Daric interleukin receptors

Abstract

The present disclosure provides improved compositions for adoptive T cell therapies for treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith. 129 20 24 26 46 39 14 N ov 2 02 4 A B S T R A C T O F T H E D I S C L O S U R E 2 0 2 4 2 6 4 6 3 9 1 4 N o v 2 0 2 4

Description

CROSS REFERENCE TO RELATED APPLICATIONS This is a divisional application of Australian patent application No. 2018385699, the entire contents of which are incorporated herein by reference. 5 STATEMENT REGARDING SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is presented in ST.26 format and is based on the Sequence Listing filed for the parent application entitled "Daric interleukin receptors”. BACKGROUND 10 Technical Field The present disclosure relates to improved adoptive cell therapies. More particularly, the disclosure relates to improved chemically regulated signaling molecules, cells, and methods of using the same for modulating spatial and temporal control of cellular signal initiation and downstream responses during adoptive immunotherapy. 15 Description of the Related Art The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer related deaths in 2012. The most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder 20 cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is projected to rise to 22 million within the next two decades. 2024264639   14 Nov 2024 Adoptive cellular therapy is emerging as a powerful paradigm for delivering complex biological signals to treat cancer. In contrast to small molecule and biologic drug compositions, adoptive cell therapies have the potential to execute unique therapeutic tasks owing to their myriad sensory and response programs and increasingly defined 5 mechanisms of genetic control. To achieve such therapeutic value, cells need to be outfitted with machinery for sensing and integrating chemical and / or biological information associated with local physiological environments. BRIEF SUMMARY The present disclosure generally relates, in part, to IL DARIC compositions, 10 polynucleotides, polypeptides, and methods of making and using the same. In various embodiments, the present disclosure contemplates, in part, a fusion polypeptide comprising: a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and a first immune receptor intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising a second 15 multimerization domain, a second transmembrane domain; and a second immune receptor intracellular signaling domain. In certain embodiments, the first multimerization domain and the second multimerization domain are the same. In some embodiments, the first multimerization domain and the second 20 multimerization domain are different. In some embodiments, the first multimerization domain and the second multimerization domain associate with a bridging factor selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative 25 thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof. 2024264639   14 Nov 2024 In particular embodiments, the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FKBP12-rapamycin binding (FRB) or variants thereof; FKBP and calcineurin or variants thereof; FKBP and cyclophilin or variants thereof; FKBP and bacterial dihydrofolate 5 reductase (DHFR) or variants thereof; calcineurin and cyclophilin or variants thereof; PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1) or variants thereof; and GIB1 and GAI or variants thereof. In various embodiments, the first multimerization domain comprises a FKBP polypeptide or variant thereof, and the second multimerization domain comprises a FRB 10 polypeptide or variant thereof. In certain embodiments, the first multimerization domain comprises a FRB polypeptide or variant thereof, and the second multimerization domain comprises a FKBP polypeptide or variant thereof. In particular embodiments, the first and second multimerization domains are 15 selected from FRB T2098L and FKBP12; and the bridging factor is AP21967. In particular embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. In some embodiments, the first transmembrane domain and the second 20 transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, a CD154 transmembrane domain, and a CD71 transmembrane domain. In certain embodiments, the first transmembrane domain and the second 25 transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain are the same. 2024264639   14 Nov 2024 In various embodiments, the first transmembrane domain and the second transmembrane domain are different. In certain embodiments, the first immune receptor intracellular signaling domain and the second immune receptor intracellular signaling domain are isolated from a cytokine 5 receptor, an interleukin receptor, a pattern recognition receptor, or a toll-like receptor. In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-12R02 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-12R01 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain 10 comprises an IL-12R01 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-12R02 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain comprises an IL-7Ra intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. 15          In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-7Ra intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an IL-2R0 intracellular signaling domain and the second immune receptor 20 intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2R0 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain 25 comprises an IL-21R intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-21R intracellular signaling domain. 