high capacity for antigen presentation, and stimulate macrophage function and differentiation. Several studies also demonstrate that IFNs-I promote CD8* T cell survival and functions. In some instances, it may be desirable to tune the expression of Type I IFNs using biocircuits of the present invention to avoid immunosuppression caused by long-term treatment with IFNs.

[00156] New anticancer immunotherapies are being developed that use recombinant type I IFN proteins, type I IFN transgene, type I IFN-encoding vectors and type I IFN-expressing cells. For example, IFN-a has received approval for treatment of several neoplastic diseases, such as melanoma, RCC and multiple myeloma. Though type I IFNs are powerful tools to directly and indirectly modulate the functions of the immune system, side effects of systemic long-term treatments and lack of sufficiently high efficacy have dampened the interest of IFN-a for clinical use in oncology. It is believed that if IFN levels are tightly regulated at the malignant tissues, type I IFNs are likely more efficacious. Approaches for intermittent delivery are proposed according to the observation that intetmittency at an optimized pace may help to avoid signaling desensitizing mechanisms (negative feedback mechanisms) induced by IFNs-I (i.e., because of SOCS1 induction) in the responding immune cells. In accordance with the present invention, the effector module may comprise a DD-IFN fusion polypeptide. The DD and its ligand control the expression of IFN to induce an antiviral and antitumor immune responses and in the meantime, to minimize the side effects caused by long-term exposure of IFN.

[00157] In some embodiments, payloads of the present invention may comprise members of tumor necrosis factor (TNF) superfamily. The term "TNF superfamily" as used herein refers to a group of cytokines that can induce apoptosis. Members of TNF family include TNF-alpha, TNF- beta (also known as lymphotoxin-alpha (LT-a)), lymphotoxin-beta (LT-β), CD40L(CD154), CD27L (CD70), CD30L(CD153), FASL(CD178), 4-1BBL (CD137L), OX40L, TRAIL (TNF- related apoptosis inducing ligand), APRIL (a proliferation-inducing ligand), TWEAK,

TRANCE, TALL-1, GITRL, LIGHT and TNFSF1 to TNFSF20 (TNF ligand superfamily member 1 to 20). In one embodiment, the payload of the invention may be TNF-alpha. TNF- alpha can cause cytolysis of tumor cells, and induce cell proliferation differentiation as well. In one aspect, the effector module of the present invention may comprise a DD-TNF alpha fusion polypeptide.

[00158] In some embodiments, payloads of the present invention may comprise inhibitor)' molecules that block inhibitory cytokines. The inhibitors may be blocking antibodies specific to an inhibitory cytokine, and antagonists against an inhibitory cytokine, or the like.

[00159] In some aspects, payloads of the present invention may comprise an inhibitor of a secondary cytokine IL35. IL35 belongs to Ihe interleukin-12 (IL12) cytokine family, and is a heterodimer composed of the IL27 β chain Ebi3 and the IL12 a chain p35. Secretion of bioactive IL35 has been described only in forkhead box protein 3 (Foxp3) ⁺ regulatory T cells (Tregs) (resting and activated Tregs). Unlike other membranes in the family, IL35 appears to function solely in an anti-inflammatory fashion by inhibiting effector T cell proliferation and perhaps other parameters (Collison et al., Nature, 2007, 450(7169): 566-569).

[00160] In some embodiments, payloads of the present invention may comprise inhibitors that block the transforming growth factor beta (TGF-β) subtypes (TGF-βΙ , TGF^2 and TGF^3). TGF-β is secreted by many cell types, including macrophages and is often complexed with two proteins LTBP and LAP. Serum proteinases such as plasmin catalyze the release of active TGF-β from the complex from the activated macrophages. It has been shown that an increase in expression of TGF-β correlates with the malignancy of many cancers. The immunosuppressive activity of TGF-β in the tumor microenvi ronment contributes to oncogenesis.

[00161] In some embodiments, payloads of the present invention may comprise inhibitors of IDO enzyme.

[00162] In some embodiments, payloads of the present invention may comprise chemokines and chemokine receptors. Chemokines are a family of secreted small cytokines, or signaling proteins that can induce directed chemotaxis in nearby responsive cells. The chemokine may be a SCY (small cytokine) selected from the group consisting of SCYAl-28 (CCLl-28), SCYBl-16 (CXCLl-16), SCYCl-2 (XCLl-2), SCYD-1 and SCYE-1; or a C chemokine selected from XCL1 and XCL2; or a CC chemokine selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27 and CCL28; or a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17; or a CX3C chemokine CX3CL1. In some aspects, the chemokine receptor may be a receptor for the C chemokines including XCR1; or a receptor for the CC chemokines including CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10; or a receptor for the CXC chemokines including CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5; or a CX3C chemokine receptor CX3CR1.

[00163] In some embodiments, payloads of the present invention may comprise other immunomodulators that play a critical role in immunotherapy, such as GM-CSF (Granulocyte- macrophage colony stimulating factor), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-l, intracellular adhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF), Interleukin-1 receptor activating kinase (iRAK-1), lactotransferrin, and granulocyte colony stimulating factor (G-CSF).

[00164] In some embodiments, the payload of the present invention may comprise

Amphiregulin. Amphiregulin (AREG) is an EGF-like growth factor which binds to the EGFR receptor and enhances CD4+ regulatory T cells (Tregs) function. AREG promotes immune suppression in the tumor environment. Thus, in some embodiment, the payloads of the present invention may comprise Amhiregulin to dampen immune response during immunotherapy'.

[00165] In some embodiments, payloads of the present invention may comprise fusion proteins wherein a cytokine, chemokine and/or other soluble factor may be fused to other biological molecules such as antibodies and or ligands for a receptor. Such fusion molecules may increase the half-life of the cytokines, reduce systemic toxicity, and increase local concentration of the cytokines at the tumor site. Fusion proteins containing two or more cytokines, chemokines and or other soluble factors may be utilized to obtain synergistic therapeutic benefits. In one embodiment, payload may be a GM-CSML2 fusion protein.

[00166] In some embodiments, the present invention provides methods for tuning the expression and function of an immune-therapeutic agent by operably linking it to an SRE within the effector module. Tuning of the immune-therapeutic agent may be invitro in cells or in vivo in a subject. In one embodiment, the immunotherapeutic agent is IL12. In some embodiments, the SRE is a DD. The immunotherapeutic agent may be stabilized by the stabilization ratio of between 1 and 100. In some embodiments, the DD destabilizes the immunotherapeutic agent by a destabilization ratio of between 0 and 0.1. In one embodiment, the destabilization ration may be between 0 and 0.01 As used herein, the term "tune^'" means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments. In some embodiments, the compositions of the present invention may be used to tune the expression or function of the payload to match the expression of function achieved by a constitutively expressed construct.

[00167] In some embodiments, the SREs of the present invention may be used to achieve pulsatile expression of the compositions of the invention. As used here, "pulsatile" refers to a plurality of payload expression at spaced apart time intervals. Generally, upon administration of the stimulus, the expression of the payload is increased causing the first pulse; following the withdrawal of the stimulus, the expression of the payload decreases and this represents the interval time between the first exposure and the next exposure to the stimulus, after which the second exposure to the stimulus is initiated. Compositions of the invention may be used in varying doses to avoid T cell energy, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present invention 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 invention to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present invention may be delivered in a pulsatile fashion to reduce tonic 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 invention, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C -reactive protein, IL6, IFN-γ, and TNF-a are elevated. Doses for pulsatile expression may be spaced apart in time intervals measured in seconds, hours, days, or months.

3. Additional effector module features

[00168] The effector module of the present invention may further comprise a signal sequence which regulates the distribution of the payload of interest, a cleavage and/or processing feature which facilitate cleavage of the payload from the effector module construct, a targeting and/or penetrating signal which can regulate the cellular localization of the effector module, a tag, and/or one or more linker sequences which link different components of the effector module. Signal sequences

[00169] In addition to the SRE (e.g., DD) and payload region, effector modules of the invention may further comprise one or more signal sequences. Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the effector module of the present invention) to their designated cellular and/or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.

[00170] A signal sequence is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location. Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases. Signal sequences derived from human proteins can be incorporated as a regulatory module of the effector module to direct the effector module to a particular cellular and/or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang et al., Protein Sci. 2004, 13:2819-2824).

[00171] In some embodiments, a signal sequence may be, although not necessarily, located at the N-terminus or C -terminus of the effector module, and may be, although not necessarily, cleaved off the desired effector module to yield a "mature" payload, i.e., an immune-therapeutic agent as discussed herein.

[00172] In some examples, a signal sequence may be a secreted signal sequence derived from a naturally secreted protein, and its variant thereof. In some instances, the secreted signal sequences may be cytokine signal sequences such as, but not limited to, IL2 signal sequence comprising amino acid of SEQ ID NO. 127, encoded by the nucleotide of SEQ ID NO. 132-135 and/or p40 signal sequence comprising the amino acid sequence of SEQ ID NO. 52, encoded by the nucleotide of SEQ ID NO. 69-77. [00173] In some instances, signal sequences directing the pay load of interest to the surface membrane of the target cell may be used. Expression of the payload on the surface of the target cell may be useful to limit the diffusion of the payload to non-target in vivo environments, thereby potentially improving the safety profile of the payloads. Additionally, the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payload for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence. Signal sequences may be selected based on their compatibility with the secretory pathway of the cell type of interest so that the payload is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence comprising amino acid SEQ ID NO. 148 and nucleotide sequence of SEQ ID NO. 159, 388, and/or 389, or CD8a signal sequence (also referred to as CD8a leader) comprising amino acid SEQ ID NO. 177 and nucleotide sequence of SEQ ID NO. 178-182.

[00174] Other examples of signal sequences include, a variant may be a modified signal sequence discussed in U.S. Pat. NOs. 8, 148, 494; 8,258,102; 9,133,265; 9,279,007; and U.S. patent application publication NO. 20070141666; and International patent application publication NO. WO1993018181; the contents of each of which are incorporated herein by reference in their entirety.

[00175] In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as virus, yeast and bacteria, which can direct an effector module to a particular cellular site, such as a nucleus (e.g., EP 1209450). Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U. S. Pat. NO. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT application publication NO. WO199109952 to Lau and Rioux), E.coli enterotoxin II signal peptides (e.g., U.S. Pat. NO. 6,605,697 to Kwon et al.), Kcoli secretion signal sequence (e.g., U.S. patent publication NO. US2016090404 to Malley et al.), a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat. NO. 8,975,041), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat. NO. 4,965,197); the contents of each of which are incorporated herein by reference in their entirety.

[00176] Signal sequences may also include nuclear localization signals (NLSs), nuclear export signals (NESs), polarized cell tubulo-vesicular structure localization signals (See, e.g., U.S. Pat. NO. 8, 993,742; Cour et al., Nucleic Acids Res. 2003, 31(1): 393-396; the contents of each of which are incorporated herein by reference in their entirety),extracellular localization signals, signals to subcellular locations (e.g. lysosome, endoplasmic reticulum, golgi, mitochondria, plasma membrane and peroxisomes, etc.) (See, e.g., U.S. Pat. NO. 7,396,811; andNegi et al., Database, 2015, 1-7; the contents of each of which are incorporated herein by reference in their entirety).

[00177] In some embodiments, signal sequences of the present invention, include without limitation, any of those taught in Table 7 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Cleavage sites

[00178] In some embodiments, the effector module comprises a cleavage and/or processing feature. The effector module of the present invention may include at least one protein cleavage signal/site. The protein cleavage signal/site may be located at the N-terminus, the C-terminus, at any space between the N- and the C- tennini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.

[00179] The effector module may include one or more cleavage signal(s)/site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopeptidase, a dipeptidase, a metalloproteinase, a glutamic proteinase, a threonine proteinase and an aspartic proteinase. In some aspects, the cleavage site may be a signal sequence of furin, actinidain, calpain-1, carboxypeptidase A, carboxypeptidase P, carboxypeptidase Y, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, cathepsin C, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin S, cathepsin V, clostripain, chymase, chymotrypsin, elastase, endoproteinase, enterokinase, factor Xa, formic acid, granzyme B, Matrix metallopeptidase-2. Matrix metallopeptidase-3 , pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin and TAGZyme.

[00180] In one embodiment, the cleavage site is a furin cleavage site comprising the amino acid sequence SARNRQKRS (SEQ ID NO. 55), encoded by nucleotide sequence of SEQ ID NO. 85; or a revised furin cleavage site comprising the amino acid sequence ARNRQKRS (SEQ ID NO. 56), encoded by nucleotide sequence of SEQ ID NO. 86; or a modified furin site comprising the amino acid sequence ESRRVRRNKRSK (SEQ ID NO. 57), encoded by nucleotide sequence of SEQ ID NO. 87-89. [00181] In some embodiments, cleavage sites of the present invention, include without limitation, any of those taught in Table 7 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International PubUcation WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Protein tags

[00182] In some embodiments, the effector module of the invention may comprise a protein tag. The protein tag may be used for detecting and monitoring the process of the effector module. The effector module may include one or more tags such as an epitope tag (e.g., a FLAG or hemagglutinin (HA) tag). A large number of protein tags may be used for the present effector modules. They include, but are not limited to, self-labeling polypeptide tags (e.g., haloalkane dehalogenase (halotag2 or halotag7), ACP tag, clip tag, MCP tag, snap tag), epitope tags (e.g., FLAG, HA, His, and Myc), fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and its variants), bioluminescent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GST) tag), immunogenic affinity tags (e.g., protein A/G, IRS, AU1, AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress and V5), and other tags (e.g., biotin (small molecule), StrepTag (StrepII), SBP, biotin carboxyl carrier protein (BCCP), eXact, CBP, CYD, HPC, CBD intein-chitin binding domain, Trx, NorpA, and NusA.

[00183] In other embodiments, a tag may also be selected from those disclosed in U.S. Pat. NOs. 8,999,897; 8,357,511; 7,094, 568; 5,011,912; 4,851,341; and 4,703,004; U.S patent application publication NOs. US2013115635 and US2013012687; and International application publication NO. WO2013091661; the contents of each of which arc incorporated herein by reference in their entirety.

[00184] In some aspects, a multiplicity of protein tags, either the same or different tags, may be used; each of the tags may be located at the same N- or C-terminus, whereas in other cases these tags may be located at each terminus.

[00185] In some embodiments, protein tags of the present invention, include without limitation, any of those taught in Table 8 of copending commonly owned U.S. Provisional Patent

Apphcation No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Targeting peptides [00186] In some embodiments, the effector module of the invention may further comprise a targeting and/or penetrating peptide. Small targeting and/or penetrating peptides that selectively recognize cell surface markers (e.g. receptors, trans-membrane proteins, and extra-cellular matrix molecules) can be employed to target the effector module to the desired organs, tissues or cells. Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides, or analogs, variants, derivatives thereof, may be incorporated into the effector module for homing the effector module to the desired organs, tissues and cells, and/or subcellular locations inside the cells.

[00187] In some embodiments, a targeting sequence and/or penetrating peptide may be included in the effector module to drive the effector module to a target organ, or a tissue, or a cell (e.g., a cancer cell). In other embodiments, a targeting and/or penetrating peptide may direct the effector module to a specific subcellular location inside a cell.

[00188] A targeting peptide has any number of amino acids from about 6 to about 30 inclusive. The peptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Generally, a targeting peptide may have 25 or fewer amino acids, for example, 20 or fewer, for example 15 or fewer.

[00189] Exemplary targeting peptides may include, but are not limited to, those disclosed in the art, e.g., U.S. Pat. NOs. 9,206,231; 9,110,059; 8,706,219; and 8,772,449; and U.S. application publication NOs. 2016089447; 2016060296; 2016060314; 2016060312; 2016060311;

2016009772; 2016002613; 2015314011 and 2015166621; and International application publication NOs. WO2015179691 and WO2015183044; the contents of each of which are incorporated herein by reference in their entirety.

[00190] In some embodiments, targeting peptides of the present invention, include without limitation, any of those taught in Table 9 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Linkers

[00191] In some embodiments, the effector module of the invention may further comprise a linker sequence. The linker region serves primarily as a spacer between two or more

polypeptides within the effector module. The "linker" or "spacer", as used herein, refers to a molecule or group of molecules that connects two molecules, or two parts of a molecule such as two domains of a recombinant protein. [00192] In some embodiments, "Linker" (L) or "linker domain" or "linker region" or "linker module" or "peptide linker" as used herein refers to an oligo- or polypeptide region of from about 1 to 100 amino acids in length, which links together any of the domains/regions of the effector module (also called peptide linker). The peptide linker may be 1-40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length. Linker length may also be optimized depending on the type of payload utilized and based on the crystal structure of the payload. In some instances, a shorter linker length may be preferably selected. In some aspects, the peptide linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids: Glycine (G), Alanine (A), Valine (V), Leucine (L), lsoleucine (I), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), Lysine (K), Arginine (R), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q). One or more of these amino acids may be glycosylated, as is understood by those in the art. In some aspects, amino acids of a peptide linker may be selected from Alanine (A), Glycine (G), Proline (P), Asparagine (R), Serine (S), Glutamine (Q) and Lysine (K).

[00193] In one example, an artificially designed peptide linker may preferably be composed of a polymer of flexible residues like Glycine (G) and Serine (S) so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with one another. The choice of a particular linker sequence may concern if it affects biological activity, stability, folding, targeting and/or pharmacokinetic features of the fusion construct. Examples of peptide linkers include, but are not limited to: MH, SG, GGSG (SEQ ID NO. 183; encoded by the nucleotide sequence SEQ ID NO. 184), GGSGG (SEQ ID NO. 53; encoded by any of the nucleotide sequences SEQ ID NO. 78, 79, 185-187), GGSGGG (SEQ ID NO. 188; encoded by any of the nucleotide sequences SEQ ID NO. 189-190), SGGGS (SEQ ID NO. 191; encoded by the nucleotide sequence SEQ ID NO. 192, 208, 405), GGSGGGSGG (SEQ ID NO. 193; encoded by the nucleotide sequence SEQ ID NO. 194), GGGGG (SEQ ID NO. 195), GGGGS (SEQ ID NO. 196) or (GGGGS)n (n=l (SEQ ID NO. 196), 2 (SEQ ID NO. 197), 3 (SEQ ID NO. 54; encoded by the nucleotide sequence SEQ ID NO. 80-84, 217)), 4 (SEQ ID NO. 198), 5 (SEQ ID NO. 199), or 6 (SEQ ID NO. 200)), SSSSG (SEQ ID NO. 201) or (SSSSG)n (n=l (SEQ ID NO. 201), 2 (SEQ ID NO. 202), 3 (SEQ ID NO. 203), 4 (SEQ ID NO. 204), 5 (SEQ ID NO. 205), or 6 (SEQ ID NO. 206)), SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO. 149; encoded by the nucleotide sequence SEQ ID NO. 160, 400-404), EFSTEF (SEQ ID NO. 128; encoded by any of the nucleotide sequences SEQ ID NO. 136-137), GKSSGSGSESKS (SEQ ID NO. 209),

GGSTSGSGKSSEGKG (SEQ ID NO. 210), GSTSGSGKSSSEGSGSTKG (SEQ ID NO. 211), GSTSGSGKPGSGEGSTKG (SEQ ID NO. 212), VDYPYDVPDYALD (SEQ ID NO. 213; encoded by nucleotide sequence SEQ ID NO. 214), EGKSSGSGSESKEF (SEQ ID NO. 215), SGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO. 406; encoded by SEQ ID NO. 407), SGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 408; encoded by SEQ ID NO. 409), GS (encoded by GGTTCC), SG (encoded by AGCGGC), or GSG (encoded by

GGATCCGGA or GGATCCGGT).

[00194] In other examples, a peptide linker may be made up of a majority of amino acids mat are sterically unhindered, such as Glycine (G) and Alanine (A). Exemplary linkers are polyglycines (such as (G)4 (SEQ ID NO. 509), (G)5 (SEQ ID NO. 510), (G)8 (SEQ ID NO. 511)), poly(GA), and polyalanines. The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention.

[00195] A linker sequence may be a natural linker derived from a multi-domain protein. A natural linker is a short peptide sequence that separates two different domains or motifs within a protein.

[00196] In some aspects, linkers may be flexible or rigid. In other aspects, linkers may be cleavable or non- cleavable. As used herein, the terms "cleavable linker domain or region" or "cleavable peptide linker" are used interchangeably. In some embodiments, the linker sequence may be cleaved enzymatically and/or chemically. Examples of enzymes (e.g.,

proteinase/peptidase) useful for cleaving the peptide linker include, but are not limited, to Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, trypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10). Chemical sensitive cleavage sites may also be included in a linker sequence. Examples of chemical cleavage reagents include, but are not limited to, cyanogen bromide, which cleaves methionine residues; N-chloro succinimide, iodobenzoic acid or BNPS-skatole (2-(2-nitrophenylsulfenyl)-3-methyhndole), which cleaves tryptophan residues; dilute acids, which cleave at aspartyl-prolyl bonds; and e aspartic acid- proline acid cleavable recognition sites (i.e., a cleavable peptide linker comprising one or more D-P dipeptide moieties). The fusion module may include multiple regions encoding peptides of interest separated by one or more cleavable peptide linkers.

[00197] In other embodiments, a cleavable linker may be a "self-cleaving" linker peptide, such as 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. Other linkers will be apparent to those skilled in the art and may be used in connection with alternate embodiments of the invention.

[00198] As a non-limiting example, the P2A cleavable peptide may be

GATNFSLLKQAGDVEENPGP (SEQ ID NO. 216; encoded by SEQ ID NO. 217).

[00199] In some embodiments, the biocircuits of the present invention may include 2A peptides. The 2A peptide is a sequence of about 20 amino acid residues from a virus that is recognized by a protease (2A peptidases) endogenous to the cell. The 2A peptide was identified among picomaviruses, a typical example of which is the Foot-and Mouth disease virus (Robertson BH, et. al., J Virol 1985, 54:651-660). 2A-like sequences have also been found in Picornaviridae like equine rhinitis A virus, as well as unrelated viruses such as porcine teschovirus- 1 and the insect Thosea asigna virus (TaV). In such viruses, multiple proteins are derived from a large polyprotein encoded by an open reading frame. The 2A peptide mediates the co-translational cleavage of this polyprotein at a single site that forms the junction between the virus capsid and replication polyprotein domains. The 2A sequences contain the consensus motif D-V/I-E-X-N-P- G-P. These sequences are thought to act co-translationally, preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping of the next codon (Donnelly ML et al. (2001). J Gen Virol, 82:1013-1025). After cleavage, the short peptide remains fused to the C -terminus of the protein upstream of the cleavage site, while the proline is added to the N-terminus of the protein downstream of the cleavage site. Of the 2A peptides identified to date, four have been widely used namely FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); porcine teschovirus- 1 2A (P2A) and Thoseaasigna virus 2A (T2A). In some embodiments, the 2A peptide sequences useful in the present invention are selected from SEQ ID NO.8-11 of International Patent Publication WO2010042490, the contents of which are incorporated by reference in its entirety.

[00200] The linkers of the present invention may also be non-peptide linkers. For example, alkyl linkers such as— NH— (CH₂) a-C(O)— , wherein a=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-Ce) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.

[00201] In some aspects, the linker may be an artificial linker from U.S. Pat. NOs. 4,946,778; 5, 525, 491; 5,856,456; and International patent publication NOs. WO2012/083424; the contents of each of which are incorporated herein by reference in their entirety. [00202] In some embodiments, linkers of the present invention, include without limitation, any of those taught in Table 11 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

[00203] In one embodiment, the linker may be a spacer region of one or more nucleotides. Non- limiting examples of spacers are TCTAGATAATACGACTCACTAGAGATCC (SEQ ID NO. 410), TATGGCCACAACCATG (SEQ ID NO. 411),

AATCTAGATAATACGACTCACTAGAGATCC (SEQ ID NO. 412), TCGCGAATG, or TCGCGA.