2024264639   14 Nov 2024 In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-18R1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-18RAP intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain 5 comprises an IL-18RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-18R1 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain comprises an IL-1R1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain. 10          In various embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1R1 intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RL2 intracellular signaling domain and the second immune receptor 15 intracellular signaling domain comprises an IL-1RAP intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1RL2 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain 20 comprises an IFNAR1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IFNAR2 intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IFNAR2 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IFNAR1 intracellular signaling domain. 25          In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR1 intracellular signaling domain. 2024264639   14 Nov 2024 In some embodiments, the first immune receptor intracellular signaling domain comprises an TLR2 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR2 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain 5 comprises an TLR3 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR3 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an TLR4 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR4 intracellular signaling domain. 10          In certain embodiments, the first immune receptor intracellular signaling domain comprises an TLR5 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR5 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an TLR6 intracellular signaling domain and the second immune receptor 15 intracellular signaling domain comprises an TLR6 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR7 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR7 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain 20 comprises an TLR8 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR8 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR9 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR9 intracellular signaling domain. 25          In some embodiments, the first immune receptor intracellular signaling domain comprises an TLR10 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR10 intracellular signaling domain. In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide. 2024264639   14 Nov 2024 In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus 5 (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In certain embodiments, the first multimerization domain localizes extracellularly when the first polypeptide is expressed and the second multimerization domain localizes 10 extracellularly when the second polypeptide is expressed. In particular embodiments, a composition comprising a fusion polypeptide contemplated herein is provided. In various embodiments, the present disclosure contemplates, in part, a composition comprising a first polypeptide comprising a first multimerization domain, a first 15 transmembrane domain, and a first immune receptor intracellular signaling domain; and a second polypeptide comprising a second multimerization domain, a second transmembrane domain, and a second immune receptor intracellular signaling domain. In some embodiments, the first multimerization domain and the second multimerization domain are the same. 20         In various embodiments, the first multimerization domain and the second multimerization domain are different. In particular embodiments, the first multimerization domain and the second multimerization domain associate with a bridging factor selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a 25 derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof. 2024264639   14 Nov 2024 In various embodiments, the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FKBP12-rapamycin binding (FRB) or variants thereof; FKBP and calcineurin or variants thereof; FKBP and cyclophilin or variants thereof; FKBP and bacterial dihydrofolate 5 reductase (DHFR) or variants thereof; calcineurin and cyclophilin or variants thereof; PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1) or variants thereof; and GIB1 and GAI or variants thereof. In particular embodiments, the first multimerization domain comprises a FKBP polypeptide or variant thereof, and the second multimerization domain comprises a FRB 10 polypeptide or variant thereof. In some embodiments, the first multimerization domain comprises a FRB polypeptide or variant thereof, and the second multimerization domain comprises a FKBP polypeptide or variant thereof. In certain embodiments, the first and second multimerization domains are selected 15 from FRB T2098L and FKBP12; and the bridging factor is AP21967. In various embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. In various embodiments, the first transmembrane domain and the second 20 transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, a CD154 transmembrane domain, and a CD71 transmembrane domain. In some embodiments, the first transmembrane domain and the second 25 transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain. In certain embodiments, the first transmembrane domain and the second transmembrane domain are the same. 2024264639   14 Nov 2024 In various embodiments, the first transmembrane domain and the second transmembrane domain are different. In particular embodiments, first immune receptor intracellular signaling domain and the second immune receptor intracellular signaling domain are isolated from a cytokine 5 receptor, an interleukin receptor, a pattern recognition receptor, or a toll-like receptor. In various embodiments, the first immune receptor intracellular signaling domain comprises an IL-12R02 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-12R01 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain 10 comprises an IL-12R01 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-12R02 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-7Ra intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. 