[00204] In one embodiment, the linker may be a BamHI site. As a non-limiting example, the BamHI site has the amino acid sequence GS and/or the DNA sequence GGATCC.

Embedded stimulus, signals and other regulatory features

[00205] In some embodiments, the effector module of the present invention may further comprise one or more microRNAs, microRNA binding sites, promoters and tunable elements. In one embodiment, microRNA may be used in support of the creation of tunable biocircuits. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, a destabilizing domain may alter cleavage sites or dimerization properties or half-life of the payload, and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits and effector modules may be engineered to contain one, two, three, four or more tuned features. In some embodiments, micro RNA sequences of the present invention, include without limitation, any of those taught in Table 13 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Polynucleotides

[00206] The term "polynucleotide" or "nucleic acid molecule" in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides, e.g., linked nucleosides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β- D-ribo configuration, α-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2 '-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof.

[00207] In some embodiments, polynucleotides of the invention may be a messenger RNA (mRNA) or any nucleic acid molecule and may or may not be chemically modified. In one aspect, the nucleic acid molecule is a mRNA. As used herein, the term "messenger RNA

(mRNA)" refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[00208] 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 poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing payload constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide, for example tenability of function. 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. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.

[00209] In some embodiments, polynucleotides of the present invention may harbor 5'UTR sequences which play a role in translation initiation. 5'UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have 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 the features that are typically found in abundantly expressed genes of target cells or tissues, the stability and protein production of the polynucleotides of the invention can be enhanced. [00210] Further provided are polynucleotides, which may contain an internal ribosome entry- site (IRES) which play an important role in initiating protein synthesis in the absence of 5' cap structure in the polynucleotide. An IRES may act as the sole ribosome binding site, or may serve as one of the multiple binding sites. Polynucleotides of the invention containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes giving rise to bicistronic and/or multicistronic nucleic acid molecules.

[00211] In some embodiments, polynucleotides encoding biocircuits, effector modules, SREs and payloads of interest such as immunotherapeutic agents may include from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). In some aspects, polynucleotides of the invention may include more than 10,000 nucleotides.

[00212] Regions of the polynucleotides which encode certain features such as cleavage sites, linkers, trafficking signals, tags or other features may range independently from 10-1,000 nucleotides in length (e.g., greater than 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[00213] In some embodiments, polynucleotides of the present invention may further comprise embedded regulatory moieties such as microRNA binding sites within the 3'UTR of nucleic acid molecules which when bind to microRNA molecules, down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-142 and miR-146 binding sites may be removed to improve protein expression in the immune cells. In some embodiments, any of the encoded payloads may be may be regulated by an SRE and then combined with one or more regulatory sequences to generate a dual or multi-tuned effector module or biocircuit system.

[00214] In some embodiments, polynucleotides of the present invention may encode fragments, variants, derivatives of polypeptides of the inventions. In some aspects, the variant sequence may keep the same or a similar activity. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to the start sequence. Generally, variants of a particular polynucleotide or polypeptide of the invention 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 skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen et al., Gapped BLAST and PSI-BLAST: anew generation of protein database search programs. Nucleic Acids Res., 1997, 25:3389-3402.)

[00215] In some embodiments, polynucleotides of the present invention may be modified. As used herein, the terms "modified", or as appropriate, "modification" refers to chemical modification with respect to A, G, U (T in DNA) or C nucleotides. Modifications may be on the nucleoside base and/or sugar portion of the nucleosides which comprise the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar. Modifications to the polynucleotides of the present invention may include any of those taught in, for example, International Publication NO. WO2013052523, the contents of which are incorporated herein by reference in its entirety.

[00216] As described herein "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as '"nucleobase"). As described herein, "nucleotide" is defined as a nucleoside including a phosphate group.

[00217] In some embodiments, the modification may be on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases '"phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another intemucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications which may be used are taught in, for example, International Application NO. WO2013052523, the contents of which are incorporated herein by reference in their entirety.

[00218] Chemical modifications and/or substitution of the nucleotides or nucleobases of the polynucleotides of the invention which are useful in the present invention include any modified substitutes known in the art, for example, (±) 1 -(2-Hydroxypropy l)pseudouridine TP, (2R)-l-(2- Hy(iroxypropyl)pseudouridine TP, l-(4-Memoxy-phenyl)pseudo-LnT,,2'-0-dimemyladenosine, l,2'-0-dimethylguanosine, l,2'-0-dimethylinosine, 1-Hexyl-pseudo-UTP, 1- Homoallylpseudouridine TP, 1-Hydroxymethylpseudouridine TP, 1 -iso-propyl-pseudo-UTP, 1- Me-2-thio-pseudo-UTP, l-Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP, 1-Me-GTP, 2'-Amino-2'-deoxy-ATP, 2'-Amino-2'-deoxy-CTP, 2'-Amino-2'-deoxy-GTP, 2'-Amino-2'- deoxy-UTP, 2'-Azido-2'-deoxy-ATP, tubercidine, under modified hydroxywybutosine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, wybutosine, wyosine, xanthine, Xanthosine-5 '-TP, xylo-adenosine, zebularine, a-thio-adenosine, a-thio-cytidine, a-thio- guanosine, and/or a-thio-uridine.

[00219] Polynucleotides of the present invention may comprise one or more of the

modifications taught herein. Different sugar modifications, base modifications, nucleotide modifications, and/or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide of the invention. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modifications) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from l%to 70%, from l%to 80%, from l%to 90%, from l%to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[00220] In some embodiments, one or more codons of the polynucleotides of the present invention may be replaced with other codons encoding the native amino acid sequence to tune the expression of the SREs, through a process referred to as codon selection. mRNA codon, and tRNA anticodon pools tend to vary spatiotemporally i.e. among organisms, cell types, sub cellular locations and over time. Thus, the codon selection described herein is a spatiotemporal (ST) codon selection.

[00221] In some embodiments of the invention, certain polynucleotide features may be codon optimized. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of the native sequence with codons that are most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage may 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 the CAI can be calculated by EMBOSS CAI program (http://emboss.sourcefbrge.net/). Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias nucleotide content to alter stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein signaling sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), OptimumGene (GenScript,

Piscataway, NJ), algorithms such as but not limited to, DNA Works v3.2.3 and/or proprietary methods. In one embodiment, a polynucleotide sequence or portion thereof is codon optimized using optimization algorithms. Codon options for each amino acid are well-known in the art as are various species table for optimizing for expression in that particular species.

[00222] In some embodiments of the invention, certain polynucleotide features may be codon optimized. For example, a preferred region for codon optimization may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon optimization of the payload encoding region or open reading frame (ORF).

[00223] After optimization (if desired), the polynucleotide components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

[00224] Spatiotemporal codon selection may impact the expression of the polynucleotides of the invention, since codon composition determines the rate of translation of the mRNA species and its stability. For example, tRNA anticodons to optimized codons are abundant, and thus translation may be enhanced. In contrast, tRNA anticodons to less common codons are fewer and thus translation may proceed at a slower rate. Presnyak et al. have shown that the stability of an mRNA species is dependent on the codon content, and higher stability and thus higher protein expression may be achieved by utilizing optimized codons (Presnyak et al. (201S) Cell 160, 1111-1124; the contents of which are incorporated herein by reference in their entirety). Thus, in some embodiments, ST codon selection may include the selection of optimized codons to enhance the expression of the SRES, effector modules and biocircuits of the invention. In other embodiments, spatiotemporal codon selection may involve the selection of codons that are less commonly used in the genes of the host cell to decrease the expression of the compositions of the invention. The ratio of optimized codons to codons less commonly used in the genes of the host cell may also be varied to tune expression.

[00225] In some embodiments, certain regions of the polynucleotide may be preferred for codon selection. For example, a preferred region for codon selection may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon selection of the payload encoding region or open reading frame (ORF).

[00226] The stop codon of the polynucleotides of the present invention may be modified to include sequences and motifs to alter the expression levels of the SREs, payloads and effector modules of the present invention. Such sequences may be incorporated to induce stop codon readthrough, wherein the stop codon may specify amino acids e.g. selenocysteine or pyrtolysine. In other instances, stop codons may be skipped altogether to resume translation through an alternate open reading frame. Stop codon read through may be utilized to tune the expression of components of the effector modules at a specific ratio (e.g.as dictated by the stop codon context). Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N is either C or U. Polynucleotide modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis and recombinant technology. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

[00227] In some embodiments, polynucleotides of the invention may comprise two or more effector module sequences, or two or more pay loads of interest sequences, which are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different effector module component.

[00228] In yet another embodiment, polynucleotides of the invention may comprise two or more effector module component sequences with each component having one or more SRE sequences (DD sequences), or two or more payload sequences. As a non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the regions. As another non-limiting example, the sequences may be in a pattern such as ABABAB or

AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire polynucleotide. In these patterns, each letter, A, B, or C represent a different sequence or component.

[00229] According to the present invention, polynucleotides encoding distinct biocircuits, effector modules, SREs and payload constructs may be linked together through the 3 '-end using nucleotides which are modified at the 3'-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. Polynucleotides can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, (MPEG)₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins,

carbohydrates, vitamins, cofactors, or a drug. As non-limiting examples, they may be conjugates with other immune conjugates.

[00230] In some embodiments, polynucleotides of the present invention may encode a fusion polypeptide comprising a destabilizing domain (DD) and at least one immunotherapeutic agent taught herein. The DD domain may be an ecDHFR mutant encoded by nucleotide sequence of SEQ ID NO. 145, 163-164, 236-237, a FKBP mutant encoded by nucleotide sequence of SEQ ID NO. 111-116, 168, hDHFR mutant encoded by nucleotide sequence of SEQ ID NO. 117, 169, 170, 221- 230, and/or 238-285.

[00231] In some embodiments, the polynucleotides of the invention may encode effector modules comprising IL12 as the payload encoded by the nucleotide sequence SEQ ID NO. 118- 126, or IL 15 as the payload comprising the nucleotide sequence of SEQ ID NO. 146-147 or 395- 397, or IL15/IL15Ra fusion polypeptide as the payload encoded by the nucleotide sequence of SEQ ID NO. 152, 171-176, 446-448, 450-454, 457^70, 479 or 482.

QsM

[00232] In accordance with the present invention, cells genetically modified to express at least one biocircuit, SRE (e. g, DD), effector module and immunotherapeutic agent of the invention, are provided. Cells of the invention may include, without limitation, immune cells, stem cells and tumor cells. In some embodiments, immune cells are immune effector cells, including, but not limiting to, T cells such as CD8⁺ T cells and CD4⁺ T cells (e.g., Thl, Th2, Thl7, 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 (TILs), cytotoxic T lymphocytes (CTLs), regulatory T cells (Tregs), and dendritic cells (DCs), other immune cells that can elicit an effector function, or the mixture thereof. T cells may be Ταβ cells and Τγδ cells. In some embodiments, stem cells may be from human embryonic stem cells, mesenchymal stem cells, and neural stem cells. In some embodiments, T cells may be depleted endogenous T cell receptors (See US Pat. NOs. 9, 273, 283; 9, 181, 527; and 9,028, 812; the contents of each of which are incorporated herein by reference in their entirety).

[00233] In some embodiments, cells of the invention may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

[00234] In some embodiments, cells of the invention may be mammalian cells, particularly human cells. Cells of the invention may be primary cells or immortalized cell lines. [00235] In some embodiments, cells of the invention may include expansion factors as payload to trigger proliferation and expansion of the cells. Exemplary payloads include RAS such as KRAS, NRAS, RRAS, RRAS2, MRAS, ERAS, and HRAS, DIRAS such as DIRASl, DIRAS2, and DIRAS3, NKIRAS such as NKIRAS1, and NK1RAS2, RAL such as RALA, and RALB, RAP such as RAP1A, RAP1B, RAP2A, RAP2B, and RAP2C, RASD such as RASD1, and RASD2, RASL such as RASLIOA, RASL10B, RASL11A, RASL1 IB, and RASL12, REM such as REM1, and REM2, GEM, RERG, RERGL, and RRAD.

[00236] Engineered immune cells can be accomplished by transducing a cell compositions with a polypeptide of a biocircuit, an effector module, a SRE and/or a payload of interest (i.e., immunotherapeutic agent), or a polynucleotide encoding said polypeptide, or a vector comprising said polynucleotide. The vector may be a viral vector such as a lentiviral vector, a gamma-retro viral vector, a recombinant AAV, an adenoviral vector and an oncolytic viral vector. In other aspects, non-viral vectors for example, nanoparticles and liposomes may also be used. In some embodiments, immune cells of the invention are genetically modified to express at least one immunotherapeutic agent of the invention which is tunable using a stimulus. In some examples, two, three or more immunotherapeutic agents constructed in the same biocircuit and effector module are introduced into a cell. In other examples, two, three, or more biocircuits, effector modules, each of which comprises an immunotherapeutic agent, may be introduced into a cell.

[00237] In one embodiment, the Chimeric antigen receptor expressing T cell (including TCR T cell) may be an "armed" CAR T cell which is transformed with a CAR and an effector module comprising a cytokine. The inducible or constitutively secrete active cytokines further armor CAR T cells to improve efficacy and persistence. In this context, such CAR T cell is also referred to as "armored CAR T cell". The "armor" molecule may be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecule may be a stimulatory factor such as IL2, IL12, IL15, IL18, type I IFN, CD40L and 4-1BBL which have been shown to further enhance CAR T cell efficacy and persistence in the face of a hostile tumor microenvironment via different mechanisms (Y eku et al., Biochem Soc Trans., 2016, 44(2): 412-418). In one embodiment, the cytokine may be IL12. Such T cells, after CAR mediated activation in the tumor, release inducible IL12 which augments T-cell activation and attracts and activates innate immune cells to eliminate CD 19- negative cancer cells.

[00238] Natural killer (NK) cells are members of the innate lymphoid cell family and characterized in humans by expression of the phenotypic marker CD56 (neural cell adhesion molecule) in the absence of CD3 (T-cell co-receptor). NK cells are potent effector cells of the innate immune system which mediate cytotoxic attack without the requirement of prior antigen priming, forming the first line of defense against diseases including cancer malignancies and viral infection.

[00239] Several prc-clinical and clinical trials have demonstrated that adoptive transfer of NK cells is a promising treatment approach against cancers such as acute myeloid leukemia (Ruggeri et al. Science; 2002, 295: 2097-2100; and Geller et al., Immunotherapy, 2011, 3: 1445-1459). Adoptive transfer of NK cells expressing CAR such as DAP12-Based Activating CAR revealed improved eradication of tumor cells (Topfer et al., J Immunol. 2015; 194:3201-3212). NK cell engineered to express a CS-1 specific CAR also displayed enhanced cytolysis and interferon^ (IFN-γ) production in multiple myeloma (Chu et al., Leukemia, 2014, 28(4): 917-927).

[00240] NK cell activation is characterized by an array of receptors with activating and inhibitory functions. The important activation receptors on NK cells include CD94/NKG2C and NKG2D (the C-type lectin-like receptors), and the natural cytotoxicity receptors (NCR) NKp30, NKp44 and NKp46, which recognize ligands on tumor cells or virally infected cells. NK cell inhibition is essentially mediated by interactions of the polymorphic inhibitory killer cell immunoglobulin-like receptors (KIRs) with their cognate human-leukocyte-antigen (HLA) ligands via the alpha- 1 helix of the HLA molecule. The balance between signals that are generated from activating receptors and inhibitory receptors mainly determines the immediate cytotoxic activation.

[00241] NK cells may be isolated from peripheral blood mononuclear cells (PBMCs), or derived from human embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs). The primary NK cells isolated from PBMCs may be further expanded for adoptive immunotherapy. Strategies and protocols useful for the expansion of NK cells may include interleukin 2 (IL2) stimulation and the use of autologous feeder cells, or the use of genetically modified allogeneic feeder cells. In some aspects, NK cells can be selectively expanded with a combination of stimulating ligands including IL15, IL21, IL2, 41BBL, IL12, IL18, MCA, 2B4, LFA-1, and BCM1/SLAMF2 (e.g., US patent publication NO. US20150190471).

[00242] In some embodiments, cells of the present invention may be dendritic cells that are genetically modified to express the compositions of the invention. Such cells may be used as cancer vaccines.

ΠΙ. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[00243] The present invention further provides pharmaceutical compositions comprising one or more biocircuits, effector modules, SREs (e.g., DDs), stimuli and payloads of interest (i.e., immunotherapeutic agents), vectors, cells and other components of the invention, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.

[00244] As used herein the term "pharmaceutical composition" refers to a preparation of biocircuits, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, cells and described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components such as physiologically suitable carriers and excipients. The pharmaceutical compositions of the invention comprise an effective amount of one or more active compositions of the invention. The preparation of a pharmaceutical composition that contains at least one composition of the present invention and/or an additional active ingredient will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.

[00245] The term "excipient" or "inert ingredient" refers to an inactive substance added to a pharmaceutical composition and formulation to further facilitate administration of an active ingredient. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to any one or more biocircuits, effector modules, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, and cells to be delivered as described herein. The phrases "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.

[00246] In some embodiments, pharmaceutical compositions and formulations are administered to humans, human patients or subjects. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs and non-human primates. It will be understood that, for human administration, preparations should meet sterility, pyrogenicity , general safety and purity standards as required by FDA Office of Biological Standards.

[00247] A pharmaceutical composition and formulation in accordance with the invention may 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 discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be 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.

[00248] The compositions of the present invention may be formulated in any manner suitable for delivery. The formulation may be, but is not limited to, nanoparticles, poly flactic-co- glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.

[00249] In one embodiment, the formulation is a nanoparticle which may comprise 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 lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.

[00250] For polynucleotides of the invention, the formulation may be selected from any of those taught, for example, in International Application PCT/US2012/069610, the contents of which are incorporated herein by reference in its entirety.

[00251] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient or inert ingredient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1 and 100, e.g., between 0.5 and 50, between 1-30, between 5-80, at least 80 (w/w) active ingredient.

[00252] Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of compositions of the present invention, "effective against" for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer. [00253] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10 in a measurable parameter of disease, and preferably at least 20, 30, 40, 50 or more can be indicative of effective treatment. Efficacy for a given composition or formulation of the present invention can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed.

IV. APPLICATIONS

[00254] In one aspect of the present invention, methods for reducing a tumor volume or burden are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit system and effector module having any

immunotherapeutic agent as described herein may be in forms of a polypeptide, or a

polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).

[00255] In another aspect of the present invention, methods for inducing an anti-tumor immune response in a subject are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a

polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).

[00256] The methods, according to the present invention, may be adoptive cell transfer (ACT) using genetically engineered cells such as immune effector cells of the invention, cancer vaccines comprising biocircuit systems, effector modules, DDs, payloads of interest (i.e., immunotherapeutic agents) of the invention, or compositions that manipulate the tumor immunosuppressive microenvironment, or the combination thereof. These treatments may be further employed with other cancer treatment such as chemotherapy and radiotherapy. 1. Adoptive cell transfer (adoptive immunotherapy)

[00257] In some embodiments, cells which are genetically modified to express at least one biocircuit system, effector module, DD, and/or payload of interest (immunotherapeutic agent) may be used for adoptive cell therapy (ACT). As used herein. Adoptive cell transfer refers to the administration of immune cells (from autologous, allogenic or genetically modified hosts) with direct anticancer activity. ACT has shown promise in clinical application against malignant and infectious disease. For example, T cells genetically engineered to recognize CD 19 have been used to treat follicular B cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099-4102; and Kochenderfer and Rosenberg, Nat Rev Clin Oncol, 2013, 10(5): 267-276) and ACT using autologous lymphocytes genetically-modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21: 233- 240).

[00258] According to the present invention, the biocircuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. Certain effector modules useful in cell therapy are given in Figures 7-12. The biocircuits, their components, effector modules and their SREs and payloads may be used in cell therapies 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 the manipulation or regulation of TILs, in allogeneic cell therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines), or to enhance T cells other than TCRs (e.g. by introducing cytokine genes).

[00259] Provided herein are methods for use in adoptive cell therapy. The methods involve preconditioning a subject in need thereof, modulating immune cells with SRE, biocircuits and compositions of the present invention, administering to a subject, engineered immune cells expressing compositions of the invention and the successful engraftment of engineered cells within the subject.

[00260] In some embodiments, SREs, biocircuits and compositions of the present invention 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 in order to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to total body irradiation and/or lymphodepleting chemotherapy. Adoptive therapy clinical trials without preconditioning have failed to demonstrate any clinical benefit, indicating its importance in ACT. Yet, preconditioning is associated with significant toxicity and limits the subject cohort that is suitable for ACT. In some instances, immune cells for ACT may be engineered to express payloads of the invention such as IL12 and IL15 as payload using SR£s of the present invention to reduce the need for preconditioning (Pengram et al. (2012) Blood 119 (18): 4133-41; the contents of which are incorporated by reference in their entirety).

[00261] In some embodiments, immune cells for ACT may 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 (CDC) cells, and any combination thereof. In other embodiments, immune stimulatory cells for ACT may be generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). In some embodiments, autologous or allogeneic immune cells are used for ACT.

[00262] In some embodiments, the compositions of the present invention may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein may be utilized to change the ratio of CD4 positive cells to CD8 positive populations. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. Payloads of the invention e.g. IL12 may be used to expand CD4 and/or CD8 populations of TILs to enhance TIL mediated immune response. In some embodiments, compositions of the present invention may be used to enhance anti-tumor activity of chimeric antigen receptor e.g. CD19 CAR and MUC16 CAR and prolong survival in tumor bearing subjects (Koneru, et al. Oncoimmunology 2015 Mar; 4(3): e994446; the contents of which are incorporated by reference in its entirety).

[00263] In some embodiments, NK cells engineered to express the present compositions may be used for ACT. NK cell activation induces perforin/granzyme-dependent apoptosis in target cells. NK cell activation also induces cytokine secretion such as IFN-γ, TNF-a and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity, and augment the adaptive immune response via up-regulation of antigen presentation by antigen presenting cells such as dendritic cells (DCs) (Reviewed by Vivier et al., Nat. Immunol., 2008, 9(5): 503-510).

[00264] NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, using CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors may enhance the anti-tumor capacity of NK cells.

[00265] In some embodiments, tumor specific CD8+ T cells may be engineered to express regulatable IL12 to eradicate pre-established tumors and/or as cancer vaccine. Such methods are described by Kerkar SP, et al. (2010). Cancer Research 70(17): 6725-6734 and Kerkar SP et al. (2011) J Clin Invest 121(12): 4746-4757; the contents of each of which are incorporated by reference in their entirety.

[00266] Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating and expanding cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531, 451; US Patent Publication NO. US20160348072 A 1 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 is described in US Patent Publication NO. US20150152387A1, U.S. Patent NO. 7,435, 596; and Oyer, J.L. (2016). Cytotherapy.l8(5):653-63; the contents of each of which are incorporated by reference herein in its entirety. Specifically, human primary NK cells may be expanded in the presence of feeder cells e.g. a myeloid cell line that has been genetically modified to express membrane bound IL15, IL21, IL12 and 4-1BBL.

[00267] In some instances, sub populations of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication NO.

WO2015039100A1. In another example, T cells positive for B and T lymphocyte attenuator marker BTLA) may be used to enrich for T cells that are anti-cancer reactive as described in U.S. Pat. NO. 9,512,401 (the content of each of which are incorporated herein by reference in their entirety).

[00268] In some embodiments, immune cells for ACT may be depleted of select sub populations to enhance T cell expansion. For example, immune cells may be depleted of Foxp3+ T lymphocytes to minimize the ant-tumor immune response using methods taught in US Patent Publication NO. US 20160298081 A 1; the contents of which are incorporated by reference herein in their entirety.

[00269] In some embodiments, activation and expansion of T cells for ACT is achieved by a transiently expressed Chimeric Antigen Receptor (CAR) on the cell surface. Such activation methods are taught in International Patent NO. WO2017015427, the content of which are incorporated herein by reference in their entirety.

[00270] In some embodiments, immune cells may be activated by antigens associated with antigen presenting cells (APCs). In some embodiments, the APCs may be dendritic cells, macrophages or B cells that antigen specific or nonspecific. The APCs may autologous or homologous in their organ. In some embodiments, the APCs may be artificial antigen presenting cells (aAPCs) such as cell based aAPCs or acellular aAPCs. Cell based aAPCs are may be selected from either genetically modified allogeneic cells such as human erythroleukemia cells or xenogeneic cells such as murine fibroblasts and Drosophila cells. Alternatively, the APCs maybe be acellular wherein the antigens or costimulatory domains are presented on synthetic surfaces such as latex beads, polystyrene beads, lipid vesicles or exosomes.

[00271] In some embodiments, adoptive cell therapy is carried out by autologous transfer, wherein the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject. In other instances, ACT may involve allogenic transfer wherein the cells are isolated and/or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy. The donor and recipient subject may be genetically identical, or similar or may express the same HLA class or subtype.

[00272] In some embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by the same biocircuit system. In one example, a cytokine such as IL12 and a CAR construct such as CD19 CAR are linked to the same hDHFR destabilizing domain. The expression of IL12 and CD 19 CAR is tuned using TMP simultaneously. In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by different biocircuit systems. In one example, a cytokine such as IL12 and a IL15 or IL15/IL15Ra are linked to different DDs in two separate effector modules, thereby can be tuned separately using different stimuli. In another example, a suicide gene and a CAR construct may be linked to two separate effector modules.

[00273] Following genetic modulation using SREs, biocircuits and compositions of the invention, cells are administered to the subject in need thereof. Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No.

4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338; the contents of each of which are incorporated herein by reference in their entirety.

[00274] In some embodiments, immune cells for ACT may also be modified to express one or more immunotherapeutic agents which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions. The immunotherapeutic agents may be a CAR or TCR specific to a different target molecule; a cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed.

[00275] In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients. One example is to introduce effector modules of the invention comprising cytokines such as gamma-cytokines (IL15) into immune cells to promote immune cell proliferation and survival. Transduction of cytokine genes (e.g., IL15) into cells will be able to propagate immune cells without addition of exogenous cytokines and cytokine expressing NK cells have enhanced tumor cytotoxicity.

[00276] NK cells may also be modified to become insensitive to suppressive cytokines such as TGF-β, thereby preserving their cytotoxicity. For example, NK cells can be genetically modified to express the dominant negative mutant form of TGF-β type Π receptor (DNTfSRII) on their surface that render NK cells resistant to the suppressive effects of TGF-β.

[00277] In some embodiments, biocircuits, their components, SREs or effector modules may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the stepwise 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 immunotherapies. Exhausted T cells have low proliferative and cytokine producing capabilities concurrent with high rates of apoptosis and high surface expression of multiple inhibitor)' receptors. T cell activation leading to exhaustion may occur either in the presence or absence of the antigen.

[00278] In some embodiments, effector modules of the present invention, useful for immunotherapy may be placed under the transcriptional control of the T cell receptor alpha locus constant (TRAC) locus in the T cells. Eyquem et al. have shown that expression of the CAR from the TRAC locus prevents T cell exhaustion and the accelerated differentiation of T cells caused by excessive T cell activation (Eyquem J. et al (2017) Nature.543(7643): 113-117; the contents of which are incorporated herein by reference in their entirety).

[00279] In some embodiments, payloads of the invention may be used in conjunction with antibodies or fragments that target T cell surface markers associated with T cell exhaustion. T- cell surface markers associated with T cell exhaustion that may be used include, but are not limited to, CTLA-l, PD-1, TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96. 2. Cancer vaccines

[00280] In some embodiments, biocircuits, effector modules, payloads of interest

(immune-therapeutic agents), vectors, cells and compositions of the present invention may be used in conjunction with cancer vaccines. In one aspect, dendritic cells are modified to express the compositions of the invention and used as cancer vaccines.

[00281] In some embodiments, cancer vaccine may comprise peptides and/or proteins derived from tumor associated antigen (TAA). Such strategies may be utilized to evoke an immune response in a subject, which in some instances may be a cytotoxic T lymphocyte (CTL) response. Peptides used for cancer vaccines may also modified to match the mutation profile of a subject. For example, EGFR derived peptides with mutations matched to the mutations found in the subject in need of therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. Oct 7;5(12): el238539; the contents of which are incorporated herein by reference in their entirely).

[00282] In one embodiment, cancer vaccines of the present invention may superagonist altered peptide ligands (APL) derived from TAAs. These are mutant peptide ligands deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than native epitopes. These alterations may allow the peptide to bind better to the restricting Class I MHC molecule or interact more favorably with the TCR of a given tumor- specific CTL subset. APLs may be selected using methods taught in US Patent Publication NO. US20160317633A1, the contents of which are incorporated herein by reference in their entirety.

3. Combination treatments

[00283] In some embodiments, it is desirable to combine compositions, vectors and cells of the invention for administration to a subject. Compositions of the invention comprising different immunotherapeutic agents may be used in combination or in conjunction with known immune-therapeutic agents for enhancement of immunotherapy.

[00284] In some embodiments, it is desirable to combine compositions of the invention with adjuvants, that can enhance the potency and longevity of antigen-specific immune responses. Adjuvants used as immunostimulants in combination therapy include biological molecules or delivery carriers that deliver antigens. As non-limiting examples, the compositions of the invention may be combined with biological adjuvants such as cytokines, Toll Like Receptors, bacterial toxins, and/or saponins. In other embodiments, the compositions of the present invention may be combined with delivery carriers. Exemplary delivery carriers include, polymer microspheres, immune stimulating complexes, emulsions (oil-in-water or water-in-oil), aluminum salts, liposomes or virosomes.

[00285] In some embodiments, immune effector cells modified to express biocircuits, effector modules, DDs and payloads of the invention may be combined with the biological adjuvants described herein. Dual regulation of CAR and cytokines and ligands to segregate the kinetic control of target-mediated activation from intrinsic cell T cell expansion. Such dual regulation also minimizes the need for pre-conditioning regimens in patients. As a non-limiting example, CAR e.g. CD19 CAR may be combined with DD regulated cytokines e.g. IL12 to enhance the anti-tumor efficacy of the CAR (Pegram H. J., et al. Tumor-targeted T cells modified to secrete IL12 eradicate systemic tumors without need for prior conditioning. Blood.2012;l 19:4133-41; the contents of each of which are incorporated herein by reference in their entirety).

[00286] In some embodiments, immune effector cells modified to express one or more antigen- specific TCRs or CARs may be combined with compositions of the invention comprising immunotherapeutic agents that convert the immunosuppressive tumor microenvironment.

[00287] In one aspect, effector immune cells modified to express payloads of the invention may be combined with CARs specific to different target molecules on the same cell may be combined. In another aspect, different immune cells modified to express the CAR construct such as NK cells and T cells may be used in combination with immune cells of the invention for a tumor treatment, for instance, a T cell modified to express a CD 19 CAR may be combined with a NK cell modified to express the same DD-1L12 to treat B cell malignancy. In other

embodiments, immune cells modified to express compositions of the invention may be combined with checkpoint blockade agents.

[00288] In some embodiments, immune effector cells modified to express biocircuits, effector modules, DDs and payloads of the invention may be combined with cancer vaccines of the invention.

[00289] In some embodiments, an effector module comprising a cytokines may be used in combination with an effector module comprising a different cytokine, or an effector module comprising a safety switch, or an effector module comprising a metabolic factor, or an effector module comprising a homing receptor.

[00290] In some embodiments, methods of the invention may include combination of the compositions of the invention with other agents effective in the treatment of cancers, infection diseases and other immunodeficient disorders, such as anti-cancer agents. As used herein, the term "anti-cancer agent" refers to any agent which is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[00291] In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surgery, or any other therapeutic agent which, in combination with the present invention, improves the therapeutic efficacy of treatment.

[00292] In one embodiment, an effector module comprising a IL12 may be used in combination with amino pyrimidine derivatives such as the Burkit's tyrosine receptor kinase (BTK) inhibitor using methods taught in International Patent Application NO. WO2016164580, the contents of which are incorporated herein by reference in their entirety.

[00293] In some embodiments, compositions of the present invention may be used in combination with immunotherapeutics other than the inventive therapy described herein, such as antibodies specific to some target molecules on the surface of a tumor cell.

[00294] Exemplary chemotherapies include, without limitation, Acivicin; Aclarubicin;

Acodazole hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin;

Ametantrone acetate; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperrin, Sulindac, Curcumin, alkylating agents including: Nitrogen mustards such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BC U), lomustine (CCNU), and semustine (methyl-CC U); thylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),

hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (IJnC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrrolidine analogs such as 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'- difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin

(pentostatin), eiythrohydroxjiionyladenine (EUNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2- CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, 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 angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF/VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N- methylhydrazine (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutcthimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin- releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab; Sdi 1 mimetics; Semustine; Senescence derived inhibitor 1; Sparfosic acid; Spicamycin D;

Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; and Zinostatin stimalamer; ΡΙ3Κβ small-molecule inhibitor, GSK2636771 ; pan-PI3K inhibitor (BKM120); BRAF inhibitors. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or any analog or derivative and variant of the foregoing.

[00295] Radiotherapeutic agents and factors include radiation and waves that induce DNA damage for example, γ-irradiation. X-rays, UV-irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[00296] In some embodiments, the chemotherapeutic agent may be an immunomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance antitumor functions of CAR modified T cells (Otahal et al., Oncoimmunology, 2015, 5(4): el 115940). Some examples of anti-tumor antibodies include tocilizumab, siltuximab.

[00297] Other agents may be used in combination with compositions of the invention may also include, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion such as focal adhesion kinase (FAKs) inhibitors and Lovastatin, or agents that increase the sensitivity of the hyper proliferative cells to apoptotic inducers such as the antibody C225.

[00298] The combinations may include administering the compositions of the invention and other agents at the same time or separately. Alternatively, the present immunotherapy may precede or follow the other agent/therapy by intervals ranging from minutes, days, weeks to months.

4. Diseases

[00299] Provided in the present invention is a method of reducing a tumor volume or burden in a subject in need, the method comprising introducing into the subject a composition of the invention.

[00300] The present invention also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell genetically modified to express at least one effector module of the invention.

Cancer

[00301] Various cancers may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term "cancer" refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those 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 marrow, tailbone, testicles, thyroid and uterus.

[00302] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/lenkemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.

[00303] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[00304] As a non-limiting example, the carcinoma which may be treated may be Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous leukemia,

Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-Cell lymphoma ), Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer. Brain cancer. Brain stem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer,

Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastases, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ. Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, MeduUoblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma,

Mesenchymous, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas. Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma, Non-Hodgkin's lymphoma, Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma,

Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer. Peritoneal cancer, Pharyngeal cancer,

Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumor, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma ), Testicular cancer, Throat cancer, Thymoma / thymic carcinoma. Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer. Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer.

Infectious diseases

[00305] In some embodiment, biocircuits of the invention may be used for the treatment of infectious diseases. Biocircuits of the invention may be introduced in cells suitable for adoptive cell transfer such as macrophages, dendritic cells, natural killer cells, and or T cells. Infectious diseases treated by the biocircuits of the invention may be diseases caused by viruses, bacteria, fungi, and/or parasites. ILlS-ILlSRa payloads of the invention may be used to increase immune cell proliferation and/or persistence of the immune cells useful in treating infectious diseases.

[00306] 'Infection diseases" herein refer to diseases caused by any pathogen or agent mat infects mammalian cells, preferably human cells and causes a disease condition. Examples thereof include bacteria, yeast, fungi, protozoans, mycoplasma, viruses, prions, and parasites. Examples include those involved in (a) viral diseases such as, for example, diseases resulting from infection by 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 molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), apapovavirus (e.g., papillomaviruses, 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., a lentivirus such as HIV); (b) bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium,

Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia,

Haemophilus, or Bordetella; (c) other infectious diseases, such chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, parasitic diseases including but not limited to malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection and prions that cause human disease such as Creutzfeldt- Jakob Disease (CJD), variant Creutzfeldt- Jakob Disease (vCJD), Gerstmann-Straixssler-Scheinker syndrome, Fatal Familial Insomnia and kuru.

5. Microbiome

[00307] Alterations in the composition of the microbiome may impact the action of anti-cancer therapies. A diverse community of symbiotic, commensal and pathogenic microorganisms exist in all environmentally exposed sites in the body and is herein referred to as the "Microbiome." Environmentally exposed sites of the body that may be inhabited by a microbiome include the skin, nasopharynx, the oral cavity, respiratory tract, gastrointestinal tract, and the reproductive tract.

[00308] In some embodiments, microbiome native or engineered with immunotherapeutic agents may be used to improve the efficacy of the anti-cancer immunotherapies. Methods of using microbiome to improve responsive to immunotherapeutic agents have been described by Sivan et al. (Sivan A., et al.2015. Science; 350: 1084-9; the contents of which are incorporated herein by reference in their entirety). In other embodiments, the microorganisms may be delivered along with immunotherapeutic compositions of the present invention to improve the efficacy of immunotherapy.

6. Tools and agents for making therapeutics

[00309] Provided in the present invention are tools and agents that may be used in generating immunotherapeutics for reducing a tumor volume or burden in a subject in need. A considerable number of variables are involved in producing a therapeutic agent, such as structure of the payload, type of cells, method of gene transfers, method and time of ex vivo expansion, pre- conditioning and the amount and type of tumor burden in the subject. Such parameters may be optimized using tools and agents described herein.

Cell lines

[00310] The present disclosure provides a mammalian cell that has been genetically modified with the compositions of the invention. 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 ΝΓΗ 3T3, human colorectal carcinoma cell line HCT116, ovarian carcinoma cell line SKOV-3, immortalized T cell lines Jurkat cells and SupTl cells, lymphoma cell line 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 instances, the cell is not an immortalized cell line, but instead a cell obtained from an individual and is herein referred to as a primary cell. For example, the cell is a T lymphocyte 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.

Tracking SREs, biocircuits and cell lines

[00311] In some embodiments, it may be desirable to track the compositions of the invention or the cells modified by the compositions of the invention. Tracking may be achieved by reporter moieties, which, as used herein, refers to any protein capable of creating a detectable signal, in response to an input. Examples include alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, glucuronidase, peroxidase, β-lactamase, catalytic antibodies, bioluminescent proteins e.g. luciferase, and fluorescent proteins such as Green fluorescent protein (GFP).

[00312] Reporter moieties may be used to monitor the response of the DD upon addition of the ligand corresponding to the DD. In other instances, reporter moieties may be used to track cell survival, persistence, cell growth, and/or localization in vitro, in vivo, or ex vivo.

[00313] In some embodiments, the preferred reporter moiety may be luciferase proteins. In one embodiment, the reporter moiety is the Renilla luciferase (SEQ ID NO. 231, encoded by nucleic acid sequence of SEQ ID NO. 232), or a firefly luciferase (SEQ ID NO. 233, encoded by nucleic acid sequence of SEQ ID NO. 234).

Animal models

[00314] The utility and efficacy of the compositions of the present invention may be tested in vivo animal models, preferably mouse models. Mouse models used to may be syngeneic mouse models wherein mouse cells are modified with compositions of the invention and tested in mice of the same genetic background. Examples include pMEL-1 and 4T1 mouse models.

Alternatively, xenograft models where human cells such as tumor cells and immune cells are introduced into immunodeficient mice may also be utilized in such studies. Immunodeficient mice used may be CByJ.Cg-Foxnlnu/J, B6;129S7-RagltmlMom/J, B6.129S7-RagltmlMom/J, B6. CB17-Prkdcscid/SzJ, NOD.129S7(B6)-RagltmlMom/J, NOD.Cg- RagltmlMomPrfltmlSdz/Sz, NOD.CB17-Prkdcscid/SzJ, NOD.Cg-PrkdcscidB2mtmlUnc/J, NOD-scid IL2Rgnull, Nude (nu) mice, SCID mice, NOD mice, RAG1/RAG2 mice, NOD-Scid mice, Beige mouse, IL2rgnull mice, b2mnuU mice, NOD-scid IL2rynull mice, NOD-sc/c/- Blmmill mice, and HLA transgenic mice.

Cellular assays

[00315] In some embodiments, the effectiveness of the compositions of the inventions as immune-therapeutic agents may be evaluated using cellular assays. Levels of expression and/or identity of the compositions of the invention may be determined according to any methods known in the art for identifying proteins and/or quantitating proteins levels. In some

embodiments, such methods may include Western Blotting, flow cytometry, and immunoassays.

[00316] Provided herein are methods for functionally characterizing cells expressing SRE, biocircuits and compositions of the invention. In some embodiments, functional characterization is carried out in primary immune cells or immortalized immune cell lines and may be 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, CD1 lb, CD16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNgamma, PD1, ΊΊΜ3 and LAG3. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC Class Π, CD40, CD45, B7-1, B7-2, IFN-γ receptor and IL2 receptor, ICAM-1 and/or Fey receptor. Examples of 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 the like; while in some cases also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56, and/or CD57. 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.

V. DELIVERY MODALITIES AND/OR VECTORS

Vectors

[00317] The present invention also provides vectors that package polynucleotides of the invention encoding biocircuits, effector modules, SREs (DDs) and payload constructs, and combinations thereof. Vectors of the present invention may also be used to deliver the packaged polynucleotides to a cell, a local tissue site or a subject. These vectors may be of any kind, 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, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.

[00318] In general, vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.

[00319] As used herein a promoter is defined as a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the polynucleotide sequence of the present invention. Vectors can comprise native or non-native promoters operably linked to the polynucleotides of the invention. The promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage-specific, and/or organism specific. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of polynucleotide sequence that is ope rati vely linked to it. Another example of a preferred promoter is Elongation Growth Factor- 1. Alpha (EF-1. alpha). 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter as well as human gene promoters including, but not limited to the phosphoglycerate kinase (PGK) promoter, actin promoter, the myosin promoter, the hemoglobin promoter, the Ubiquitin C (Ubc) promoter, the human U6 small nuclear protein promoter and the creatine kinase promoter. In some instances, inducible promoters such as but not limited to metallothionine promoter, glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter may be used. In some embodiments, the promoter may be selected from the SEQ ID NO. 49-51, 312.

[00320] In some embodiments, the optimal promoter may be selected based on its ability to achieve minimal expression of the SREs and payloads of the invention in the absence of the ligand and detectable expression in the presence of the ligand.

[00321] Additional promoter elements e.g. enhancers may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10-100 base pairs upstream or downstream of the start site. In some instances, two or more promoter elements may be used to cooperatively or independently activate transcription.

[00322] In some embodiments, the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.

l. Lenti viral vectors

[00323] In some embodiments, lentiviral vectors/particles may be used as vehicles and delivery modalities. Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell. Some examples of lenti virus include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, the Simian

Immunodeficiency Virus (SIV), feline immunodeficiency virus (FTV), 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).

[00324] Typically, lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as "'self-inactivating"). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463). Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe. Correspondingly, lentiviral vehicles, for example, derived from HIV-l/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non- dividing cells. As used herein, the term "recombinant" refers to a vector or other nucleic acid containing both lentiviral sequences and non-lentiviral retroviral sequences.

[00325] Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three (in second generation lentiviral systems) or four separate plasmids (in third generation lentiviral systems). The producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector). In general, the plasmids or vectors are included in a producer cell line. The plasmid s/vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art. As non-limiting example, the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

[00326] The producer cell produces recombinant viral particles that contain the foreign gene, for example, the effector module of the present invention. The recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art. The recombinant lentiviral vehicles can be used to infect target cells.

[00327] Cells that can be used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11 : 452- 459), FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T- based producer cell lines (e.g., Stewart et al., Hum Gene Ther._20l 1, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al., ii/οοίί 2009, 113(21): 5104-5110; the contents of each of which are incorporated herein by reference in their entirety).

[00328] In some aspects, the envelope proteins may be heterologous envelop proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelop proteins. The VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Pity virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LTV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV). The gp64 or other baculoviral env protein can be derived from Autographa califomica

nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhidrosis virus, Bombyx mori nuclear polyhidrosis virus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana

nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus.

[00329] Additional elements provided in lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5 ' or 3^' terminus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof. Other elements include central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer. The effector module is linked to the vector.

[00330] Methods for generating recombinant lentiviral particles are discussed in the art, for example, U.S. Pat. NOs. 8, 846, 385; 7,745, 179; 7,629,153; 7,575,924; 7,179, 903; and 6, 808, 905; the contents of each of which are incorporated herein by reference in their entirety.

[00331 ] Lenti virus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJMl, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJMl-EGFP, pULTRA, plnducer20, pHTV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionll.

[00332] Lentiviral vehicles known in the art may also be used (See, U.S. Pat. NOs. 9, 260, 725; 9,068,199; 9,023,646; 8,900,858; 8,748,169; 8,709,799; 8,420,104; 8,329,462; 8,076,106;

6,013,516; and 5,994,136; International Patent Publication NO. WO2012079000; the contents of each of which are incorporated herein by reference in their entirety).

2. Retroviral vectors (γ-retro viral vectors)

[00333] In some embodiments, retroviral vectors may be used to package and deliver the biocircuits, biocircuit components, effector modules, SREs or payload constructs of the present invention. Retroviral vectors (RVs) allow the permanent integration of a transgene in target cells. In addition to lentiviral vectors based on complex HIV- 1/2, retroviral vectors based on simple gamma-retro viruses have been widely used to deliver therapeutic genes and demonstrated clinically as one of the most efficient and powerful gene delivery systems capable of transducing a broad range of cell types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).

[00334] In some embodiments, gamma-retro viral vectors derived from a mammalian gamma- retrovirus such as murine leukemia viruses (MLVs), are recombinant. The MLV families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are able to infect only murine cells using mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xprl) receptor, but differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses such as focus-forming viruses (MCF) infect murine, human and other species.

[00335] Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasmids including one encoding the retroviral structural and enzymatic (gag- pol) polyprotein, one encoding the envelope (env) protein, and one encoding the vector mRNA comprising polynucleotide encoding the compositions of the present invention that is to be packaged in newly formed viral particles.

[00336] In some aspects, the recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated in the outer lipid layer of the viral particles which can increase/alter the cell tropism. Exemplar}' envelop proteins include the gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or Simian endogenous retrovirus envelop protein, or Measles Virus H and F proteins, or Human immunodeficiency virus gpl20 envelope protein, or cocal vesiculovirus envelop protein (See, e.g., U.S. application publication NO. 2012/164118; the contents of which are incorporated herein by reference in its entirety). In other aspects, envelope glycoproteins may be genetically modified to incorporate targeting/binding ligands into gamma-retroviral vectors, binding ligands including, but not limited to, peptide ligands, single chain antibodies and growth factors (Waehler et al., Nat. Rev. Genet. 2007, 8(8):573-587; the contents of which are incorporated herein by reference in its entirety). These engineered glycoproteins can retarget vectors to cells expressing their corresponding target moieties. In other aspects, a "molecular bridge" may be introduced to direct vectors to specific cells. The molecular bridge has dual specificities: one end can recognize viral glycoproteins, and the other end can bind to the molecular determinant on the target cell. Such molecular bridges, for example ligand-receptor, avidin-biotin, and chemical conjugations, monoclonal antibodies and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Y ang et al., Biotechnol. Bioeng., 2008, 101(2): 357-368; and Maetzig et al., Viruses, 2011, 3, 677-713; the contents of each of which are incorporated herein by reference in their entirety).