15          In some embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-7Ra intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain comprises an IL-2R0 intracellular signaling domain and the second immune receptor 20 intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2R0 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain 25 comprises an IL-21R intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-2Ry intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-21R intracellular signaling domain. 2024264639   14 Nov 2024 In various embodiments, the first immune receptor intracellular signaling domain comprises an IL-18R1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-18RAP intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain 5 comprises an IL-18RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-18R1 intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-1R1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain. 10          In particular embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1R1 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RL2 intracellular signaling domain and the second immune receptor 15 intracellular signaling domain comprises an IL-1RAP intracellular signaling domain. In certain embodiments, the first immune receptor intracellular signaling domain comprises an IL-1RAP intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IL-1RL2 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain 20 comprises an IFNAR1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IFNAR2 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an IFNAR2 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an IFNAR1 intracellular signaling domain. 25          In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR1 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR1 intracellular signaling domain. 2024264639   14 Nov 2024 In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR2 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR2 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain 5 comprises an TLR3 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR3 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an TLR4 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR4 intracellular signaling domain. 10          In certain embodiments, the first immune receptor intracellular signaling domain comprises an TLR5 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR5 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR6 intracellular signaling domain and the second immune receptor 15 intracellular signaling domain comprises an TLR6 intracellular signaling domain. In various embodiments, the first immune receptor intracellular signaling domain comprises an TLR7 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR7 intracellular signaling domain. In some embodiments, the first immune receptor intracellular signaling domain 20 comprises an TLR8 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR8 intracellular signaling domain. In particular embodiments, the first immune receptor intracellular signaling domain comprises an TLR9 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR9 intracellular signaling domain. 25          In various embodiments, the first immune receptor intracellular signaling domain comprises an TLR10 intracellular signaling domain and the second immune receptor intracellular signaling domain comprises an TLR10 intracellular signaling domain. In particular embodiments, the composition further comprises a cell. 2024264639   14 Nov 2024 In certain embodiments, a polypeptide complex comprising a first and second polypeptide contemplated herein is provided. In particular embodiments, a polynucleotide encoding a fusion polypeptide or a first and second polypeptide contemplated herein is provided. 5         In various embodiments, a cDNA encoding a fusion polypeptide or a first and second polypeptide contemplated herein is provided. In some embodiments, an RNA encoding a fusion polypeptide or a first and second polypeptide contemplated herein is provided. In various embodiments, vector comprising a polynucleotide contemplated herein is 10 provided. In certain embodiments, a cell comprising a fusion polypeptide, a first and second polypeptide, a polynucleotide, or a vector contemplated herein is provided. In particular embodiments, the cell is a hematopoietic cell. In various embodiments, the cell is a T cell. 15         In some embodiments, the cell is a CD3+, CD4+, and / or CD8+ cell. In various embodiments, the cell is an immune effector cell. In particular embodiments, the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells. In some embodiments, the cell is a natural killer (NK) cell or natural killer T (NKT) 20 cell. In particular embodiments, the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors. In various embodiments, the cell further comprises an engineered antigen receptor. 25          In certain embodiments, the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a DARIC receptor or components thereof, and a chimeric cytokine receptor. In various embodiments, a composition comprising a fusion polypeptide, a first and second polypeptide, a polynucleotide, a vector, or a cell contemplated herein is provided. 2024264639   14 Nov 2024 In various embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a composition comprising a fusion polypeptide, a first and second polypeptide, a polynucleotide, a vector, or a cell contemplated herein is provided. 5          In various embodiments, a method of treating a subject in need thereof comprising administering the subject an effective amount of a composition contemplated herein is provided. In various embodiments, a method of treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, 10 and immunodeficiency, or condition associated therewith, comprising administering to the subject an effective amount of a composition contemplated herein is provided. In various embodiments, a method of treating a solid cancer comprising administering to the subject an effective amount of a composition contemplated herein is provided. 