[00337] In some embodiments, the recombinant gamma-retroviral vectors are self-inactivating (SIN) gammaretro viral vectors. The vectors are replication incompetent. SIN vectors may harbor a deletion within the 3' U3 region initially comprising enhancer/promoter activity. Furthermore, the 5' U3 region may be replaced with strong promoters (needed in the packaging cell line) derived from Cytomegalovirus or RSV, or an internal promoter of choice, and/or an enhancer element. The choice of the internal promoters may be made according to specific requirements of gene expression needed for a particular purpose of the invention.

[00338] In some embodiments, polynucleotides encoding the biocircuit, biocircuit components, effector module, SRE are inserted within the recombinant viral genome. The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling of a more effective promoter from a different retrovirus or virus in place of the wild-type promoter and the like). In some examples, the recombinant gamma-retroviral vectors may comprise modified packaging signal, and/or primer binding site (PBS), and/or 5'-enhancer/promoter elements in the U3-region of the 5'- long terminal repeat (LTR), and/or 3 -SIN elements modified in the US- region of the 3' -LTR. These modifications may increase the titers and the ability of infection.

[00339] Gamma retroviral vectors suitable for deUvering biocircuit components, effector modules, SREs or payload constructs of the present invention may be selected from those disclosed in U.S. Pat. NOs. 8,828,718; 7,585,676; 7,351,585; U.S. application publication NO. 2007/048285; PCT application publication NOs. WO2010/113037; WO2014/121005;

WO2015/056014; and EP Pat. NOs. EP1757702; EP1757703 (the contents of each of which are incorporated herein by reference in their entirety).

3. Adeno-associated viral vectors (AAV)

[00340] In some embodiments, polynucleotides of present invention may be packaged into recombinant adeno-associated viral (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids. The serotype capsids may include capsids from any identified AAV serotypes and variants thereof, for example, AAVl, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVl 1, AAV12 and AAVrhlO.

[00341] In one embodiment, the AAV serotype may be or have a sequence as described in United States Publication No. US20030138772, herein incorporated by reference in its entirety, such as, but not limited to, AAVl (SEQ ID NO. 6 and 64 of US20030138772), AAV2 (SEQ ID NO. 7 and 70 of US20030138772), AAV3 (SEQ ID NO. 8 and 71 of US20030138772), AAV4 (SEQ ID NO. 63 of US20030138772), AAV5 (SEQ ID NO. 114 of US20030138772), AAV6 (SEQ ID NO. 65 of US20030138772), AAV7 (SEQ ID NO. 1-3 of US20030138772), AAV8 (SEQ ID NO. 4 and 95 of US20030138772), AAV9 (SEQ ID NO. 5 and 100 of

US20030138772), AAV10 (SEQ ID NO. 117 of US20030138772), AAVl 1 (SEQ ID NO. 118 of US20030138772), AAV12 (SEQ ID NO. 119 of US20030138772), AAVrhlO (amino acids 1 to 738 of SEQ ID NO. 81 of US20030138772) or variants thereof. Non-limiting examples of variants include SEQ ID NOs. 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99, 101-113 of US20030138772, the contents of which are herein incorporated by reference in their entirety.

[00342] In one embodiment, the AAV serotype may have a sequence as described in Pulicherla et al. (Molecular Therapy, 2011, 19(6): 1070-1078), U.S. Pat. NOs. 6,156,303; 7,198,951; U.S. Patent Publication NOs. US2015/0159173 and US2014/0359799; and International Patent Publication NOs. WO1998/011244, WO2005/033321 and WO2014/14422; the contents of each of which are incorporated herein by reference in their entirety.

[00343] AAV vectors include not only single stranded vectors but self-complementary AAV vectors (scAAVs). scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[00344] The rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.

[00345] The biocircuits, biocircuit components, effector modules, SREs or payload constructs may be encoded in one or more viral genomes to be packaged in the AAV capsids taught herein.

[00346] Such vectors or viral genomes may also include, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements necessary 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, and the like.

[00347] In some embodiments, more than one effector module or SRE (e.g. DD) may be encoded in a viral genome.

4. Oncolytic viral vector

[00348] In some embodiments, polynucleotides of present invention may be packaged into oncolytic viruses, such as vaccine viruses. Oncolytic vaccine viruses may include viral particles of amymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector sufficient to induce oncolysis of cells in the tumor (e.g., US Pat. NO. 9,226,977).

5. Messenger RNA (mRNA)

[00349] In some embodiments, the effector modules of the invention may be designed as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA molecules may have the structural components or features of any of those taught in International Application number PCT/US2013/030062, the contents of which are incorporated herein by reference in its entirety.

[00350] Polynucleotides of the invention may also be designed as taught in, for example, Ribostem Limited in United Kingdom patent application serial number 0316089.2 filed on July 9, 2003 now abandoned, PCT application number PCT/GB2004/002981 filed on July 9, 2004 published as WO2005005622, United States patent application national phase entry serial number 10/563,897 filed on June 8, 2006 published as US20060247195 now abandoned, and European patent application national phase entry serial number EP2004743322 filed on July 9, 2004 published as EP1646714 now withdrawn; Novozymes, Inc. in PCT application number PCT/US2007/88060 filed on December 19, 2007 published as WO2008140615, United States patent application national phase entry serial number 12/520,072 filed on July 2, 2009 published as US20100028943 and European patent application national phase entry serial number EP2007874376 filed on July 7, 2009 published as EP2104739; University of Rochester in PCT application number PCT/US2006/46120 filed on December 4, 2006 published as

WO2007064952 and United States patent application serial number 11/606,995 filed on

December 1, 2006 published as US20070141030; BioNTech AG in European patent application serial number EP2007024312 filed December 14, 2007 now abandoned, PCT application number PCT/EP2008/01059 filed on December 12, 2008 published as WO2009077134, European patent application national phase entry serial number EP2008861423 filed on June 2, 2010 published as EP2240572, United States patent application national phase entry serial number 12/,735,060 filed November 24, 2010 published as US20110065103, German patent application serial number DE 10 2005 046 490 filed September 28, 2005, PCT application PCT/EP2006/0448 filed September 28, 2006 published as WO2007036366, national phase European patent EP1934345 published March, 21, 2012 and national phase US patent application serial number 11/992,638 filed August 14, 2009 published as 20100129877; Immune Disease Institute Inc. in United States patent application serial number 13/088,009 filed April 15, 2011 published as US20120046346 and PCT application PCT/US2011/32679 filed April 15, 2011 published as WO20110130624; Shire Human Genetic Therapeutics in United States patent application serial number 12/957,340 filed on November 20, 2010 published as US20110244026; Sequitur Inc. in PCT application PCT/US1998/019492 filed on September 18, 1998 published as WO1999014346; The Scripps Research Institute in PCT application number PCT/US2010/00567 filed on February 24, 2010 published as WO2010098861, and United States patent application national phase entry serial number 13/203,229 filed November 3, 2011 published as US20120053333; Ludwig- Maximillians University in PCT application number PCT/EP2010/004681 filed on July 30, 2010 published as WO2011012316; Cellscript Inc. in United States patent number 8,039,214 filed June 30, 2008 and granted October 18, 2011, United States patent application serial numbers 12/962,498 filed on December 7, 2010 published as US20110143436, 12/962,468 filed on December 7, 2010 published as US20110143397, 13/237,451 filed on September 20, 2011 published as US20120009649, and PCT applications PCT/US2010/59305 filed December 7, 2010 published as WO2011071931 and PCT/US2010/59317 filed on December 7, 2010 published as WO2011071936; The Trustees of the University of Pennsylvania in PCT application number PCT/US2006/32372 filed on August 21, 2006 published as WO2007024708, and United States patent application national phase entry serial number 11/990,646 filed on March 27, 2009 published as US20090286852; Curevac GMBH in German patent application serial numbers DE102001 027 283.9 filed June 5, 2001, DE102001 062480.8 filed December 19, 2001, and DE 20 2006 051 516 filed October 31, 2006 all abandoned, European patent numbers EP1392341 granted March 30, 2005 and EP1458410 granted January 2, 2008, PCT apphcation numbers PCT/EP2002/06180 filed June 5, 2002 published as WO2002098443, PCT/EP2002/14577 filed on December 19, 2002 published as WO2003051401,

PCT/EP2007/09469 filed on December 31, 2007 published as WO2008052770,

PCT/EP2008/03033 filed on April 16, 2008 published as WO2009127230, PCT/EP2006/004784 filed on May 19, 2005 published as WO2006122828, PCT/EP2008/00081 filed on January 9, 2007 published as WO2008083949, and United States patent application serial numbers 10/729,830 filed on December 5, 2003 published as US20050032730, 10/870,110 filed on June 18, 2004 published as US20050059624, 11/914,945 filed on July 7, 2008 published as

US20080267873, 12/446,912 filed on October 27, 2009 published as US2010047261 now abandoned, 12/522,214 filed on January 4, 2010 published as US20100189729, 12/787,566 filed on May 26, 2010 published as US20110077287, 12/787,755 filed on May 26, 2010 published as US20100239608, 13/185,119 filed on July 18, 2011 published as US20110269950, and

13/106,548 filed on May 12, 2011 published as US20110311472 all of which are herein incorporated by reference in their entirety.

[00351] In some embodiments, the effector modules may be designed as self-amplifying RNA. "Self-amplifying RNA" as used herein refers to RNA molecules that can replicate in the host resulting in the increase in the amount of the RNA and the protein encoded by the RNA. Such self-amplifying RNA may have structural features or components of any of those taught in International Patent Application Publication No. WO2011005799 (the contents of which are incorporated herein by reference in their entirety). VI. DOSING. DELIVERY AND ADMINISTRATIONS

[00352] The compositions of the invention may be delivered to a cell or a subject through one or more routes and modalities. The viral vectors containing one or more effector modules, SREs, immunotherapeutic agents and other components described herein may be used to deliver them to a cell and/or a subject. Other modalities may also be used such as mRNAs, plasmids, and as recombinant proteins.

1. Delivery to cells

[00353] In another aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention and vectors comprising said polynucleotides may be introduced into cells such as immune effector cells.

[00354] In one aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention, may be packaged into viral vectors or integrated into viral genomes allowing transient or stable expression of the polynucleotides. Preferable viral vectors are retroviral vectors including lenti viral vectors. In order to construct a retroviral vector, a polynucleotide molecule encoding a biocircuit, an effector module, a DD or a payload of interest (i.e. an

immunotherapeutic agent) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components. The recombinant retroviral particles are secreted into the culture media, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are especially preferred as they are capable of infecting both dividing and non-dividing cells.

[00355] Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electroporation, sonoporation, hyrdoporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery' such as cationic lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors.

[00356] In some embodiments, the polypeptides of the invention may be delivered to the cell directly. In one embodiment, the polypeptides of the invention may be delivered using synthetic peptides comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the invention are co introduced into the cell with the ELD-CLD- synthetic peptide. ELDs facilitate the escape of proteins that are trapped in the endosome, into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the ait. CPDs allow the transport of proteins across the plasma membrane and have also been described in the art. The ELD-CLD fusion proteins synergist cally increase the transduction efficiency when compared to the co-transduction with either domain alone. In some embodiments, a histidine rich domain may optionally be added to the shuttle construct as an additional method of allowing the escape of the cargo from the endosome into the cytosol. The shuttle may also include a cysteine residue at the N or C terminus to generate multimers of the fusion peptide. Multimers of the ELD-CLD fusion peptides generated by the addition of cysteine residue to the terminus of the peptide show even greater transduction efficiency when compared to the single fusion peptide constructs. The polypeptides of the invention may also be appended to appropriate localization signals to direct the cargo to the appropriate sub-cellular location e.g. nucleus. In some embodiments any of the ELDs, CLDs or the fusion ELD-CLD synthetic peptides taught in the International Patent Publication, WO2016161516 and

WO2017175072 may be useful in the present invention (the contents of each of which are herein incorporated by reference in their entirety).

2. Dosing

[00357] The present invention provides methods comprising administering any one or more compositions for immunotherapy to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating a clinical condition such as cancer, infection diseases and other immunodeficient diseases.

[00358] Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, or prophylactically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, previous or concurrent therapeutic interventions and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00359] Compositions of the invention may be used in varying doses to avoid T cell energy, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present invention 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 invention to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present invention may be delivered in a pulsatile fashion to reduce tonic 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 invention, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN7, and TNF-o are elevated.

[00360] In some embodiments, the compositions of the invention may initially be delivered in a low priming single dose followed by a multiple dose regimen to limit the toxicity associated with IL12 (Lasek W, et al. (2014) Cancer Immunol Immunother. 63:419-35).

3. Administration

[00361] In some embodiments, the compositions for immunotherapy may be administered to cells ex vivo and subsequently administered to the subject. Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531, 451; the contents of each of which are incorporated herein by reference in their entirety. Isolation of NK cells is described in U.S. Pat. NOs. 7,435, 596; the contents of which are incorporated by reference herein in its entirety.

[00362] In some embodiments, compositions of the present invention, may be administered by any of the methods of administration taught in the copending commonly owned U.S. Provisional Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

[00363] In some embodiments, depending upon the nature of the cells, the cells may be introduced into a host organism e.g. a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation. In some aspects, the cells of the invention may be introduced at the site of the tumor. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like. The cells may be in a physiologically-acceptable medium.

[00364] In some embodiments, the cells of the invention may be administrated in multiple doses to subjects having a disease or condition. The administrations generally effect an improvement in one or more symptoms of cancer or a clinical condition and/or treat or prevent cancer or clinical condition or symptom thereof. [00365] In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, polypeptides of the present invention comprising biocircuits, effector molecules, SREs, payloads of interest (immune-therapeutic agents) and compositions of the invention may be delivered in vivo to the subject. In vivo delivery of immunotherapeutic agents is well described in the art. For example, methods of delivery of cytokines are described in the E.P. Pat. NO. EP0930892 Al, the contents of which are incorporated herein by reference. Routes of delivery

[00366] The pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs (e.g., DDs), payloads (i.e. immunotherapeutic agents), vectors and cells of the present invention may be administered by any route to achieve a therapeutically effective outcome.

[00367] These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracranial (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intrasinal infusion, intravitreal, (through the eye), intravenous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-ammotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-ammotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracistemal (within the cistema magna

cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route

administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering 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 tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal.

VII. DEFINITIONS

[00368] At various places in the present specification, features or functions of the compositions of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.

[00369] Activity: As used herein, the term "activity^'' refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve one or more biological events. In some embodiments, biological events may include cell signaling events. In some embodiments, biological events may include cell signaling events associated protein interactions with one or more corresponding proteins, receptors, small molecules or any of the biocircuit components described herein. [00370] Adoptive cell therapy (ACT): The terms "Adoptive cell therapy" or "Adoptive cell transfer", as used herein, refer to a cell therapy involving in the transfer of cells into a patient, wherein cells may have originated from the patient, or from another individual, and are engineered (altered) before being transferred back into the patient. The therapeutic cells may be derived from the immune system, such as Immune effector cells: CD4+ T cell; CD8+ T cell, Natural Killer cell (NK cell); and B cells and tumor infiltrating lymphocytes (TILs) derived from the resected tumors. Most commonly transferred cells are autologous anti-tumor 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 (TCR) or chimeric antigen receptor (CAR).

[00371] 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 molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a receptor, and soluble factor.

[00372] Agonist: the term "agonist" as used herein, refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular receptor or family of receptors, e.g., agonist of a co-stimulatory receptor.

[00373] Antagonist the term "antagonist" as used herein refers to any agent that inhibits or reduces the biological activity of the target(s) it binds.

[00374] Antigen: the term "antigen" as used herein is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itself. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells such as cytotoxic T lymphocytes and T helper cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. In the context of the invention, the terms "antigens of interest" or "desired antigens" refers to those proteins and/or other biomolecules provided herein that are immunospecifically bound or interact with antibodies of the present invention and/or fragments, mutants, variants, and/or alterations thereof described herein. In some embodiments, antigens of interest may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof. [00375] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately'' or "about" refers to a range of values that fall 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 than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100 of a possible value).

[00376] Associated with: As used herein, the terms "associated with," "conjugated," "linked," "attached," and "tethered," when used with respect to two or more moieties, mean that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serve as Unking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An "association" need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the "associated" entities remain physically associated.

[00377] Autologous: the term "autologous" as used herein is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[00378] Barcode: the term "barcode" as used herein refers to polynucleotide or amino acid sequence that distinguishes one polynucleotide or amino acid from another.

[00379] Cancer: the term "cancer" as used herein refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues ultimately metastasize to distant parts of the body through the lymphatic system or bloodstream.

[00380] Co-stimulatory molecule: As used herein, in accordance with its meaning in immune T cell activation, refers to a group of immune cell surface receptor/ligands which engage between T cells and A PCs and generate a stimulatory signal in T cells which combines with the stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs

[00381] Cytokines: the term "cytokines", as used herein, refers to a family of small soluble factors with pleiotropic functions that are produced by many cell types that can influence and regulate the function of the immune system.

[00382] Delivery: the term "delivery" as used herein refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload. A "delivery agent" refers to any agent which facilitates, at least in part, the in vivo delivery of one or more substances (including, but not limited to a compounds and/or compositions of the present invention) to a cell, subject or other biological system cells.

[00383] Destabilized: As used herein, the term "destable," "destabilize," "destabilizing region" or "destabilizing domain" means a region or molecule that is less stable than a starting, reference, wild-type or native form of the same region or molecule.

[00384] Engineered: As used herein, embodiments of the invention are "engineered" when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

[00385] Expression: 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 an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; (4) folding of a polypeptide or protein; and (5) post-translational modification of a polypeptide or protein.

[00386] Feature: As used herein, a "feature" refers to a characteristic, a property', or a distinctive element.

[00387] Formulation: As used herein, a "formulation" includes at least a compound and/or composition of the present invention and a delivery agent.

[00388] Fragment. A "fragment," as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein. In some embodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. In some embodiments, fragments of an antibody include portions of an antibody.

[00389] Functional: As used herein, a "functional" biological molecule is a biological entity with a structure and in a form in which it exhibits a property and/or activity by which it is characterized.

[00390] Immune cells: the term "an immune cell", as used herein, refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a T γδ cell, a Ταβ cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[00391] Immunotherapy : the term "immunotherapy" as used herein, refers to a type of treatment of a disease by the induction or restoration of the reactivity of the immune system towards the disease.

[00392] Immunotherapeutic agent: the term "immunotherapeutic agent" as used herein, refers to the treatment of disease by the induction or restoration of the reactivity of the immune system towards the disease with a biological, pharmaceutical, or chemical compound.

[00393] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[00394] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[00395] Linker: As used herein, a linker refers to a moiety that connects two or more domains, moieties or entities. In one embodiment, a linker may comprise 10 or more atoms. In a further embodiment, a linker may comprise a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. In some embodiments, a linker may comprise one or more nucleic acids comprising one or more nucleotides. In some embodiments, the linker may comprise an amino acid, peptide, polypeptide or protein. In some embodiments, a moiety bound by a linker may include, but is not limited to an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, a protein complex, a payload (e.g., a therapeutic agent), or a marker (including, but not limited to a chemical, fluorescent, radioactive or bioluminescent marker). The linker can be used for any useful purpose, such as to form multimers or conjugates, as well as to administer a payload, as described herein. Examples of chemical groups mat can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomelic units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bonds include an amido bond which may be cleaved for example by the use of tris(2- carboxyethyl) phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond which may be cleaved for example by acidic or basic hydrolysis.

[00396] Checkpoint/factor: As used herein, a checkpoint factor is any moiety or molecule whose function acts at the junction of a process. For example, a checkpoint protein, ligand or receptor may function to stall or accelerate the cell cycle.

[00397] Metabolite: Metabolites arc the intermediate products of metabolic reactions catalyzed by enzymes that naturally occur within cells. This term is usually used to describe small molecules, fragments of larger biomolecules or processed products.

[00398] Modified: As used herein, the term "'modified" refers to a changed state or structure of a molecule or entity as compared with a parent or reference molecule or entity. Molecules may be modified in many ways including chemically, structurally, and functionally. In some embodiments, compounds and/or compositions of the present invention are modified by the introduction of non-natural amino acids.

[00399] Mutation: As used herein, the term "mutation" refers to a change and/or alteration. In some embodiments, mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and/or alterations to a protein and/or nucleic acid sequence. Such changes and/or alterations may comprise the 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, wherein mutations comprise the addition and/or substitution of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides. The resulting construct, molecule or sequence of a mutation, change or alteration may be referred to herein as a mutant.

[00400] Neoantigen: the term "neoantigen", as used herein, refers to a tumor antigen that is present in tumor cells but not normal cells and do not induce deletion of their cognate antigen specific T cells in thymus (i.e., central tolerance). These tumor neoantigens may provide a "foreign" signal, similar to pathogens, to induce an effective immune response needed for cancer immunotherapy. A neoantigen may be restricted to a specific tumor. A neoantigen be a peptide/protein with a missense mutation (missense neoantigen), or a new peptide with long, completely novel stretches of amino acids from novel open reading frames (neoORFs). The neoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in DNA mismatch repair causing micro-satellite instability), gene-fusion, read-through mutations in stop codons, or translation of improperly spliced RNA (e.g., Saeterdal et al., Proc Natl Acad Sci USA, 2001, 98: 13255-13260).

[00401] Off-target: As used herein, "off target" refers to any unintended effect on any one or more target, gene, cellular transcript, cell, and/or tissue.

[00402] Operably linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

[00403] Payload or pay load of interest (POI): the terms "payload" and "payload of interest (POI)", as used herein, are used interchangeable. A payload of interest (POI) refers to any protein or compound whose function is to be altered. In the context of the present invention, the POI is a component in the immune system, including both innate and adaptive immune systems. Payloads of interest may be a protein, a fusion construct encoding a fusion protein, or non- coding gene, or variant and fragment thereof. Payload of interest may, when amino acid based, may be referred to as a protein of interest.

[00404] Pharmaceutically acceptable excipients: the term "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and/or dissolving active agents. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplar}' excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pynolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pynolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[00405] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts of the compounds described herein are forms of the disclosed compounds wherein the acid or base moiety is in its salt form (e.g., as generated by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammomum, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts, for example, from non-toxic inorganic or organic acids. In some embodiments a pharmaceutically acceptable salt is prepared from a parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al. Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. Pharmaceutically acceptable solvate: The term "pharmaceutically acceptable solvate," as used herein, refers to a crystalline form of a compound wherein molecules of a suitable solvent are incorporated in the crystal lattice. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N'-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1,3-dimethyl- 2-imidazolidinone (DMEU), l,3-dimethyl-3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate." In some embodiments, the solvent incorporated into a solvate is of a type or at a level that is physiologically tolerable to an organism to which the solvate is administered (e.g., in a unit dosage form of a pharmaceutical composition).

[00406] Stable: As used herein "stable" refers to a compound or entity that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

[00407] Stabilized: As used herein, the term "stabilize", "stabilized," "stabilized region" means to make or become stable. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondary status or state or to a reference compound or entity.

[00408] Standard CAR: As used herein, the term "standard CAR" refers to the standard design of a chimeric antigen receptor. The components of a CAR fusion protein including the extracellular scFv fragment, transmembrane domain and one or more intracellular domains are linearly constructed as a single fusion protein.

[00409] Stimulus response element (SRE): the term "stimulus response element (SRE), as used herein, is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the "responsive" nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus. Further, the response of any SRE to a stimulus may be a matter of degree or kind. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1 and 100 or a factored increase or decrease such as 2- fbld, 3-fold, 4-fold, 5-fbld, 10-fbld or more. One non-limiting example of an SRE is a destabilizing domain (DD).

[00410] Subject: As used herein, the term "subject" or "patient" refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

[00411] T cell: A T cell is an immune cell that produces T cell receptors (TCRs). T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). TMcan be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD 127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cell and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD 127 as compared to naive T cells or TCM). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Other exemplary T cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8+CD28- and Qa-1 restricted T cells.

[00412] T cell receptor. T cell receptor (TCR) refers to an immunoglobulin superfamily member having a variable antigen binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, which is capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and β chains (also known as TCRa and TCRp\ respectively), or γ and δ chains (also known as TCRy and TCR6, respectively). The extracellular portion of TCR chains (e.g., a-chain, β-chain) contains two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or Vo, β-chain variable domain or Ύβ) at the N-terminus, and one constant domain (e.g., a-chain constant domain or Ca and β-chain constant domain or Cp,) adjacent to the cell membrane. Similar to immunoglobulin, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs). A TCR is usually associated with the CD3 complex to form a TCR complex. As used herein, the term 'TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD36 chain, two CD3e chains, a homodimer of CD3ζ chains, a TCRa chain, and a TCRp^* chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD36 chain, two CD3e chains, a homodimer of Οϋ3ζ chains, a TCRy chain, and a TCR6 chain. A "component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, TCRp\ TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3E or CD3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRp\ a complex of TCRy and TCR5, a complex of CD3E and CD36, a complex of CD3y and CD3E, or a sub-TCR complex of TCRa, TCRp\ CD3y, CD35, and two CD3e chains.

[00413] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.

[00414] Treatment or treating: As used herein, the terms "treatment" or "treating" denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.

[00415] Tune: As used herein, the term "tune" means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments.

EQUIVALENTS AND SCOPE

[00416] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[00417] In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in or otherwise relevant to a given product or process.

[00418] It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of is thus also encompassed and disclosed.

[00419] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that arc expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[00420] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[00421] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

EXAMPLES

Example 1, Generation of novel ligand responsive SREs or DDs by mutagenesis screening

Study design

[00422] To engineer constructs that display ligand dependent stability, a candidate ligand binding domain (LBD) is selected and a cell-based screen using yellow fluorescent protein (YFP) as a reporter for protein stability is designed to identify mutants of the candidate LBD possessing the desired characteristics of a destabilizing domain: low protein levels in the absence of a ligand of the LBD, (i.e., low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation (Banaszynski, etal., (2006) Cell; 126(5): 995-1004). The candidate LBD binds to a desired ligand but not endogenous signaling molecules.

[00423] The candidate LBD sequence (as a template) is first mutated using a combination of nucleotide analog mutagenesis and error-prone PCR, to generate libraries of mutants based on the template candidate domain sequence. The libraries generated are cloned in-frame at either the 5'- or 3'-ends of the YFP gene, and a retroviral expression system is used to stably transduce the libraries of YFP fusions into NIH3T3 fibroblasts.

[00424] The transduced NIH3T3 cells are subjected to three to four rounds of sorting using fluorescence-activated cell sorting (FACS) to screen the libraries of candidate DDs. Transduced NIH3T3 cells are cultured in the absence of the high affinity ligand of the ligand binding domain (LBD), and cells that exhibit low levels of YFP expression are selected through FACS.

Screening Strategy I

[00425] The selected cell population is cultured in the presence of the high affinity ligand of the ligand binding domain for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells that exhibit high levels of YFP expression are selected through FACS and the selected cell population is split into two groups and treated again with the high affinity ligand of the ligand binding domain at different concentrations; one group is treated with the lower concentration of the ligand and the other is treated with a high concentration of the ligand, for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells expressing mutants that are responsive to lower concentrations of the ligand are isolated.

[00426] The isolated cells responsive to the lower concentration of the ligand are treated with the ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media. This fourth sorting is designed to enrich cells that exhibit fast kinetics of degradation (Iwamoto etal, Chem Biol. 2010 Sep 24; 17(9): 981-988).

Screening Strategy Π

[00427] The selected cell population is subject to additional one or more sorts by FACS in the absence of high affinity ligand of LBD and cells that exhibit low levels of YFP expression are selected for further analysis. Cells are treated with high affinity ligand of the ligand binding domain, for a period of time (e.g. 24 hours), and sorted again by FACS. Cells expressing high levels of YFP are selected for through FACS. Cells with high expression of YFP are treated with ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media to enrich cells that exhibit fast kinetics of degradation. Any of the sorting steps may be repeated to identify DDs with ligand dependent stability.

[00428] The cells are recovered after sorting. The identified candidate cells are harvested and the genomic DNA is extracted. The candidate DDs are amplified by PCR and isolated. The candidate DDs are sequenced and compared to the LBD template to identify the mutations in candidate DDs. Example 2. DP regulated recombinant IL12 expression

[00429] FKBP (DD)-IL12 and DHFR (DD)-IL12 constructs were packaged into pLVX IRES- Puro lentiviral vectors with CMV, EFla, or PGK promoters or without a promoter. The IL12 consists of two subunits, p40 and p35 which are separated by a linker. A p40 signal sequence was inserted next to the DD or IL12. In several constructs, a furin protease cleavage site or a modified furin site was included.

[00430] HEK293T cells were transiently transfected with 200ng or Ιμ% of FKBP-IL12 plasmids (OT-IL12-001 to OT-IL12-005), and subsequently treated with 10μΜ Shield-1 or vehicle control for 6 hours. Culture media was collected from transfected cells and diluted 1 :50 to measure IL12 levels using p40 ELISA. The stabilization ratio was defined as fold change in IL12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. The average IL12 ELISA readings and stabilization ratio are presented in Table 7.

Table 7; Ligand dependent IL12 induction

[00431] OT-IL12-002 and OT- IL12-004 showed low level of IL12 expression in the absence of ligand when compared to IL12 levels in HEK 293T parental cells. Treatment with Shield-1 resulted in an increase in IL12 levels in OT-IL12-002, OT-IL12-004, and OT-IL12-005 constructs and a stabilization ratio between 2 and 4. These data show that OT-IL 12-002 and OT- IL 12-004 are destabilized in the absence of these constructs are stabilized by Shield-1.

[00432] 1L12 expression was measured in cells following stable transduction. 500,000 cells stably transduced with OT-IL 12-004 were plated in a 12 well plate and incubated overnight in growth media consisting of Dulbecco's Modified Eagle medium (DMEM) and 10 fetal bovine serum (FBS). The next day, cells were treated with luM Shield-1 or vehicle control for 6 or 24 hours. Following treatment with Shield-1, growth media was collected from the cells and diluted 10, 40, 160 or 640 fold and IL12 levels were quantified using IL12-p40 ELISA. The stabilization ratio was defined as fold change in IL12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. The average IL12 ELISA readings and stabilization ratio at 6 hours arc presented in Table 8.

Table 8; Ligand dependent IL12 induction (6 hours)

[00433] IL12 stabilization ratio greater than 1 was observed at 10, 40 and 160-fold dilutions of media, indicating that IL12 is stabilized by Shield- 1 treatment at these dilutions at 6 hours.

[00434] The average IL12 ELISA readings and stabilization ratio at 24 hours are presented in Table 9.

Table 9: Ligand dependent IL12 induction (24 hours)

[00435] IL12 stabilization ratio greater than 1 at all media dilutions tested and the highest stabilization ratio was observed at 40-fold dilution of media at 24 hours, suggesting ligand dependent stabilization.

[00436] To evaluate Shield-1 dependent FKBP-1L12 induction over time, 2 million cells were plated in growth medium and incubated overnight in the presence of ΙμΜ Shield-1 or vehicle control. Cells were then incubated for with the ligand for 2, 4,6, 8, 24, 48, or 72 hours and growth media was collected for the cells at all time points. Growth media was diluted 400-fold and IL12 levels were measured using IL12 p40 ELISA. The stabilization ratio was defined as fold change in IL12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. Average IL12 ELISA readings and stabilization ratio are presented in Table 10. Table 10: IL12 induction over time

[00437] Stabilization ratio increased over time and peaked at 48 hours, suggesting that IL12 is stabilized by Shield-1 with increasing duration of ligand treatment.

[00438] To evaluate the dependence of FKBP-IL12 production on Shield-1 dose levels, OT- IL12-004 transduced HEK293T cells were plated at different densities (40,000 cells, 20,000 cells, 10,000 cells or 5,000 cells per well) onto a 96-well plate. Following overnight incubation, cells were treated with growth medium containing 0 to 10μΜ Shield-1 for 24 hours. Media was then collected, diluted 400-fold and FKBP-IL12 levels were measured using IL12-p40 ELISA. Average IL12 ELISA readings are presented in Table 11.

Table 11: Dose and cell number dependent IL12 induction

[00439] A dose dependent IL12 induction was observed at all cell numbers tested. IL12 induction increased with Shield-1 up to a dose of luM; following which IL12 induction plateaued. Notably, greater IL12 induction was observed at 2000 and 4000 cells/well.

Example 3. DP regulated recombinant IL12 mediated functions in HEK293T cells

[00440] HEK-Blue sensor cells (InvivoGen, San Diego, CA) were utilized to evaluate whether DD regulated IL12 is capable of regulating signaling downstream of IL12. In these cells, the IL12 receptor, STAT4 and downstream transcriptional elements are linked to a reporter gene such that IL 12 signaling can be monitored. One million HEK 293T were transfected with 200ng of OT-IL 12-003 plasmid using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). 48 hours after transfection, cells were treated with growth media containing ΙΟμΜ Shield- 1, incubated for another 24 hours, following which, media was collected. 50,000 HEK 293 Blue sensor cells were plated onto 96 well plates and incubated overnight with media (at different dilutions) from Shield-1 treated OT-IL12-003 expressing HEK293T cells. After overnight incubation, 20 μΐ media was removed from each well and incubated with 180 μΐ Quanti-Blue reagent (InvivoGen, San Diego, CA) for 30 minutes at 37°C. Absorption was measured at 620 nm using a spectrophotometer. To generate a standard curve, 180 μΐ Quanti-Blue reagent was mixed with 20 μΐ of recombinant IL12 at following concentrations 500, 250, 125, 62.5, 31.25, 15.62, 7.8 and 3.9 pg/ml. Functional IL12 concentrations were determined by comparing the optical density of each sample with IL12 standard curve. Measurable levels of functional IL12 were reached with 640-fold dilutions of IL12 containing growth media and further plateaued at higher concentrations of the media (Figure 13A).

[00441] The dependence of functional IL12 production on the dose of Shield-1 used was measured. 10,000 HEK293T cells stably transduced with OT-IL12-004 were plated onto 96 well plates and treated with growth media coritaining 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.005, 0.002 or 0 μΜ Shield-1 for 24 hours. Following Shield-1 treatment, media from cells was diluted 200-fold and 20uL of the diluted media was added to HEK Blue sensor cells. After overnight incubation, 20 μΐ of media was removed from each well and incubated with 180 μΐ Quanti-Blue reagent (mvivoGen, San Diego, CA) for 30 minutes at 37°C. Absorption was measured at 620 nm using a spectrophotometer. To generate a standard curve, 180 μΐ Quanti-Blue reagent was mixed with 20 μΐ of recombinant IL12 at following concentrations 500, 250, 125, 62.5, 31.25, 15.62, 7.8 and 3.9 pg/ml. Functional IL12 concentrations were determined by comparing the optical density of each sample with IL12 standard curve. A dose dependent increase in the levels of functional IL12 levels was observed (Figure 13B).

Example 4. DP regulated recombinant IL12 expression in vivo

[00442] SKOV3 tumor cells expressing FBP regulated-ILl 2 (#OT-ILl 2-009) or parental cells were implanted into SCID Beige mice (Day 0). Mice implanted with FKBP IL12 were dosed intraperitoneally with Shield-1 (lOmg/kg) or vehicle control on Day 2 and Day 7, while the parental cells were left untreated. Blood samples were collected at 0, 2, 4, 6, 8 and 24 hours after Shield-1 dosing and plasma human IL12 levels were measured using ELISA. The average adjusted concentration of plasma IL12 is presented in Figure 13C. At Day 2, 1L12 levels increased in Shield-1 treatment and the levels were higher than vehicle control at 4, 6, 8, and 24 hours. Maximum IL 12 levels were detected in Shield-1 treated mice at 8 hours following treatment. In contrast, at day 7, IL12 levels were very low and almost comparable to the IL12 levels in parental SKOV3 cells.

[00443] The experiment was repeated 28 days following implantation of SKOV3 tumor cells. Mice were split into three groups, with the groups receiving 1, 2 or 3 doses of ligand or vehicle control. Mice received multiple doses with a two-hour interval. Blood samples were collected right before the first dose (0 hours), and 6 hours and 24 hours after the first dosing. Plasma IL12 levels were measured and average IL12 concentrations are shown in Figure 13D. The two dose and three dosing scheme resulted in higher plasma IL12 levels when compared to vehicle treated samples. Peak plasma IL12 levels was detected at 6 hours following shield- 1 treatment with all dosing schemes, and the highest IL12 plasma levels were detected with the three-dose regimen. This demonstrates the ligand dependent stabilization of IL12 in vivo.

Example 5. DP regulated IL15

[00444] To test ligand dependent IL15 production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10% FBS and incubated overnight at 37°C at 5% C02. Cells were then transfected with lOOng of OT-IL15-001(constitutive) or OT-IL15- 002 (ecDHFR-IL15) using Lipofectamine 2000 and incubated for 48 hrs. Following the incubation, media was exchanged for growth medium with 10uM Trimethoprim or vehicle control and further incubated for 24 hrs. Media was collected and the undiluted samples or samples diluted 4, 16, 256, 1024, 4096 or 16384-fold were tested using human IL15 ELISA. The stabilization ratio was defined as fold change in IL15 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. Average IL15 ELISA readings and stabilization ratio are presented in Table 12.

Table 12: DD-IL15 induction

[00445] The 16-fold, 4-fold diluted, and undiluted media samples showed stabilization ratio greater than 1.5, suggesting a Trimethoprim dependent stabilization of 1L15 at these dilutions. Example 6. DP regulated expression of IL15-IL15Ra fusion molecule

[00446] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subunit (IL15Ra) and DDs such as ecDHFR (DD), FKBP (DD), or human DHFR (DD). These fusion molecules were cloned into pLVX-EF la-lRES-Puro vector.

[00447] To test ligand dependent ILlS-ILlSRa production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5 %C02. Cells were then transfected with lOOng of constitutive IL15-IL15Ra (OT- IL15-008) or DD linked IL15-IL15Ra (OT-IL15-006, OT-IL15-007, OT-IL15-009, OT-IL15- 010, OT-IL15-01 1) using Lipofectamine 2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 10uM Trimethoprim or 1 uM Shield- 1 and further incubated for 24 hrs. Cells were harvested and IL15 levels are analyzed via western blotting using human IL15 antibody (Abeam, Cambridge, UK). OT-IL15-009 showed the strong ligand (Trimethoprim) dependent stabilization of IL15, while OT-IL15-006 and OT- IL15-007 showed modest ligand dependent stabilization of IL15 (Figure 14A).

[00448] Surface expression of membrane bound IL15-IL15Ra constructs (OT-IL15-006, OT- IL15-007, OT-IL15-008, OT-IL15-009, OT-IL15-010, OT-IL15-011) was determined by FACS using anti-IL15 and anti-IL15Ra antibodies. HEK293T cells were transfected with IL15-IL15Ra constructs and then treated with suitable ligand (Shield- 1 or Trimethoprim). 48 hours after transfection, cells were analyzed using FACS. As expected, constitutive IL15-IL15Ra construct OT-IL 15-008 showed high surface expression of IL15 and IL15Raboth in the presence and absence of ligand. Consistent with the results from the western blot, OT-IL15-009 showed the strong ligand (Trimethoprim) dependent surface expression of IL15 and IL15Ra (Figure 14B, Figure 14C).

[00449] Membrane bound-IL15-IL15Ra constructs (OT-IL15-008 to OT IL15-011) were transduced into human colorectal carcinoma cell line, HCT-116 and stable integrants were selected with 2μg of puromycin. Stably integrated cells were then incubated for 24 hours in the presence or absence of 10uM Trimethoprim or luM Methotrexate.

[00450] Surface expression of IL15 -IL15Ra fusion constructs was examined by staining with PE- conjugated IL15Ra antibody (Cat no. 330207, Biolegend, San Diego, CA). The median fluorescence intensity obtained with the different constructs in the presence or absence of the corresponding ligand is presented in Table 13. Table 13: Surface expression of IL15-IL15Ra fusion constructs

[00451] The stabilization ratio was calculated as the fold change in GFP intensity in ligand treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilization ratio was calculated as the fold change in GFP intensity in the DD regulated constructs compared to the constitutive construct (OT-IL15-008) in the absence of the ligand. Destabilization ratios less than 1 and stabilization ratios greater than 1 are desired in DDs. The ratios are presented in Table 14.

Table 14: ILlS-IL15Ra destabilization and stabilization ratios

[00452] Destabilization ratios less than one was observed with OT-IL 15-006 (ecDHFR (R12H, E129K)) and OT-IL15-011(hDHFR (Q36F, N65F, Y122I)) indicating a strong destabiUzation in the absence of ligand. Stabilization ratio greater than 1 was observed with all constructs with TMP treatment and with both OT-IL 15-010 and 11 with MTX treatment. These data show that OT-IL 15-006 and OT-IL 15-011 are both strongly destabilized in the absence of ligand and strongly stabilized in the presence of ligand.

[00453] The expression and ligand-dependent stabilization of IL15-IL15Ra constructs (OT- IL 15-008 to OT-IL15-011) was measured in HCT-l 16. Cells were incubated with

10μΜ Trimethoprim or ΙμΜ Methotrexate or DMSO for 24 hours. Following incubation, cells were harvested and cell extracts were prepared. Cell extracts were run on SDS-PAGE and western blotted with anti-IL15 antibody (Catalog No. 7213, Abeam, Cambridge, UK). As shown in Figure 14D, the IL15/IL15Ra constitutive construct (OT-IL15-008) showed ligand independent IL15 expression while the DD regulated constructs (OT-IL 15-009 to OT-IL15-011) showed ligand dependent IL15 expression. The identity of the IL15 bands was also confirmed by immunoblotting with the anti-human DHFR antibody (Catalog No. 117705, Genetex, Irvine, CA). As shown in Figure 14D, both IL15-IL15Ra fusion constructs (OT-IL15-010 and 011) showed ligand dependent expression of DHFR expression.

[00454] To evaluate the dose dependence of ligand induced stabilization, IL15-IL15Ra fusion constructs namely, OT-IL15-009 (ecDHFR (R12Y, Υ100Γ)), OT-IL15-010 (hDHFR (Y122I, Al 25F)), and OT-IL15-011 (hDHFR (Q36F, N65F, Y122I)) were stably transduced into HCT- 116 cells and incubated with increasing concentrations of Trimethoprim for 24 hours. Surface expression of IL15-IL15Ra fusion construct was quantified by FACS using IL15Ra- PE antibody. The median fluorescence intensity with increasing doses of TMP is represented in Table 15.

Table IS: Surface expression of ILlS-ILlSRa

[00455] As shown in Table 15, all three constructs showed a dose dependent increase in median fluorescence intensity indicating a dose dependent increase in surface expression of IL15- IL15Ra fusion upon addition of DD stabilizing ligand.

[00456] The time course of ligand dependent stabilization of IL15-IL15Ra fusion constructs was measured in HCT-116 cells. Cells were transduced with OT-IL15-009 (ecDHFR (R12Y, Y100I) construct and incubated with 10uM Trimethoprim for 0, 12, 16, 24, 48 or 72 hours. Following incubations, surface expression of IL15-IL15Ra fusion construct was quantified by FACS using lL15Ra- PE antibody and compared to parental untransfected cells. The median fluorescence intensity (MFI) over time is represented in Table 16. Table 16: Time course of IL15-IL15Ra surface expression

[0045η As shown in Table 16, OT-IL15-009 (ecDHFR (R12Y, Yl 001) showed a time- dependent increase in median fluorescence intensity indicating that the surface expression of IL15-IL15Ra fusion increased with increased duration of treatment with DD stabilizing ligand. Example 7. In vitro T cell assay development

[00458] The goal of the study was to determine the T cell stimulation regimen and dose of IL12 needed to maximize T cell persistence and T cell differentiation in vitro, to mimic an in vivo adoptive cell transfer therapy regimen. The study recapitulates the design of the adoptive cell therapy regimen wherein the T cells were initially exposed to the antigen in vitro which results in activation followed by a resting phase and finally in vivo transfer where the T cells encounter the antigen again. T cells were stimulated CD3/CD28 beads or soluble CD3/CD28 on day 0 and the CD3/CD28 stimulus was washed off at the end of 48 hours. Cells were treated with a dose of IL12 ranging from 0.01- 1000 ng/mL. On day 9, the Thl phenotype of the cells was evaluated by examining the frequency of IFNgamma positive CD4+ cells and CD8+ cells. On day 14, cells were divided into two groups- one group received a second CD3/CD28 stimulation and a second group that was not stimulated. On day 16, the Thl phenotype was evaluated in both groups using FACS. The results for day 16 are presented in Figure 15. IFN gamma expression was higher in cells that received a CD3/CD28 restimulation on day 14 compared to cells that did not receive second stimulation. This indicates that both antigen restimulation and IL12 exposure were required for the Thl phenotype. Further, as little as 0.1 ng/mL of IL12 was able to cause Thl- skewing and IFN gamma production from T cells in vitro, and higher doses of IL12 further improved this effect.

Example 8. Measuring human T cells responses in vitro and in vivo

[00459] IL12 promotes the differentiation of naive T cells into Thl cells which results in the secretion of IFN gamma from T cells. Human T cells were treated with IL12 or left untreated and analyzed by flow cytometry for the expression of IFN gamma and T cell marker CD3. Treatment with IL12 resulted in the differentiation of T cells as measured by an increase in the percentage of 1FN gamma positive T cells from 0.21 to 22.3 (see inset of Figure 16A).

[00460] To test if membrane bound IL15/IL15Ra fusion protein (OT-IL15-008) can induce human T cell expansion, human T cells were transduced with the construct. T cell proliferation was measured by evaluating forward and side scatter of the T cell population using flow cytometry. Transduction with membrane bound IL15/IL15Ra fusion construct resulted in the expansion of human T cells (58.9) compared to control untransfected cells (37.8) (Figure 16B).

[00461] Tracking T cells following their adoptive transfer is critical to determine their distribution at different sites in the host, their identity and persistence overtime. Human T cells were stimulated with CD3/CD28 beads and incubated with 50U/ml of IL2. Cells were expanded in vitro for 7 days with IL2 supplementation on day 3 and day 5. On day 5, the CD3/CD28 beads were removed and the cells were cultured for two days. On day 7, cells were washed to remove IL2 and 5 million human T cells were injected intravenously into immune compromised, WOO£g-Pttok!^KUII2rgf^m'^w9^,/8zS mice. Blood samples were obtained 4, 24, 120 and 168 hours after cell transfer. Mice were euthanized 168 hours after cell transfer and the bone marrow and spleen were harvested. Immune cells were isolated from all samples and analyzed for the presence of human T cells using CD3 and CD45 cell surface markers. As shown in Figure 16C, the percentage of CD3 positive, CD45 positive human T cells in the blood was higher in animals injected with human T cells, especially at 120 and 168 hours. CD3 positive, CD45 positive human T cells were also detected in the spleen and bone marrow of animals injected with human T cells. As expected no CD3 positive, CD45 positive human T cells were detected in control animals that were not injected with human T cells.

[00462] To determine the identity of the T cells following adoptive transfer, blood samples were collected from mice 48 hours after injection. CD4 and CD8 T cells were analyzed for surface expression of CD45RA and CD62L. Both markers are highly expressed in naive T cells but are lost as the T cells become antigen exposure. As shown in Figure 16D, human CD4 and CD8 T cells showed high surface expression of both markers prior to injecting into mice, but was lost 48 hours after in vivo cell transfer indicating that the human T cells are exposed to the antigen in vivo.