15          In particular embodiments, the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer. In certain embodiments, the solid cancer is a pancreatic cancer, a lung cancer, or a 20 breast cancer. In various embodiments, the present disclosure contemplates, in part, a method of treating a hematological malignancy comprising administering to the subject an effective amount of a composition contemplated herein. In particular embodiments, the hematological malignancy is a leukemia, lymphoma, 25 or multiple myeloma. BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS Figure 1A shows a cartoon of IL-18R DARIC and IL-18R DARIC-GFP constructs and polypeptides. 2024264639   14 Nov 2024 Figure 1B shows GFP expression in donor PBMCs transduced with LVV encoding IL-18R-DARIC-GFP. Figure 1C shows growth curves for untransduced donor PBMCs and PBMCs transduced with LVV encoding IL-18R-DARIC-GFP. 5        Figure 1D shows CD62L and CD45Ra expression in untransduced donor PBMCs and PBMCs transduced with LVV encoding IL-18R-DARIC-GFP. Figure 2 shows IFNy production in untransduced donor PBMCs and PBMCs transduced with LVV encoding IL-18R-DARIC-GFP and pre-treated with 50 ng / mL human recombinant IL-12 for 24 hours, and then subsequently cultured in medium only, 10 medium with 100 ng / mL human recombinant IL-18 or medium with 1 nM rapamycin. BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS SEQ ID NO: 1 sets forth the amino acid sequence for an IL-18R DARIC signaling component. SEQ ID NO: 2 sets forth the amino acid sequence for an IL-18R DARIC signaling 15 component. SEQ ID NO: 3 sets forth the amino acid sequence for an IL-18R DARIC binding component. SEQ ID NO: 4 sets forth the amino acid sequence for an IL-18R DARIC binding component. 20         SEQ ID NO: 5 sets forth the amino acid sequence for an IL-18R DARIC polyprotein comprising an IL-18R DARIC binding component and signaling component separated by a viral P2A domain. SEQ ID NO: 6 sets forth the amino acid sequence for an IL-18R DARIC -GFP polyprotein comprising an IL-18R DARIC binding component, signaling component, and 25 GFP, each separated by a viral P2A domain. SEQ ID NOs: 7-17 set forth the amino acid sequences of various linkers. SEQ ID NOs: 18-42 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites. 2024264639   14 Nov 2024 DETAILED DESCRIPTION A.   Overview The present disclosure generally relates to chemically regulatable polypeptides that transduce immunostimulatory signals to cells expressing the polypeptides. Without 5 wishing to be bound by any particular theory, the polypeptides contemplated herein are fusion polypeptides comprising a chemically inducible multimerization domain linked to one or more immunostimulatory endodomains that stimulate immune effector cell activity and function. Coexpression of immunostimulatory fusion polypeptides in immune effector cells in the presence of a factor that induces multimerization and activation of the fusion 10 polypeptides renders the cells resistant to the immunosuppressive impacts of the TME by restoring or increasing proinflammatory cytokine secretion. In particular preferred embodiments, a fusion polypeptide is referred to as a DARIC immune receptor. In various embodiments, the present disclosure contemplates, in part, polypeptides that convert a chemical signal to an immunostimulatory signal mediated through or by 15 multimerization of intracellular domains of immune receptors. In various embodiments, the present disclosure contemplates, in part, polypeptides that convert a chemical signal to an immunostimulatory signal mediated through or by multimerization of intracellular domains of cytokine receptors. In various embodiments, the present disclosure contemplates, in part, polypeptides 20 that convert a chemical signal to an immunostimulatory signal mediated through or by multimerization of intracellular domains of interleukin receptors. In various embodiments, the present disclosure contemplates, in part, polypeptides that convert a chemical signal to an immunostimulatory signal mediated through or by multimerization of intracellular domains of pattern recognition receptors. 25          In various embodiments, the present disclosure contemplates, in part, polypeptides that convert a chemical signal to an immunostimulatory signal mediated through or by multimerization of intracellular domains of toll-like receptors. 2024264639   14 Nov 2024 In particular embodiments, the present disclosure contemplates, in part, a polypeptide comprising an inducible multimerization domain, a transmembrane domain and one or more intracellular domains of one or more immune receptors; and a polypeptide comprising another inducible multimerization domain, a transmembrane domain and one or 5 more intracellular domains of one or more immune receptors. In one embodiment, the polypeptides are linked to each other by a polypeptide cleavage signal, e.g., a 2A polypeptide cleavage signal. In particular embodiments, the present disclosure contemplates, in part, an immune effector cell, e.g., CAR T cell or engineered TCR T cell, that expresses a polypeptide 10 comprising an inducible multimerization domain, a transmembrane domain and one or more intracellular domains of one or more immune receptors; and a polypeptide comprising another inducible multimerization domain, a transmembrane domain and one or more intracellular domains of one or more immune receptors. In particular embodiments, the transmembrane domains are isolated from a receptor 15 expressed on an immune effector cell; and intracellular signaling domains are isolated from an IL-12 receptor, an IL-7 receptor, an IL-15 receptor, an IL-21 receptor, an IL-2 receptor, an IL-1 receptor, an IL-18 receptor, an IL-36 receptor, a type I IFN receptor, a TLR1 receptor, a TLR2 receptor, a TLR3 receptor, a TLR4 receptor, a TLR5 receptor, a TLR6 receptor, a TLR7 receptor, a TLR8 receptor, a TLR9 receptor, or a TLR10 receptor. 