Example 9. DP regulated and IL12 mediated functions

[00463] DD-IL12 function is characterized in vivo by evaluating the ability of tumor cells expressing these constructs to establish tumors and proliferate under the treatment of corresponding synthetic ligands e.g. Shield- 1, Trimethoprim or Methotrexate. 2-10 million HCT- 116 cells stably transduced with the constructs are subcutaneously xenografted with 50 matrigel into mice capable of producing functional B and NK cells. Approximately, two weeks after injection, when the tumors reach a size of approximately 300 cubic mm, mice are dosed with corresponding stabilizing ligands e.g. Shield- 1, Trimethoprim or Methotrexate at varying concentrations every two days. Shield- 1 is injected with a carrier consisting of 10%

Dimethylacetamide, 10% Solutol HS15, and 80% saline. Tumor volume and body weight are monitored twice a week and the experiment is terminated once the tumors reach 1000 cubic mm in size. Plasma and tumor samples are collected 8 hours after the last dose of the ligand and IL12 as well as the ligand levels are measured.

[00464] To evaluate the ability of IL12 expressing cells to form tumors, HCT-116 cells stably transduced with DD-IL12 constructs are pretreated with corresponding stabilizing ligands, Shield- 1, Trimethoprim or Methotrexate and subsequently xenografted into mice. Reduction in tumor growth and a concomitant increase in IL12 levels in ligand treated mice compared to untreated controls is indicative conditional regulation of IL12 in vivo.

Example 10. DP regulated recombinant IL12 mediated functions in T cells

[00465] Functional responses to DD-IL12 is evaluated in primary human T cells and in human cell lines/transformed hematopoietic cell lines e.g. Raji cells. Human T cells are purified from peripheral blood mononuclear cells (PBMCs) by negative selection using CD4+ T- cell isolation kit (Miltenyi Biotec, Germany). T cells are treated with growth media from HEK 293T cells expressing DD-IL12 constructs for 5 days. Cells are then activated with beads conjugated with- CD3/CD28 beads (Thermo Fisher Scientific, Waltham, MA) at the ratio of 3 beads per T cell and cultured for 3 days. Functional response to DD-IL12 is determined by measuring Interferon gamma in CD3 positive cells using flow cytometry. IL12 promotes the differentiation of naive T cells into Thl cells which results in the secretion of IFN gamma from T cells.

[00466] To evaluate IL12 induced phosphorylation of STAT4 (Signal transducer and activator of transcription 4), human T-cells are isolated from PBMCs and activated with

phytohemagglutinin (PHA, 2μg/ml) for 3 days followed by treatment with 50 IU/ml of

Interleukin-2 (IL2) for 24hrs. Cells are then washed, resuspended in fresh media and rested for 4 hrs. Supernatant from DD-IL12 expressing HEK293T cells is added to the primary cells, followed by incubation for 30 minutes. Cells are then harvested and STAT4 phosphorylation is analyzed using STAT4 antibody (Cell Signaling Technology, Danvers, MA).

Example 11. Functional analysis of DP regulated IL15-IL15Ra fusion molecule

[00467] Activation via IL15 can sustain T cell persistence by conferring a survival advantage. In addition, IL15/IL15Ra fusion molecule has been shown to confer a memory phenotype on T cells and increase proliferation of NK cells (Hurton (2016), PNAS, 113: E7788-7797; the contents of which are incorporated herein by reference in their entirety).

[00468] To evaluate signaling by DD regulated IL15-IL15Ra fusion constructs, NK-92 cells are incubated with HCT-116 cells expressing DD regulated ILlS-ILlSRa fusion constructs. Trans signaling by IL15/IL15Ra is expected to increase STAT5 phosphorylation in NK92, which is measured by western blotting, and by FACS. ProUferation of NK92 cells is also measured.

[00469] To evaluate the effect of DD regulated IL15-IL15Ra fusion constructs on primary T cells, cells are transduced with the fusion constructs. T cell proliferation in the absence of exogenous IL15 supplementation is measured. The T cell memory phenotype is measured by quantifying CD62L expression by FACS.

[00470] To assess if DD-IL15/1L 15R expressing T cells maintain prolonged persistence in vivo, DD modified T cells are injected into mice. Constructs are tagged with luciferase reporter to allow in vivo tracking in mice. Mice are treated with vehicle control or corresponding ligand, Shield- 1, Trimethoprim or Methotrexate depending on the construct utilized and monitored over a period of 40-50 days using bioluminescent imaging (PerkinElmer, Massachusetts). Mice treated with ligand are expected to retain T cells expressing DD-IL15/IL15Ra while T cells in vehicle control treated animals are not expected to persist.

Example 12. Evaluation of antitumor response of DD regulated pavloads in syngeneic mouse models

[00471 ] The efficacy of cancer immunotherapy in organisms with intact immune cells is evaluated using syngeneic mouse models e.g. pMEL-1 and 4T1 mouse models. Immune cells such as T cells and NK cells are isolated from syngeneic mice and transduced with DD regulated payloads such as DD-IL12 or DD-IL12 with DD-IL15 or DDlL15-IL15Ra,. Cells are then injected into mice bearing subcutaneous syngeneic tumors and treated with varying

concentrations of ligand, Shield- 1, Trimethoprim or Methotrexate, depending on the DD used. Mice treated with immune cells transduced with DD regulated payload are expected to have a reduced tumor burden when compared to control animals.

Example 13. Optimizing workflow for discovery of DD-regulated immunotherapeutic agents

[00472] To identify DD-IL12 constructs suitable for immunotherapy, constructs are introduced into cell lines e.g. HEK293T cells and Jurkat cells. The expression of the construct in the presence or absence of the corresponding ligand is tested. Constructs which show low basal expression in the absence of ligand and ligand-dose responsive regulation are selected for further analysis. If no DD- IL12 constructs show ligand-dependent expression, then constructs are redesigned and the experiment is repeated till a regulatable construct is identified Next, the ligand dependent regulation of the DD-IL12 constructs is tested in vitro in primary T cells. If the constructs show low basal expression in the absence of the ligand and ligand dose responsive expression, they are subject to in vivo PK/PD proof of concept experiments. The constitutively expressing IL12 constructs are transduced into T cells and IL12 expression is measured in parallel to the regulated construct. If no expression is detected in vitro, efforts are refocused on testing DD-IL12 constructs in vitro in T cells. In contrast, if the constitutive constructs show expression, then the expression of IL12 is measured in vivo.

[00473] To test in vivo PK/PD, mice are injected with T cells expressing DD-IL12 constructs and the test group is dosed with the ligand corresponding to the DD, while the control group is dosed with the appropriate vehicle control. IL12 expression is measured in the plasma of animals. Constructs that display ligand-dependent expression of IL12 are selected for in vivo functional proof of concept experiments. Parallel experiments are also conducted using the constitutive IL12 constructs. If constitutive IL12 expression is detected in vivo, then the constructs are selected for functional experiments.

[00474] The functional analysis in vivo is performed by testing if the constitutive and DD regulated IL12 cause a detectable increase in IFN gamma production in the plasma in a constitutive or ligand dependent manner respectively. If yes, then in vivo proof of concept is achieved and constructs suitable for immunotherapy are identified. If none of the DD regulated constructs show IFNgamma, then alternate dosing regimens are explored. If the constitutive IL12 constructs do not produce IFNgamma, then efforts are focused on identifying DD-IL12 constructs that show in vivo expression in T cells.

Example 14. Co-expression of DD regulated oavloads

[00475] The co-expression of DD regulated payloads has the potential to confer greater antitumor activity than the single agent alone. Cells are co-transfected with DD-IL12 and DD-IL15 or DD-IL15/IL15Ra constructs. Transfected cells are treated with stabilizing ligands depending on the DD utilized. DD-IL12, DD-IL15 and DD-IL15/IL15Ra expression in the media is measured by ELISA.

Example 15. IL12 expression in vivo

[00476] SKOV3 tumor cells expressing FKBP regulated-IL12 (#OT-IL12-009) or parental cells were implanted into SCID Beige mice (Day 0). Mice implanted with FKBP IL12 were dosed intraperitoneally with Shield- 1 (lOmg/kg or 30 mg/kg) or vehicle control on Day 43. Blood samples were collected prior to dosing (0 hour) as well as at 2, 6, and 24 hours after Shield- 1 dosing and serum human IL12 levels were measured using ELISA. The serum IL12

concentrations are presented in Figure 17. IL12 levels increased in Shield- 1 treated mice at 2 and 4 hours after treatment, compared with those at 0 hours. By 24 hours following treatment, the IL12 levels returned levels observed at 0 hours. The increase in 1L 12 at 2 and 4 hours after treatment was observed with both doses of Shield- 1. IL12 levels in vehicle control treated mice was generally lower than those in the Shield- 1 treated mice.

Example 16. Promoter selection for expression of IL12 constructs

[00477] The expression of SREs in a vector can be driven by either the retroviral long terminal repeat (LTR) or by cellular or viral promoters located upstream of the SRE. The activity of the promoter may vary with the cell type and thus promoter selection must be optimized for each cell type. To identify optimal promoters, IL12 fused to FKBP DD and an optional furin cleavage site was cloned into pLVX. IRES Puro vector and placed under the transcriptional control of a CMV promoter (OT-IL12-005, OT-IL12-009), a PGK promoter (OT-IL12-025, OT-IL028), an EFla promoter (OT-IL12-020, OT-IL12-026, OT-IL12-029), or without a promoter (OT-IL12- 027, OT-IL12-030). Constructs were transiently transfected into HEK293T cells and cells were treated with Shield- 1 for 24 hours. IL12 levels in the supernatant were measured using p40 ELISA and MSD immunoassay. As shown in Figure 18A and Figure 18B, among the IL12 constructs, expression of IL12 in the absence of Shield- 1 was very low to undetectable when the PGK, EFla or no promoter was utilized. In contrast, IL12 expression under the CMV promoter was detectable even in the absence of Shield- 1 suggesting that the CMV promoter is leaky. IL12 levels were increased with shield- 1 treatment with all constructs. The PGK promoter showed the smallest induction of IL12 with ligand treatment compared to other promoters.

[00478] 1L 12 constructs were also tested in HCT 116 cells and Raji cells over a range of shield- ldoses. IL12 constructs driven by the EFla promoter were transfected into HCT116 cells. Cells were incubated for 24 hours and then treated with Shield- 1 for another 24 hours. Secreted IL12 levels were then measured. As shown in Figure 18C, IL12 expression in HCT cells expressing OT-IL 12-026 (EFla) construct increased with increasing doses of Shield- 1 while the expression of IL12 was undetectable with vehicle control. OT-IL12-029 showed similar increase in IL12 expression with Shield- 1 expression, however, the levels of IL12 obtained with vehicle control treatment were comparable to shield- 1 treated cells. As expected the constitutive construct showed high levels of IL12 both in the presence and absence of Shield- 1, while the parental HCT116 cells did not secrete any ILl 2. Intracellular IL12 levels in HCT116 cells were compared with secreted IL12 levels. Cells were dosed with luM Shield-1 for 24 hours and IL12 were measured in the supernatant and within cells using MSD immunoassay. As shown in Figure 18D, secreted IL12 levels with the OT-IL12- 026 and 029 construct was increased with Shield- 1 treatment as compared to the vehicle control. However, intracellular concentration of IL12 did not increase with both constructs in Shield- 1 treated cells. The intracellular concentrations of IL12 were also 10-fold lower than secreted IL12 levels. These data suggest that Shield- 1 may be increasing the secretion of IL12 into the media.

[00479] A Shield- 1 dose responsive increase in secreted IL12 levels was observed with the expression of OT-IL12-025 (PGK) in Raji cells (Figure 18E). Taken together, these results indicate that transcriptional control of IL12 by EFla, or no promoter results in low basal expression in the absence of ligand and strong ligand dependent expression. In contrast, the CMV promoter results in high basal expression while only low levels of 1L12 are detected with PGK promoter.

Example 17. Effect of cytokines on T cell expansion and activation

[00480] To test the requirement of IL2 and IL12 for T cells expansion and activation, T cells were stimulated with soluble CD3/CD28, CD3/CD28 Dynabeads or left unstimulated for two days. Each of these groups was further split into two sub groups. One sub group was treated with IL2 and 100 ng/ml of IL12 while the second sub group was treated with IL2 only for the duration of the stimulation. For the soluble CD3/CD28 stimulated cells, a third subgroup mat was only treated with 100 ng/ml of IL12 was also included. T cell expansion over the course of 14 days was measured and the fold change in T cells expansion is shown in Figure 19A. CD3/CD28 dynabeads plus IL2 with or without IL12 had the most profound impact on T cell expansion followed by the T cells treated with soluble CD3/CD28 plus IL2 with or without ILl 2.

Unstimulated cells and cells treated with soluble CD3/CD28 cells that did receive 1L2 treatment were unable to expand over the course of the experiment. These results show that IL2 is required for T cell expansion, but IL12 may be dispensable.

[00481] The effect of IL12 on T cell activation was measured by determining the frequency of IFNgamma positive CD4+ and CDS + T cells. IFNg is produced by activated T cells. Three different stimulation protocols were used. In the first protocol, cells were stimulated with CD3/CD28 dynabeads for 2 days, following which the beads were washed off and the cells were treated with varying concentrations of IL12 for 7 days (from day 2 to day 9). At day 9, cells were restimulated with soluble CD3/CD28 and the frequency of IFNgamma positive cells was determined by FACS. The results are presented in Figure 19B as the percentage of cells. In the second protocol, following 2 days of CD3/CD28 dynabead stimulation, T cells were maintained in culture for a longer duration of 14 days i.e. from day 2 to day 16. At day 16 cells, were restimulated with soluble CD3/CD28. At day 16, the frequency of IFNgamma positive cells was measured. The results are presented in Figure 19C as the percentage of cells. In the third protocol, T cells were initially stimulated for 2 days with CD3/CD28 dynabeads and IL2, followed by treatment with IL2 only for 9 days (i.e. from day 2 to day 11), followed by IL12 treatment for 2 to 5 days. In the last two days of the experiment, cells were also restimulated with soluble CD3/CD28. IFNgamma positive CD4 and CD8 cells were measured using FACS. The third protocol mimics the environment that is presented to T cells in adoptive cell therapy, both during in vitro transduction and T cells expansion as well as the in vivo. The results are presented in Figure 19D as the percentage of cells. In both 7-day treatment with IL12 as well as 14-day treatment with IL12, shown in Figure 19B and Figure 19C respectively, re stimulation with CD3/CD28 cells at the end of the experiment increased the percentage of IFNgamma positive cells. A half maximum effective concentration (EC50) of IL 12 observed with the first protocol for CD8 cells was 50 pg/ml. The EC50 of IL12 observed with the second protocol was 12 pg/ml for CD4 cells and 65 pg/ml for CD8 cells. Long-term culture with CD3/CD28 further increased the dependence on re-stimulation and IL12 for IFNg production.

[00482] The results obtained with the third stimulation protocol are presented in Figure 19D. Immune cells treated with IL12 for the final 5 days of the experiment combined with CD3/CD28 restimulation showed the highest percentage of IFN gamma positive cells (EC50 = 24 pg/ml for CD4 cells and 40 pg/ml for CD8 cells), followed by cells that received HJ 2 for 2 days. Thus, T cells expanded in vitro can later differentiate in response to IL 12, but restimulation may be required for IFNg production.

[00483] Taken together these results indicate that IL12 can stimulate IFN production in T cells when restimulated with CD3/CD28.

Example 18. IL12 dependent, re-stimulation independent Thl markers

[00484] T cells require T cell receptor restimulation in vivo or in vitro stimulation with CD3/CD28 to produce IFNgamma. To study the effect of IL12 activity on T cells in the absence of restimulation, several T cell markers were explored. T cells were expanded using one of the following 4 expansions strategies (i) Day 10 cytokine switch from IL2 to IL12, CD3/CD28 stimulation from day 0 to day 10 with no restimulation (ii) Day 10 cytokine switch from IL2 to IL12, CD3/CD28 stimulation from day 0 to day 10 and restimulation at with CD3/CD28 from day 12 to day 14 (iii) Day 10 cytokine switch from IL2 to IL12, CD3/CD28 stimulation from day 0 to day3 with no restimulation (iv) Day 10 cytokine switch from IL2 to IL12, CD3/CD28 stimulation from day 0 to day 3 and rcstimulation at with CD3/CD28 from day 12 to day 14. Markers tested include CD69, IFNg, Perforin, CXCR3, Granzyme B, CCR5, CXCR6, Ki-67 and T-bet. IFNg appears to be the most robust and consistent marker for IL12 activity on human T cells, but requires re-stimulation of T cells to induce production. Thl markers which increase in response to IL12 in the absence of re-stimulation and IL2 (similar to in vivo conditions) include Ki-67, T-bet, Perforin, CXCR3, CCR5.

Example 19. Regulated expression of IL15-IL15Ra in T cells

[00485] DD regulated IL15-IL15Ra constructs such as OT-IL15-009 or constitutively expressed constructs such as OT-1L15-008 were transduced into T cells such as primary T cells or SupTl cells. The transduction was carried out at two different lentivirus concentrations, 5 μΐ and 20 μΐ for the DD regulated construct using Lenti boost™ (Sirion Biotech, Germany). 4 days after transduction, cells were treated with 10μΜ TMP or DMSO control for 24 and 48 hours. Samples were analyzed with an anti IL15Ra antibody using FACS. Additional controls samples such as cells treated with Lentiboost only, untransduced cells treated with DMSO or TMP, and Isotype controls were included in the FACS analysis. The FACS results are depicted in Figure 20A for 24 hours of TMP treatment and in Figure 20B for 48 hours of TMP treatment. In both figures, DMSO-A and TMP-A indicate cells treated with 5 μΐ of lentivirus and DMSO- B and TMP-B indicate cells treated with 20 μΐ of lentivirus. Treatment of T cells expressing OT-IL15-009 with TMP for 24 hours resulted in an increase in the expression of IL15Ra in T cells with both doses of lentivirus used. Additionally, very low levels of IL15Ra were detected in the DMSO treated samples under the same conditions as well as in untransduced T cells. As expected, the constitutively expressed construct, OT-IL15-008 showed high expression of IL15Ra. TMP dependent expression of OT-IL15-009 was not observed in SupTl cells (Figure 20A). Similar results were observed for both T cells and SupTl cells at 48 hours (Figure 20B). These results show that tight regulation of IL15-IL15Ra constructs can be achieved in primary T cells.

[00486] The surface expression of IL15 and IL15Ra was measured for OT-IL 15-008 and OT- IL 15-009. The percentage of cells expressing IL15, IL15Ra or both on the cell surface is presented in Table 17.

Table 17; Surface expression of IL1S and IL15Ra

[00487] As shown in Table 17, the percentage of cells with detectable surface expression of IL15 and IL15Ra was less than 5% with both constructs. Further, the percentage of cells with surface expression of IL15Ra was much higher than the percentage of cells with detectable surface expression of IL15.

[00488] The effect of increasing doses of TMP on ILl 5Ra expression in T cells was measured using the OT-IL15-009 construct. T cells were treated with a range of doses of TMP starting from 0.156 μΜ to 160 uM for 24 hours. IL15Ra expression was measured using FACS. As shown in Figure 20C, the percentage of IL15Ra expressing T cells with OT-IL 15-009 cells was detected even at the lowest concentration of TMP and the percentage of IL15Ra positive cells at the lowest concentration of TMP was higher than the untreated control. The percentage of IL15Ra cells increased with increasing doses of TMP.

Example 20. TMP dose responsive expression of IL15-IL15Ra

[00489] IL15-IL15Ra fusion constructs, OT-IL15-008, OT-IL15-009, and OT-1L15-010 were stably expressed in HCT116 cells treated with increasing doses of TMP ranging from 1 ΟμΜ, 33uM, and ΙΟΟμΜ TMP for 24 hours. Cell lysates were immunoblotted with anti IL15Ra antibody. As shown in Figure 21, IL15Ra expression of OT-IL15-009 was virtually undetectable in the absence of TMP, and addition of increasing doses of TMP resulted in an increase in IL15Ra levels. Modest increase in IL15Ra expression was observed with OT-IL15-010 construct with the addition of TMP. As expected, the constitutive construct, OT-1L15-008 showed strong expression of ILl 5Ra both in the presence and absence of ligand.

Example 21. Effect of IL15-IL15Ra on T cell persistence and T cell memory phenotvpe

[00490] The effect of constitutively expressed IL15-IL15Ra fusion construct, OT-IL15-008 on T cell persistence was measured in NSG mice. T cells were transduced with OT-IL15-008 and 4 million cells T cells were injected intravenously into NSG mice (number of mice =3). As a control, additional mice were injected with untransduced T cells. Blood samples were obtained from mice at 2, 3, 4, 5 and 6 weeks and analyzed by FACS for the presence of CD8 and/or CD4 positive human T cells expressing IL15 and IL15Ra. The percentage of human T cells in the blood was calculated as the percentage of total T cells i.e. human T cells (measured using anti- human CD45 antibody) and the mouse T cells and endothelial cells (measured using the anti- mouse CD45 antibody). As shown in Figure 22 A, the percentage of T cells in the blood at 2 weeks was greater in mice injected with T cells transduced with OT-IL15-008 compared to control mice that were injected with untransduced T cells. This observed increase in T cells decreased over 3,4, and 5 weeks, and the percentage of T cells was comparable between the two cohorts. At 6 weeks, one of the mice injected with OT-IL15-008 transduced T cells showed a higher percentage of human T cells in the blood. Thus, at 2 weeks, the frequency of human T cells in the blood is increased in the blood of mice injected with OT-IL15-008 transduced T cells.

[00491] The number of T cells in the blood was measured by comparing the number of human T cells in SO uL of mouse blood using anti-human CD4S antibody as a marker for human T cells and anti-murine CD3 antibody as a marker for murine endothelial cells. As shown in Figure 22B, the number of human T cells in the blood increased at 2 weeks in mice injected with OT-IL15- 008 transduced T cells, as compared to mice injected with untransduced T cells. The differential between the two cohorts was diminished at 3 weeks and 4 weeks. At 6 weeks, one of the mice injected with OT-IL15-008 transduced T cells showed a higher number of human T cells in the blood. Thus, at 2 weeks, the frequency and number of human T cells in the blood is increased in the blood of mice injected with OT-IL15-008 transduced T cells. These data support the role of IL15-IL15Ra fusion proteins in T cell persistence. The increased T cell frequency and number observed at 6 weeks in one of the mice may be due to graft versus host disease.

[00492] The effect of OT-IL15-008 expression on the CD4 and CD8 subset of T cells was measured prior to injecting into mice (Week 0) and 2 weeks after injection. As shown in Figure 22C, the ratio of CD4 and CD8 cells was 1 : 1 prior to injecting into mice. However, at 2 weeks, the proportion of CD4 positive cells was much higher than the CD8 positive cells in the transduced cells, indicating that OT-IL 15-008 causes a preferential expansion of CD4 positive cells. The expression of the OT-IL15-008 construct within the CD4 and CD8 subsets was measured using anti IL15Ra antibody. As shown in Figure 22D, prior to injections, 25 % of the OT-IL15-008 transduced CD4 T cells and CD8 T cells expressed IL15Ra. At week 2, the percentage of IL15Ra positive CD4 and CD8 T cells increased to 80% indicating a preferential expansion of T cells transduced with OT-IL15-008. As expected, untransduced control T cells were negative for IL15Ra expression.

Example 22. Promoter selection for expression of SREs in T cells

[00493] The expression of SREs in a vector can be driven by either the retroviral long terminal repeat (LTR) or by cellular or viral promoters located upstream of the SRE. The activity of the promoter may vary with the cell type and thus promoter selection must be optimized for each cell type. To identify optimal promoters, AcGFP (SEQ ID NO. 235) was cloned into pLVX. IRES Puro construct with a CMV or an EFla promoter. Patient derived T cells and Sup Tl cells were transduced with the constructs and GFP expression was measured at day 3 and day 5 after transduction using FACS. As shown in Figure 23, both the CMV promoter and the EFla promoter can drive the expression of GFP in SupTl cells and T cells. The percentage of GFP positive T cells was higher when GFP expression was driven by CMV promoter compared to an EFla promoter, both at 3 days and 6 days after transduction. In contrast, the percentage of GFP positive cells was much higher when GFP expression was driven by the EFla promoter when compared to the CMV promoter. Thus, the optimum promoter suitable for expression differs based on the cell type.

Example 23. Effect of cytokines on NK cell proliferation and activation

[00494] Immune cells such as Natural Killer cells depend on cytokines such as IL15 for their proliferation and survival. This dependence on cytokines can be used to test the functionality of DD regulated or constitutively expressed cytokines and cytokine fusion proteins.

[00495] The dependency of the NK-92 cells on cytokines for activation was tested. Cells were initially cultured for 3 days with IL2, following which, cells were washed twice and cultured in media without IL2 for 7 hours. The cells were cultured for 18 hours in the presence of IL12 (10 ng/ml) or varying concentrations of IL15 (100 ng/ml, 20 ng/ml, 4 ng/ml, 0.8 ng/ml, 0.16 ng/ml, 0.032 ng/ml, 0.0064 ng/ml and 0.00128 ng/ml). NK-92 cell activation in response to IL15 and IL12 treatment was evaluated by FACS analysis using a panel of markers whose increased expression is associated with NK activation. These include NKG2D, CD71, CD69; chemokine receptors such as CCR5, CXCR4, and CXCR3, Perforin, Granzyme B and Interferon gamma (IFNg). Prior to FACS analysis for IFNg, cells were cultured for 4 hours with Brefeldin A. NK cells respond to external stimuli such as cytokines in their environment through the

phosphorylation of proteins JAK/STAT, ERK, and p38/MAPK pathways which are important for cell activation, signaling and differentiation pathways. The phosphorylation of AKT, STAT3 and STAT5 in response to cytokine addition was measured by FACS. Since phosphorylation events are transient NK-92 cells were treated with the cytokines for 15 or 60 minutes, prior to the analysis. The fold change in mean fluorescence intensities compared to untreated for IL15 treatment are presented in Table 18. Table 18: IL15 induced markers

[00496] Treatment with IL15 resulted in an increase in the expression of CD69, CXCR4, Perforin, Granzyme B, and IFNg. The effect of IL 15 on these markers was dose dependent with a higher dose of IL15 resulting in a corresponding upregulation of markers. Phosphorylation of STAT5 was increased both at 15 and 60 minutes after the addition of IL2 or IL15. Taken together, these results show that cytokines can activate NK cells.

[00497] The fold change in activation markers observed with IL12 treatment are shown in Table 19.

Table 19: IL12 induced markers

[00498] Treatment with IL 12 resulted in an increase in the expression of markers CD69, CCR5, Perforin, Granzyme B, and IFNgamma. Further, IFNg levels secreted by NK-92 cells into the media was higher upon treatment with IL12 than those of untreated controls.

Example 24, Effect pf Kgftnd φη T cell proliferation

[00499] The effect of ligands specific to the SREs of the invention on immune cell proliferation was measured to identify concentrations of the ligand that did not inhibit T cell growth or survival. T cells derived from two different donors were stimulated with CD3/CD28 and treated with ligand TMP at doses ranging from 0.04 uM to 160 μΜ or DMSO. The percentage of divided cells within the CD4 and the CD8 populations of T cells was measured using FACS. Concentrations of IMP ranging from 0.04 μΜ to 40 μΜ showed no effect on the percentage of divided cells within the CD8 and CD4 populations, while 160 μΜ concentration of TMP resulted in an 70-90% reduction in the percentage of divided cells. Thus, the optimal concentration of TMP for T cell based experiments was determined to be less than 160 μΜ.

Example 25. IL12 expression in vivo; HCT116 tumor study

[00500] The HCT116 tumor study is established to correlate tumor size with serum IL12 levels before and after dosing with ligands such as Aqua Shield. DD regulated or constitutive constructs are transduced into HCT116 cells and the cells are injected subcutaneously at 10 million per injection into the flanks of CD-I nude mice, and SCID beige mice. Mice are allowed to develop tumors that are approximately 200-300 mm³ in size and then dosed with ligands. For example, Shield- 1 is dosed orally at 50mg/kg or vehicle control. The frequency of dosing is varied to identify the optimal dosage and frequency of dosage. Blood samples are collected prior to shield- 1 dosing as well as 2, 4, 6 and 24 hours after dosing. Plasma IL12 levels are measured and correlated with tumor volume. When the tumors reach approximately 1000 mm³, mice are sacked and plasma, tumor and kidney samples are collected. Tumor growth is expected to correlate with plasma IL12 levels such that larger tumors secrete more 1L12.

Example 26. EBV tumor antigen mediated TCR re-stimulation in vivo

[00501] Human T cells engineered to express DD regulated cytokines are not antigen specific which limits their functional analysis in mice. However, functionality of T cells in vivo requires their restimulation which occurs upon engagement with the antigen. This requirement for antigen mediated restimulation can be mimicked experimentally in mice using the Epstein Barr Virus (EBV) antigen. Approximately 90% adults have a current or a previous EBV infection.

Additionally, the major histocompatibility group HLA-A02 has been associated with the decreased risk of developing EBV positive Hodgkin's lymphoma, suggesting that the CTL peptide epitopes that promote EBV clearance are presented by HLA-A02. Tumor cell lines that are HLA-A02 positive e.g. Raji cells are used for in vivo studies. Primary human T cells obtained from various donors are expanded with CD3/CD28 dynabeads. To test reactivity of T cells to the EBV antigen, EBV positive Raji cells and EBV negative Ramos cells are used. The involvement of HLA-A02 in antigen recognition is tested using anti-HLA antibodies with both cell. Cell killing assays are performed by incubating T cells with fluorescently labelled Raji cells or Ramos cells and the ability of the donor T cells to preferentially kill Raji cells is evaluated. The activation of T cells in response to interaction with EBV antigen is measured by culturing mitomycin treated Raji or Ramos cells with fluorescently labelled T cells. The activation and proliferation status of T cells is examined by measuring expression of IL2, IFNg, CD 107a, Granzyme, Perforin. Since most humans have been exposed to EBV, the donor T cells in most instances are expected to be immunoreactive to Raji cells but not to Ramos cells. It is likely that T cells reactive to Raji cells will be positive for markers of T cell activation such as IL2, Granzyme and Perforin.

Example 27. Ligand regulated expression of IL12 in T cells

[00502] T cells from human donors were thawed on day zero and stimulated with

aCD3/aCD28 beads. On day one, cells were transduced with either DD regulated IL12 constructs, OT-IL12-026 and OT-1L12-029; or were left untransduced. The puro titer of the OT- IL12-026 and OT-IL12-029 were 6e⁷ TU/mL and 5e⁷ TU/mL respectively. Cells were allowed to recover and then treated with Shield- 1 for 48 hours. IL12 levels were measured in the supernatant using an immunoassay, MSD assay. The results are shown in Table 20.

Table 20: IL12 levels in T cells

[00503] As shown in Table 20, IL12 levels were increased 50-fold in OT-IL12-026, the construct without a cleavable linker and increased 22-fold in OT-1L 12-029, the construct with the cleavable furin linker. As expected, no IL12 expression was seen in the untransduced parental T cells. Taken together, these data demonstrate that IL12 secretion can be induced in T cells transduced with DD regulated IL12 and can be regulated by the addition of ligand.

Example 28. Kinetic and concentration-dependent regulation of IL12 in human T cells

[00504] On day 0, the human donor T cells were thawed and cultured in the presence of IL2 and CD3/CD28 beads. On day 1, cells were transduced with lentivirus for construct OT-IL12-026 at MOIs of 40, 13.3 or 4.4. 24 hours following the transduction, the media was replaced with fresh media containing CD3/CD28 beads and IL2. On day 13, the cells were debeaded by centrifuging cells and resuspending the cell pellet in media with IL2. Cells were then plated at 100,000 per well on U-bottom 96-well plate. On day 14, cells were treated for 24 hours with varying concentrations of Shield-1 or vehicle control. On day 15, the supernatant was harvested and IL12 p40 levels were measured by MSD assay. The results are shown in Table 21. In the Table 21, MOI stands for multiplicity of infection. Table 21; IL12 levels in T cells

[00505] As shown in Table 21, all three MOIs showed a Shield-l dose dependent increase in IL12 levels. Additionally, IL12 levels secreted by T cells was also proportionate to the MOI, with the higher MOI showing the highest IL12 levels for any given concentration of Shield-l treatment. The dose response curve was similarly shaped for all three doses was similar, but the absolute values were different for each dose. At higher concentrations of Shield-l, i.e. greater than 5μΜ, viability was reduced as cells started to die likely due to the high concentration of vehicle. The ECso which is the half effective dose of Shield-l for each MOI is show in Table 22.

Table 22; Shield-l ECv in T cells

[00506] A time course experiment was also performed using Shield-l at a dose of 1 μΜ. T cells transduced with OT-IL12-020, OT-IL12-026 or empty vector were treated with Shield-l or vehicle control for 4, 8, 16 and 24 hours. IL12 p40 levels were measured using MSD assay. The results are shown in Table 23. The stabilization ratio was measured as the ratio of expression of IL12 in the presence of the stimulus to the expression in the absence of the stimulus.

Table 23; Time course of IL12 expression

[00507] As shown in Table 23, OT-IL 12-026 construct showed an increase in IL12 levels over the course of time, when compared to cells treated with vehicle control. At 4 hours after treatment, the stabilization ratio IL12 levels were increased 2.7-fold compared to vehicle, at 8 hours IL12 levels increased by 6-fbld; at 16 hours, the levels increased by 35-fold, and by 24 hours the IL12 levels increased to 14-fold compared to vehicle control. The constitutive construct OT-IL12-020, showed consistently high expression of IL12. A decrease in the expression of the constitutive construct was observed at 16 hours. However, the levels of IL12 were still much higher than OT-IL 12-026, both in the presence or absence of Shield- 1. As expected the vehicle control showed little to no expression of IL12. Taken together, these data demonstrate the kinetic regulation of DD-IL12 secretion in T cells with fine-tuned control compared to the elevated levels of constitutive IL12 secretion.

[00508] In another study, it was shown that CD8 cells expand more than CD4 cells, and that T cells (especially the CD8+ subset) lose IL12 expression during expansion. Restimulation of the cells with CD3/CD28 beads at day 14 increased the frequency and expression level of IL12 as compared to cells at day 14 that had not been restimulated.

[00509] To further evaluate restimulation, OT-IL12-020, OT-IL12-026, vehicle only and empty vectors were tested in T cells at day 0, 7 and 14 post transduction. The CD8+ subset increased overtime in vitro, the frequency of IL12p70+ T cells decreased over time in culture but IL12 can be re induced with CD3/CD28 and were shown to increase with restimulation on day 14. Shield- 1 was found to increase production of IL12, but not IFNy, by T cells in vitro on Day 15 post transduction. In this study, basal levels of DD-IL12 were sufficient (remained above the EC50 for Thl differentiations) to skew non-transduced cells towards a Thl phenotype during in vitro T cell expansion.

Example 29. In vivo regulation of DD-IL12 in T cells

[00510] T cells were activated, transduced with OT-IL12-020, OT-IL12-026 or empty vector and expanded over a period of 10 days as discussed in Example 28. On day 0 of the assay, T cells were injected in vivo into NSG mice, following which Aquashield was injected into mice. On day 3, Aquashield was injected at a dose of 100 mg/kg. Prior to the Aquashield injection, a blood sample was collected from the mice and served as the 0-hour time point or as the untreated control. A repeat dose of 100 mg/kg was administered at 4 hours following the first dose. Blood samples were collected from mice at 4, 8, 12 and 24 hours following the initial dose. Plasma IL12 levels were measured using MSD assay and the results are shown in Table 24.

Tflble 24; IL12 l_mls in T yyllg in vivo

[00511] As shown in Table 24, plasma IL12 levels for OT-IL 12-026 were much higher at 4 hours following first dose and peaked at 8 hours, following which, they decreased at 12 hours and decreased even further at 24 hours. Virtually no IL12 was detected in the plasma of vehicle control treated mice suggesting low basal expression of the construct. These observations were also reflected in the destabilization ratio of OT-IL 12-026, which was 0.0035, indicating strong destabilization in the absence of ligand and stabilization ratios of 18.17, 27, 29.37 and 7.65 respectively for 4, 8, 12 and 24 hours of Shield-1 treatment of OT-IL 12-026 expressing cells. The stabilization ratio was measured as the ratio of expression of IL12 in the presence of the stimulus to the expression in the absence of the stimulus. The destabilization ratio was measured as the ratio of expression of IL 12 in the absence of the shield-1 to the expression of IL12 that is expressed constitutively. As expected, plasma of mice injected with T cells expressing the constitutive construct showed high IL12 expression both at 0 and 24 hours, while no IL12 was detected in the plasma of mice injected with T cells expressing empty vector.

[00512] 1L12 levels in response to varying doses of Aquashield was also tested in vivo. 3 days after injecting 25 million T cells transduced with OT-IL12-026, NSG mice (n=4) were dosed orally with Aquashield at 50 or 100 mg/kg at 0 and 48 hours. Blood samples were collected at 0, 4, 8 and 24 hours after the first dosing. The mice were allowed to rest for 24 hours, following which, they were dosed with Aquashield again (i.e. at 48 hours since the initial dose) and blood samples were collected at time points identical to the first dose. Plasma IL12 levels were measured using p70 MSD assay. The results are shown in Table 25. The stabilization ratio was measured as the ratio of expression of IL12 in the presence of the stimulus to the expression in the absence of the stimulus. The destabilization ratio was measured as the ratio of expression of IL12 in the absence of the shield- 1 to the expression of IL12 that is expressed constitutively.

Tflbls 25; $hi¾Jtf-l flpsg r$sp9iise

[00513] Similar to the single dosing experiments, described in table 24, mice treated with lOOmg/kg showed peak plasma IL12 in Aquashield treated mice at 8 hours and declined by 24 hours, reaching levels comparable to the IL 12 levels at 0 hours, and remained low at 48 hours. 4 hours after the second dose, IL12 levels began to increase, reaching a peak level at 8 hours following the second dose and reached baseline levels by 24 hours after the second dose e.g., 72 hours after the initial dose. Mice treated with the SO mg/kg Aquashield showed peak plasma IL12 at 4 hours after dosage, which continued to decline at subsequent time points tested. These trends were also reflected in the stabilization ratios calculated for each of the time points with both doses. With higher dose of Shield- 1, higher stabilization ratios were observed at each time point suggesting the dose responsive elevation in IL12 levels. The destabilization ratios were also measured at both doses of Shield- 1 by injecting a small cohort of mice with T cells expressing the constitutive construct, OT-IL12-020. At time point zero, the stabilization ratio for 50 and 100 mpk was 0.004 for both doses, suggesting low basal expression in the absence of ligand. These data also suggest that it is possible to restimulated the T cells transplanted into mice to produce IL12. Additionally, IFNy was produced in the basal state and it was upregulated concurrent with IL12.

[00514] An additional cohort dosed at 10 mg/kg was also included to fully define the dose response curve. These results are shown in Table 26. Table 26: IL12 response to second dose of Shield- 1

[00515] Compared to the 50 and 100 mg/kg doses, the 10 mg/kg dose showed very little IL12 expression mat was comparable to the expression observed with the vehicle control treatment. A small increase in IL12 levels was observed at 4 hours with the lOmg/kg dose, but the stabilization ratio observed at this time point was much lower than observed with the 50 and 100 mpk doses. Taken together, these data demonstrate that in vivo repeat dosing of the ligand, Aquashield results in a dose dependent increase in IL12 levels. The lack of IL12 in the plasma at 24 hours after dosing indicates that IL12 is cleared from the plasma by this time point resulting in distinct peaks of circulating IL12.

Example 30. In vivo time course study of IL12 levels in mice

[00516] HCT116 parental cells or cells transduced with IL12 constructs (OT-IL12-020, OT- IL12-026, or OT-IL12-029) were injected into immune compromised CD1 nude mice (n=4 per group) according to the study design in Table 27 below.

Table 27. Study Design

[00517] The mice were bled (blood harvested for plasma PK and IL12 MSD) at day 14 after subcutaneous injection of 5x10⁶ cells (day 0), and 6, 10, and 24 hours post the day 15 dosing. At the end of the study, tumor and kidneys were minced with the razor in 500 ul PBS, spun down, and supernatant isolated for IL12 Meso Scale Diagnostic(MSD) assay. [00518] As shown in Figure 24A, the basal plasma IL12 levels of the DD constructs were high, but the OT-IL12-026 and OT-IL12-029 constructs were still 100-fold lower than the constitutive (OT-IL 12-020) construct. When Figure 24A is shown as fold change from pre-dose plasma, OT- IL 12-026 shows regulation at 6 and 10 hours. Figures 24B and 24C show that IL12 is detectable in kidney (Figure 24B) and tumor (Figure 24C) and the levels coordinate with plasma levels.

Example 31. In vivo time course study of IL12 levels in mice

[00519] HCT116 parental cells or cells transduced with IL12 constructs (OT-IL12-020, OT- IL 12-026) were injected subcutaneously into Matrigel plus in female NSG mice (implant 200 ul matrigel plug with lxlO⁷ cells) (n=4) according to the study design in Table 28 below.

Table 28. Study Design

[00520] Terminal collection of plasma (for IL12 MSD), plug supernatants and kidneys were collected. As shown in Figure 25 A, regulation of IL12 was achieved in vivo with high dose Aquashield. There was less regulation observed in the plasma (Figure 25B) and there was some flexi-IL12 detected in the kidneys (Figure 25C).

Example 32. Shield-1 Can Induce ~40-50x Increases in IL12 Production bv Primary

Human T Cells Transduced with the IL12-026 Construct

[00521] On Day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 bead:cell ratio. The next day, lentiviruses (empty vector (pLVX-EFla- IRES-Puro), OT-IL12-020 (constitutive), or OT-IL12-026 (regulated)) were added at a multiplicity of infection (MOI) of 10 in the presence of LentiBOOST and 5% FBS. On day 2, the cells were washed to remove the LentiBOOST and the bead:cell ratio was reduced to 1:3, and fresh 10% media and IL2 were added. On days 6, 9, and 13 the cells were counted for equal cell number plating, media replaced, ligand was added, and cells were either left unstimulated or restimulated with soluble ImmunoCult™ Human CD3/CD28 T Cell Activator (StemCell Technologies). After overnight incubation (on days 7, 10, and 14), the supernatants were collected for IL12p40 and p70 MSD assay, and transduction efficiency was analyzed by FACS. OT-IL12-026 T cells were found to be 7% transduced, and OT-IL12-020 (constitutive) T cells were 13% transduced on day 7. Restimulation was shown to increase the expression of IL12 (Figure 26A). Ligand increased production of 1L12 by 10-day expanded OT-IL12-026 expressing T cells by 40-50 fold (Figure 26B and Figure 26C).

Example 33. Dose Response of Shield-1 on Transduced T Cells

[00522] Human T cells were activated with CD3/CD28 Dynabeads (Life Technologies) for 1 day prior to transduction with lentiviruses (OT-IL 12-026 or vector control), followed by 12-13 days of expansion in culture. T cells that had been transduced with different amounts of virus (4-40 MOI) were exposed to either a dose response of Shield-1 for 24h (left panel). T cells that had been transduced at an MOI of 14 were treated with luM Shield-1 or vehicle control for increasing amounts of time (right panel). The levels of IL12 that had accumulated in the supernatants (from 100,000 cells per 200uL media) were measured using human IL12p40 MSD V-plex assay kits (Meso Scale Discovery).

[00523] From the analysis, it was shown that the increase in IL12 production by T cells expressing OT-IL 12-026 is dose responsive to the ligand, Shield-1 Figure 27A, and accumulates overtime Figure 27B.

Example 34. In Vivo Dose Response, and Repeat Dosing of AquaShield in NSG Mice with Transferred T Cells Expressing QT-IL12-026

[00524] Primary human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 beadxell ratio. The next day, lentiviruses (OT-IL12-020 (constitutive), OT-1L12-026 (regulated), or vector control) were added at a multiplicity of infection (MOI) of 10 in the presence of LentiBOOST and 5% FBS. The following day, T cells were washed to remove the LentiBOOST and the bead:cell ratio was reduced to 1:3, and fresh 10% media and IL2 were added. The T cells were expanded for a total of 10 days, and then 25x1ο⁶ vector control or OT- IL12-026 transduced T cells or lOxlO⁶ constitutive OT-IL12-020 transduced T cells were transferred into NSG mice (study day 0). Three days after cell transfer, the animals were dosed with either vehicle or AquaShield (10, 50 or lOOmg/kg). Blood was sampled for plasma analysis of IL12p70 by MSD assay at 0, 4, 8, and 24h post dosing (Figure 28A). Clear dose responsive increases in plasma IL12 was observed. [00525] On day 5 post T cell transfer, animals were dosed a second time with AquaShield (Figure 28B). A second increase in plasma IL12 was observed upon repeat dosing with

AquaShield.

Example 35. In Vivo Regulation of DD-12 Expressed in T Cells

[00526] To determine whether ligand can stabilize DD-IL12 in vivo upon sequential dosing of AquaShield, T cells are transduced with DD-IL12-expressing constructs (OT-IL12-020 or OT- IL 12-026) and implanted into mice (n=4 per group) (day 0) as outlined in the study design below.

Table 29. Study Design

[00527] For each group, a pre-bleed sample is collected as well as samples at 4 hours and 24 hours after each dose. At the end of the study, tissue and organ samples are collected. FACS analysis is conducted to determine cell numbers and Thl markers.

[00528] On Day 0, primary' human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 bead:cell ratio. The next day, lentiviruses (empty vector (pLVX-EFla- IRES-Puro), OT-IL12-020 (constitutive), or OT-ILl 2-026 (regulated)) were added at a multiplicity of infection (MOI) of 10 in the presence of LentiBOOST and 5% FBS. On day 2, the cells were washed to remove the LentiBOOST and the bead:cell ratio was reduced to 1:3, and fresh 10% media and IL2 were added.

[00529] In vitro evaluation of these cells is shown under Figure 26A-26C.

[00530] After 10 days of expansion, T cells were injected into NSG mice (12 x 10⁶ cells injected, cells were 15% (constitutive) and 7.5% (regulated) IL12 positive by FACS). For each group, a pre-bleed sample was collected as well as plasma samples at 4 hours and 24 hours after each dose. At the end of the study, tissue and organ samples are collected. FACS analysis was conducted to determine T cell numbers in the blood and to assess Thl phenotypic markers.

[00531] As shown in Figure 29A, IL12 expression in response to sequential pulsed doses of ligand (50 mg/kg Aquashield administered orally on day 4 and 6 (50 mpk Aquashield q48hr)) was elevated in the plasma of mice with T cells expressing OT-IL 12-026 as compared to the vehicle treated controls. T cells expressing the empty vector control did not produce IL12. T cells transduced wilh OT-IL12-020 (IL12-020), the constitutive control, produced IL12 throughout the time course.

[00532] In Figure 29B, elevated plasma IL12 expression in response to sequential pulsed doses of ligand (50 mg/kg Aquashield administered orally for 4 days (day 3-6) (50 mpk Aquashield QDx4)) was seen in mice bearing OT-IL 12-026 expressing T cells as compared to the vehicle treated controls. Cells transduced with OT-IL12-020 (IL12-020), the constitutive control, produced IL12 throughout the time course.