20          In particular embodiments, the intracellular signaling domains are isolated from IL- 12R02, IL-7Ra, IL-2Ry, IL-2R0, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide 25 synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification. See, e.g., 2024264639   14 Nov 2024 Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. 5 Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister 10 Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid The Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture 15   (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 20   1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 25   1988); Current Protocols in Immunology (Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology. 2024264639   14 Nov 2024 B.    Definitions Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Unless defined otherwise, all technical and scientific terms used herein have the 5 same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. 10          The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or one or more elements. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. 15         The term “and / or” should be understood to mean either one, or both of the alternatives. As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference 20 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, 25 weight or length. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and 30 limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” 2024264639   14 Nov 2024 indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting 5 essentially of” indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements. Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular 10 feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that 15 the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment. An “antigen (Ag)” refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a cancer-specific protein) that are injected or absorbed into an animal. 20 Exemplary antigens include but are not limited to lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. A “target antigen” or “target antigen of interest” is an antigen that a binding domain 25 contemplated herein, is designed to bind. In particular embodiments, the target antigen is selected from the group consisting of: alpha folate receptor, 5T4, av^6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, 30 fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, 2024264639   14 Nov 2024 HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL- 1 1Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, STn, TAG72, TEMs, VEGFR2, and WT-1. In one embodiment, the antigen is an MHC-peptide complex, such as a 5 class I MHC-peptide complex or a class II MHC-peptide complex. An “antibody” refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those 10 recognized by an immune cell. An “epitope” or “antigenic determinant” refers to the region of an antigen to which a binding agent binds. Antibodies include antigen binding fragments thereof, such as a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a 15 trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody) and portions of full length antibodies responsible for antigen binding. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), 20 heteroconjugate antibodies (such as, bispecific antibodies) and antigen binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. A “linker” refers to a plurality of amino acid residues between the various polypeptide domains added for appropriate spacing and conformation of the molecule. In 25 particular embodiments, the linker is a variable region linking sequence. A “variable region linking sequence,” is an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with interaction of the two subbinding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain 30 variable regions. In particular embodiments, a linker separates one or more heavy or 2024264639   14 Nov 2024 light chain variable domains, hinge domains, multimerization domains, transmembrane domains, co-stimulatory domains, and / or primary signaling domains. Illustrated examples of linkers suitable for use in particular embodiments contemplated herein include, but are not limited to the following amino acid sequences: 5 GGG; DGGGS (SEQ ID NO: 7); TGEKP (SEQ ID NO: 8) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 9) (Pomerantz et al. 1995, supra); (GGGGS)n wherein n = 1, 2, 3, 4 or 5 (SEQ ID NO: 10) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 11) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 12) (Bird et al., 1988, 10 Science 242:423-426), GGRRGGGS (SEQ ID NO: 13); LRQRDGERP (SEQ ID NO: 14); LRQKDGGGSERP (SEQ ID NO: 15); LRQKD(GGGS)2 ERP (SEQ ID NO: 16). Alternatively, flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display 15 methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 17) (Cooper et al., Blood, 101(4): 1637-1644 (2003)). A “spacer domain,” refers to a polypeptide that separates two domains. In one embodiment, a spacer domain moves an antigen binding domain away from the effector 20 cell surface to enable proper cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). In particular embodiments, a spacer domain separates one or more heavy or light chain variable domains, multimerization domains, transmembrane domains, co-stimulatory domains, and / or primary signaling domains. The spacer domain may be derived either from a natural, synthetic, semi-synthetic, or 25 recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. A “hinge domain,” refers to a polypeptide that plays a role in positioning the 30 antigen binding domain away from the effector cell surface to enable proper cell / cell 2024264639   14 Nov 2024 contact, antigen binding and activation. In particular embodiments, polypeptides may comprise one or more hinge domains between the binding domain and the multimerization domain, between the binding domain and the transmembrane domain (TM), or between the multimerization domain and the transmembrane domain. The hinge domain may be 5 derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. A “multimerization domain,” as used herein, refers to a polypeptide that preferentially interacts or associates with another different polypeptide directly or via a bridging molecule, 10   e.g., a chemically inducible dimerizer, wherein the interaction of different multimerization domains substantially contributes to or efficiently promotes multimerization (i.e., the formation of a dimer, trimer, or multipartite complex, which may be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer). A multimerization domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. 