[00533] Figure 29C shows the IL12 expression over 11 days for the constitutive construct OT- IL12-020 (IL12-020). Ligand-regulated expression of IL12 from T cells expressing DD-IL12 from the construct OT-IL 12-026 was seen in mice treated with 50 mg/kg Aquashield administered orally on day 5 and 10 (50 mpk Aquashield d5/10). T cells expressing the empty vector control did not produce IL12.

[00534] Figure 29D shows ligand-induced regulation of plasma IL12 expression from T cells expressing DD-IL12 from the construct OT-IL 12-026 when mice were treated orally with 50 mg/kg Aquashield on day 10 (50 mpk Aquashield dlO). The single ligand pulse increased plasma IL12 levels over those detected in vehicle-treated control mice harboring OT-IL12-026 expressing T cells.

[00535] Regulation of IL12 for all constructs shown in Figures 29A-29D did not impact IFNy levels, instead the levels of IFNy gradually rose over time. This is likely due to the exposure of the T cells to IL 12 in culture during the in vitro expansion phase. However, ligand-induced regulation of IL12 increased granzyme B (GrB) (Figure 29E) and perforin expression (Figure 29F) by CD8+ T cells in vivo at day 7 post in vivo T cell transfer.

Example 36. Effect of PGK Promoter and N-terminal FKBP

[00536] HEK293T cells were transiently transfected with Lipofectamine 3000 and 2ug plasmid DNA each of: OT-IL12-019 (PGK promoter), OT-IL12-020 (EFlalpha promoter), OT-IL12-025 (PGK promoter, C-terminal FKBP domain), OT-IL12-026 (EFlalpha promoter, C-terminal FKBP domain), OT-IL12-046 (N-teiminal FKBP). Ligand (luM Shield-1) was added one day after transfection, and the cells were further cultured for 2 more days. IL12 secretion into the supernatants was quantitated by IL12p40 MSD assay. Genomic DNA (gDNA) and messenger RNA (mRNA) was purified from the cells. The levels of construct DNA integration into the cellular genome and levels of IL12 mRNA expression were quantitated by qPCR using primers specific to the WPRE element and IL12 within the respective constructs. [00537] The gDNA qPCR analysis demonstrated that the FKBP DD-containing constructs had integrated to similar levels within the cellular genomes, and that the PGK promoter, as expected, generated less IL12 mRNA expression than the EF1 alpha promoter (Figure 30A).

[00538] Due to the lower levels of mRNA transcription induced by the PGK promoter, the IL12p40 MSD assay also demonstrated that the PGK promoter reduced both basal and peak IL12 levels of secretion as compared to the construct using the EF1 alpha promoter. The lower basal levels of IL12 production downstream of the PGK promoter resulted in ~2 fold improved ligand- induced IL12 regulation as compared with the construct with the EF1 alpha promoter (Figure 30B). More specifically, the ligand-induced regulation of IL12 expression increased from 6-fold to 13-fold with the change from the EF1 alpha to the PGK promoter, respectively.

[00539] Constructs containing FKBP either at the N-terminus or at the C-terminus of IL12 were integrated similarly into the cellular genome and generated similar levels of mRNA (Figure 30A). However, while C-terminal containing FKBP constructs regulate IL12 expression, the N- terminal-containing FKBP construct failed to regulate IL12 expression (Figure 30B).

Examplg 37_T Kintfiys φί ligand-flgpgnflgnt stabilization yf DD-IL15- IL15Ra

[00540] The on/off kinetics of ligand-dependent stabilization of DD-IL15-IL15Ra was measured in CD4 positive T cells. T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Lentivirus was added to wells in the presence of

LentiBoost reagent, and cells were incubated for another 24 hrs and washed. Cells were resuspended in fresh media, and media was added every 2-3 days to expand and maintain cells at O.S-lxlOVml. After 7 days of expansion, T cells transduced with the ecDHFR DD-IL15-IL15Ra fusion construct (OT-IL15-009) were treated with ΙΟΟμΜ ecDHFR ligand Trimethoprim (TMP) or vehicle control, DMSO. At multiple time points (i.e., 1, 2, 4, 6, 8, 15, 22 and 24 hrs) after TMP treatment, the transduced T cells were collected and analyzed for ILlSRa surface expression using anti-IL15Ra antibodies by flow cytometry. Untransduced T cells were used as a negative control. The T cells were sorted into CD4 positive and CD8 positive populations and the percentage of ILlSRa positive CD4 positive T cells was analyzed. Figure 31 shows the kinetics of surface expression of IL1 SRa on CD4 T cells after TMP treatment. Among the CD4 positive T cells transduced with the OT-IL15-009 construct, the proportion of cells with surface expression of IL15Ra remained similar for both TMP treated and DMSO treated cells until 2 hrs after TMP treatment, and was comparable to that of untransduced cells. However, from 4 hrs after TMP treatment, the cells transduced with the OT-IL15-009 construct and treated with TMP exhibited an increased proportion of cells with surface expression of IL15Ra. This trend was observed until 22 hours after treatment with TMP. The CD4 positive T cells with surface - expressed IL15Ra cells constituted ~1% of untransduced cells, indicating that the proportion of cells that expressed endogenous IL15Ra is low.

Example 38. Ligand-dependent stabilization of DD-IL15-IL15Ra fusion molecules in vivo

[00541] To examine whether ligand treatment induces stabilization of the DD-IL15-IL15Ra fusion molecules in vivo, HCT116 cells transduced with the OT-IL15-009 construct were implanted subcutaneously in BALB/c nude mice and treated with TMP. TMP was orally administered to mice at a dose of 100 mg/kg, twice a day for 11 days after implantation, followed by administration of TMP at the dose of 300 mg/kg, twice a day for 6 days. As a negative control, separate mice implanted with HCT116 cells transduced with the OT-IL15-009 construct were treated with the vehicle twice a day for 17 days. At 4 hrs after the last dosing of TMP or the vehicle control, tumors were harvested from the mice and analyzed for the levels of IL15-IL15Ra fusion molecules by western blotting. As shown in Figure 32, HCT116 tumors harvested from mice treated with TMP exhibited elevated levels of IL15-IL15Ra expression, compared to tumors treated with the vehicle. The GAPDH level was analyzed as a loading control. These data show that administration of ligand enabled stabilization of the DD-IL15- IL15Ra fusion molecule in vivo.

[00542] Consistent with the efficacy of TMP-dependent IL15-IL15Ra stabilization in vivo, elevated levels of TMP (399.38 ng/g tumor) were observed in HCT116 tumors harvested from mice treated with TMP for 17 days. The levels of TMP associated with HCT116 tumors were considerably higher than those observed in mouse plasma at day 3 (15.67 ng/ml plasma) and at day 17 (99.5 ng/ml plasma), indicating that the orally administered TMP was successfully delivered to and accumulated in HCT116 tumors implanted in mice.

Example 39. Shedding resistant IL15-IL15Ra constructs

[00543] To maintain the efficiency of the trans-presentation of IL 15 via the IL 15-IL 15Ra fusion molecule, the IL15-IL15Ra shedding needs to be prevented. For this purpose, new DD-IL15- IL15Ra and constitutive IL15-IL15Ra constructs are designed through a variety of modifications on the IL15-IL15Ra fusion molecule. For example, the IL15 molecule or the IL15Ra molecule is truncated or mutated to remove presumable cleavage sites. IL15Rahas a cleavage site

(PQGHSDTT from the position 168 to 175 of SEQ ID NO. 150) in the extracellular domain immediately distal to the transmembrane domain of the receptor, as described by Bergamaschi C etal. (2008). J Biol Chem ;283(7):4189-99; Anthony SM et al. (2015). PLoS One. 10(3):

eO 120274), and mternational Patent Application Publication Nos. WO2014066527 and

WO2009002562 (the contents of each of which are incorporated herein by reference in their entirety). Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) has been implicated as a protease that cleaves between glycine (at the position 170 of SEQ ID NO. 150) and histidine (at the position 171 of SEQ ID NO. 150) and generates a naturally occurring soluble form of IL15Ra. The same mechanism can be responsible for the IL15-IL15Ra shedding. Hence, the cleavage site of IL15Ra is mutated such that cleavage by an endogenous protease is prevented. The mutation of the cleavage site is introduced by substitution, insertion or deletion of amino acid residues. The IL15-IL15Ra fusion molecule is also modified such that the full- length or truncated IL15-IL15Ra fusion molecule is fused to heterologous hinge domains and/or heterologous transmembrane domains. As non-limiting examples, variants of IL15Ra can be utilized. Additionally, the length and sequence of the linkers that connect IL15 and IL15Ra are modified.

[00544] To confirm that the modifications on the IL15-IL15Ra fusion molecule prevent shedding, the new DD-IL15-IL15Ra or constitutive IL15-IL15Ra constructs are introduced into HCT-1 16 cells. Surface expression of IL15 and IL15Ra on the HCT-116 cells is examined by flow cytometry using anti-IL15 and IL15Ra antibodies to assess surface IL15-IL15Ra shedding. The presence or absence of IL15 in the cell culture supernatant is also analyzed by MSD assay. As a functional assay based on the sensitivity of NK cell activation by shed IL15 in tumor supernatant, the transwell assay is conducted using HCT-116 cells transduced with new DD- IL15-IL15Ra or constitutive IL15-IL15Ra expressing constructs and NK cells. The new DD- IL15-IL15Ra-expressing constructs that do not induce activation of NK cells in the presence of ligand and the new constitutive IL15-IL15Ra-expressing constructs that do not induce activation of NK cells are chosen for use in future experiments.

Example 40_T Rfflilafcd yxpr$ssi_Yn qf IL15-IL15Ra fr^n mylgflilg with C-ftrminfll DP

[00545] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subunit (IL15Ra) and a human DHFR (DO). These fusion molecules were cloned into pLVX- EFla-IRES-Puro vector.

[00546] To test ligand dependent IL15-IL15Ra production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5% C02. Cells were then transfected with lOOng of constitutive IL15-IL15Ra (OT- IL15-008) or DD linked IL15-IL15Ra (OT-1L15-037 or OT-IL15-040) using Lipofectamine 2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 50uM Trimethoprim (TMP) and further incubated for 48 hrs. Cells were harvested and IL15 levels are analyzed via western blotting using human IL15 antibody (Abeam, Cambridge, UK). The molecular weight of IL15Ra in OT-IL15-037 and OT-IL15-040 appeared to be the same as OT-IL15-008. [00547] To test if IL15 is shed into the media, supernatant from HEK293 cells expressing IL15- IL15Ra fusion constructs was subject to immunoassays such as MSD (Rockville, Maryland). 48 hours after transfection, cells were analyzed and, as expected, constitutive IL15-IL15Ra construct OT-IL 15-008 showed high surface expression of 1L15 in the presence and absence of ligand. OT-IL15-037 and OT-IL15-040 showed the ligand (Trimethoprim) dependent surface expression of IL15 and IL15Ra (Figure 33). The detection of membrane bound IL15-IL15Ra fusion constructs in the supernatant suggests that IL15 constructs are likely shed from the cell surface.

Example 41. Effect TMP exposure to TMP in vitro on membrane bound IL1S expression

[00548] In order to determine if the dose and time of exposure to TMP in vitro influenced membrane bound IL15 expression, an in vitro dose response study was conducted with T cells expressing OT-IL15-073.For this purpose, T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Lenti virus was added to wells. After 24 hrs, fresh media was added every 2-3 days to expand cells while maintaining cells at O.S-lxlO^ml. On day 11 of expansion. T cells treated with TMP starting at 100 uM, lOx dilutions and 9 points were analyzed after 2 hours in culture (washed 3x after TMP addition, fresh media added without TMP for 22 hours), 6 hours in culture, or 24 hours in culture and the results are shown in Figure 34A. As shown in Figure 34B and Table 30, this study showed that TMP ligand regulates membrane bound IL15 expression and the dose and time of exposure to TMP in vitro influences membrane bound IL1S expression.

Table 30. Membrane Bound IL15 Expression

Example 42, Related memprane ρομηά IL15 expression in vivo

[00549] To evaluate regulation of membrane bound IL15 in vivo, 2 constructs were selected for evaluation in vivo. Four group of T cells were used for this study and are outlined in Table 31. In Table 31, "N" represents the number of mice in each group.

Table 31. T Cell Groups

[00550] The T cells which were to be used as part of the in vivo study were evaluated 6 days post transduction, day of implant (day 9 post transduction) and 13 days post transduction and the cells in Groups 2-4 showed expression of the constructs.

[00551] T cells outlined in Table 31 were administered to mice by intravenous administration (3.9 x 10⁶ cells per mouse implanted). On day 3 the mice were dosed with 500 mg/kg of TMP 3 times (4 hours between doses) and bled 2 hours after each dose. The mice were again bled on day 4, 24 hours after the first TMP dose.

[00552] Figures 35A-35C show the expression of membrane bound IL15, 2, 6, 10, and 24 hours after the first TMP dose, using 1L15 staining (Figure 35A), IL15Ra staining (Figure 35B), and IL15/IL15Ra double ++ staining (Figure 35C). Figure 35D are FACS plots for each mouse 10 hours after the first TMP dose. Figure 35E shows the expression of membrane bound IL15 in blood 2, 6, 10, and 24 hours after the first TMP dose and Figure 35F shows the plasma TMP levels 2, 6, 10, and 24 hours after the first TMP dose.

Example 43. Effect of long term intraperitoneal (IP) or oral (PO) TMP dosing on T cell function

[00553] In this study, T cells transduced with OT-IL15-071 or OT-IL15-073 (no lentiBoost) were administered intravenously to mice (15 x 10⁶ per mouse). 6 study groups were evaluated for this study: (1) untransduced, (2) OT-IL15-071 T cells, (3) OT-IL15-073 PO vehicle, (4) OT- IL15-073 PO TMP 500 mg/kg), (5) OT-IL15-073 IP vehicle, and (6) OT-IL15-073 IP TMP 300 mg/kg. The study design is shown in Table 32. PO dosing is 500 mg/kg TMP in 0.1M citrate and IP dosing is 300 mg/kg TMP lactate in water.

Table 32. Study Design

[00554] The regulated expression in blood was analyzed 6 hours and 24 hours after the first dose, and 6 hours after the 5^th dose.

[00555] OT-1L15-071 showed expression of membrane bound IL15 and the untransduced control did not show any expression.

[00556] Regulation of membrane bound IL15 was seen with repeat PO and IP dosing. As seen in Figure 36, regulated expression of membrane bound IL15 was detected 6 hours after the first dose on day 0, and 6 hours after dosing on day S (126 hrs) with both PO and IP dosing. There was no increase in expression in mice treated with vehicle.

[00557] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

[00558] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A composition for inducing an immune response in a cell or a subject comprising a first effector module, said first effector module comprising a first stimulue response element (SRE) operably linked to a first payload, wherein said first payload comprises an immune-therapeutic agent.

2. The composition of claim 1, wherein the immunotherapeutic agent is a cytokine.

3. The composition of claim 2, wherein the first SRE is responsive to or interacts with at least one stimulus.

4. The composition of claim 3, wherein the first SRE is a first destabilizing domain (DD).

5. The composition of claim 4, wherein the first DD is derived from a parent protein or a mutant protein having one, two, three or more amino acid mutations compared to said parent protein, wherein the parent protein is selected from:

(a) human protein FKBP, comprising the amino acid sequence of SEQ ID NO. 3;

(b) human DHFR (hDHFR), comprising the amino acid sequence of SEQ ID NO. 2;

(c) E. Coli DHFR, comprising the amino acid sequence of SEQ ID NO. 1;

(d) PDE5, comprising the amino acid sequence of SEQ ID NO. 4;

(e) PPAR, gamma comprising the amino acid sequence of SEQ ID NO. 5;

(f) CA2, comprising the amino acid sequence of SEQ ID NO. 6; or

(g) NQ02, comprising the amino acid sequence of SEQ ID NO. 7.

6. The composition of claim 5, wherein the parent protein is hDHFR and the first DD comprises a mutant protein having:

(a) a single mutation selected from hDHFR (I17V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81R), hDHFR (A107V), hDHFR (Y122I), hDHFR (N127Y), hDHFR

(M140I), hDHFR (K185E), hDHFR (N186D), and hDHFR (M140I), hDHFR (Amino acid 2-187 of WT; N127Y), hDHFR (Amino acid 2-187 of WT; I17V), hDHFR (Amino acid 2-187 of WT; Y122I), and hDHFR (Amino acid 2-187 of WT; K185E);

(b) a double mutation selected from hDHFR (C7R, Y163C), hDHFR (A10V, H88Y), hDHFR (Q36K, Y122I), hDHFR (M53T, R1381), hDHFR (T57A, I72A), hDHFR (E63G, I176F), hDHFR (G21T, Y122I), hDHFR (L74N, Y122I), hDHFR (V75F, Y122I), hDHFR (L94A, T147A), DHFR (V121A, Y22I) , hDHFR (Y122I, A125F), hDHFR (H131R, E144G), hDHFR (T137R, F143L), hDHFR (Y178H, E18IG), and hDHFR (Y183H, K185E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; I17V, Y122I), hDHFR (Amino acid 2-187 of WT; Y122I, M140I), hDHFR (Amino acid 2-187 of WT; N127Y, Y122I), hDHFR (Amino acid 2-187 of WT; E162G, I176F), and hDHFR (Amino acid 2-187 of WT; H131R, E144G), and hDHFR (Amino acid 2-187 of WT; Y122I, A125F); or

(c) a triple mutation selected from hDHFR (V9A, S93R, P150L), hDHFR (I8V, K133E, Y163C), hDHFR (L23S, V121A, Y157C), hDHFR (K19E, F89L, E181G), hDHFR (Q36F, N65F, Y122I), hDHFR (G54R, M140V, S168C), hDHFR (VI 10A, V136M, K177R), hDHFR (Q36F, Y122I, A125F), hDHFR (N49D, F59S, D153G), and hDHFR (G21E, I72V, I176T), hDHFR (Amino acid 2-187 of WT; Q36F, Y122I, A125F), hDHFR (Amino acid 2-187 of WT; Y122I, H131R, E144G), hDHFR (Amino acid 2-187 of WT; E31D, F32M, VI 161), and hDHFR (Amino acid 2-187 of WT; Q36F, N65F, Y122I); or

(d) a quadruple or higher mutation selected from hDHFR (V2A, R33G, Q36R, L100P, K185R), hDHFR (Amino acid 2-187 of WT; D22S, F32M, R33S, Q36S, N65S), hDHFR (I17N, L98S, K99R, Ml 12T, E151G, E162G, E172G), hDHFR (G16S, I17V, F89L, D96G, K123E, M140V, D146G, K156R), hDHFR (K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, K177E, Y178C, K185E, N186S), hDHFR (N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L), hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G,

Gl 17D, L132P, I139V, M140I, D142G, D146G, E173G, D187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N, L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, VI 10A, II 15V, Y122D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, VI 13A, Wl 14R, Y122C, M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S), and hDHFR (A10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, Ml 12V, Wl 14R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N).

7. The composition of claim 6, wherein the stimulus is selected from Trimethoprim (TMP) and Methotrexate (MTX).

8. The composition of claim 2, wherein the cytokine is an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor B, a CC chemokine, a CXC chemokine, a CX3C chemokine or a growth factor.

9. The composition of claim 8, wherein the interleukin is selected from IL1, IL1 -alpha, ILl-beta, ILl-delta, ILl-epsilon, ILl-eta, ILl-zeta, ILRA, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL10C, IL10D, IL1 la, ILllb, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20L, IL21, IL22, IL23, IL23A, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL36a, 1Ι.36β, IL36y, IL36RN, IL37, IL37a, IL37b, IL37c, U37d, IL37e, and IL38.

10. The composition of claim 9, wherein the interleukin is the whole or a portion of IL12.

11. The composition of claim 10, wherein the IL12 comprises a p40 subunit of SEQ ID NO. 58, appended to a p35 subunit of SEQ ID NO. 59.

12. The composition of any of claims 1-11, wherein said first effector module comprises the amino acid sequence of any of SEQ ID NO. 60-68 and 318.

13. The composition of any of claims 1-12, further comprising a second effector module, said second effector module comprising a second SRE linked to an immunotherapeutic agent.

14. The composition of claim 13, wherein the immunotherapeutic agent is a cytokine selected from an IL15 and an IL15/TL15Ra fusion polypeptide.

15. The composition of any of claims 1-14, wherein said first SRE stabilizes the

immunotherapeutic agent by a stabilization ratio of 1 or more, wherein the stabilization ratio comprises the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the expression, function or level of the immunotherapeutic agent in the absence of the stimulus.

16. The composition of any of claims 1-14, wherein said first SRE destabilizes the

immunotherapeutic agent by a destabilization ratio between 0, and 0.09, wherein the destabilization ratio comprises the ratio of expression, function or level of the

immunotherapeutic agent in the absence of the stimulus specific to said first SRE to the expression, function or level of the immunotherapeutic agent that is expressed constitutively, and in the absence of the stimulus specific to sad first SRE.

17. A polynucleotide encoding either or both of said first and said second effector modules of any of claims 1-14.

18. The polynucleotide of claim 17, which is a DNA molecule, or a RNA molecule.

19. The polynucleotide of claim 18, wherein the polynucleotide is an RNA molecule and said RNA molecule is a messenger RNA.

20. The polynucleotide of claim 19, wherein the polynucleotide is chemically modified.

21. The polynucleotide of claim 18, which is a DNA molecule.

22. The polynucleotide of claim 18, wherein the polynucleotide comprises spatiotemporally selected codons.

23. The polynucleotide of claim 18, wherein the polynucleotide further comprises at least one additional feature selected from a promoter, a linker, a signal peptide, a tag, and a targeting peptide.

24. A vector comprising a polynucleotide of claim 17.

25. The vector of claim 24, wherein the vector is a viral vector, or a plasmid.

26. The vector of claim 25, which is a viral vector and said viral vector is a retroviral vector, a lenti viral vector, a gamma retroviral vector, a recombinant AAV vector, an adeno viral vector, or an oncolytic viral vector.

27. The vector of claim 26, wherein the polynucleotide is optionally placed under the transcriptional control of a promoter.

28. The vector of claim 27, wherein the promoter is selected from a CMV promoter, an EFla promoter and a PGK promoter.

29. The vector of claim 28, wherein the promoter is an EFla promoter.

30. An immune cell for adoptive cell transfer (ACT), which expresses any of the compositions of any of claims 1-16, the polynucleotides of any of claims 17-23, and/or is infected or transfected with the vector of any of claims 24-29.

31. The immune cell of claim 29, selected from a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine-induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

32. The immune cell of claim 31, which is autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

33. A method of reducing a tumor volume or burden in a subject in need thereof, comprising contacting said subject with the immune cells of any of claims 30-32.

34. A method of inducing an anti-tumor immune response in a subject comprising contacting contacting said subject with the immune cells of any of claims 30-32.

35. The method of claim 34, wherein the immune cell is selected from a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine- induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

36. A method of inducing an immune response in a subject comprising administering to the subject an effective amount of any of the compositions of claims 1-16, the polynucleotides of any of claims 17-23, the vectors of any of claims 24-29, or any of the cells of claims 30-32.

37. A method for pulsatile regulation of an immune-therapeutic agent in a cell or a subject comprising the steps of

(a) contacting the cell or subject with an effective amount of the composition of any of claims 1-16, wherein said first SRE responds to a stimulus and regulates the expression and function of the immune-therapeutic agent; and

(b) administering the stimulus to the cell or subject, followed by the withdrawal of the stimulus at selected time intervals, thereby achieving pulsatile regulation of the immunotherapeutic agent.