15          Illustrative examples of multimerization domains suitable for use in particular embodiments contemplated herein include an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP12-rapamycin binding (FRB) polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYR1-like 1 20 (PYL1) polypeptide or variants thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, a GIB1 polypeptide or variants thereof, or a GAI polypeptide or variants thereof. As used herein, the term “FKBP-rapamycin binding polypeptide” refers to an FRB polypeptide. In particular embodiments, the FRB polypeptide is an FKBP12-rapamycin 25 binding polypeptide. FRB polypeptides suitable for use in particular embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB polypeptide comprises a 93 amino acid sequence Ile-2021 through Lys -2113 and a mutation of T2098L, with reference to GenBank Accession No. L34075.1. An FRB polypeptide contemplated herein binds to an FKBP polypeptide through a 30 bridging factor, thereby forming a tripartite complex. 2024264639   14 Nov 2024 As used herein, the term “FK506 binding protein” refers to an FKBP polypeptide. In particular embodiments, the FKBP polypeptide is an FKBP12 polypeptide. In certain embodiments, an FKBP domain may also be referred to as a “rapamycin binding domain”. Information concerning the nucleotide sequences, cloning, and other aspects of various FKBP 5 species is known in the art (see, e.g., Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). An FKBP polypeptide contemplated herein binds to an FRB polypeptide through a bridging factor, thereby forming a tripartite complex. A “bridging factor” refers to a molecule that associates with and that is disposed between two or more multimerization domains. In particular embodiments, multimerization 10 domains substantially contribute to or efficiently promote formation of a polypeptide complex only in the presence of a bridging factor. In particular embodiments, multimerization domains do not contribute to or do not efficiently promote formation of a polypeptide complex in the absence of a bridging factor. Illustrative examples of bridging factors suitable for use in particular embodiments contemplated herein include, but are not limited to AP21967, 15 rapamycin (sirolimus) or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof. Rapamycin analogs (rapalogs) include but are not limited to those disclosed in U.S. Pat. 20 No. 6,649,595, which rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, a bridging factor is a rapalog with substantially reduced immunosuppressive effect as compared to rapamycin. In a preferred embodiment, the rapalog is AP21967 (also known as C-16-(S)-7-methylindolerapamycin, IC50 = 10nM, a chemically modified non-immunosuppressive rapamycin analogue). Other illustrative rapalogs suitable 25 for use in particular embodiments contemplated herein include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. A “substantially reduced immunosuppressive effect” refers to at least less than 0.1 to 0.005 times the immunosuppressive effect observed or expected for the same dose measured 2024264639   14 Nov 2024 either clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity. A “transmembrane domain” or “TM domain” is a domain that anchors a polypeptide to the plasma membrane of a cell. The TM domain may be derived either 5 from a natural, synthetic, semi-synthetic, or recombinant source. An “intracellular signaling domain” or “endodomain” refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a 10 truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. The term “effector function” or “effector cell function” refers to a specialized 15 function of an immune effector cell. Effector function includes, but is not limited to, activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses elicited with antigen binding to the receptor expressed on the immune effector cell. It is known that signals generated through the TCR alone are insufficient for full 20 activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR / CD3 complex) and co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. 25          A “primary signaling domain” refers to a signaling domain that regulates the primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Illustrative examples of ITAM containing primary signaling domains that are suitable for 2024264639   14 Nov 2024 use in particular embodiments include, but are not limited to those derived from FcRy, FcRp, CD3y, CD3S, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. As used herein, the term, “co-stimulatory signaling domain,” or “co-stimulatory domain” refers to an intracellular signaling domain of a co-stimulatory molecule. Co- 5 stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such co-stimulatory molecules from which co-stimulatory domains may be isolated include, but are not limited to: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, 10 CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. An “immune disorder” refers to a disease that evokes a response from the immune system. In particular embodiments, the term “immune disorder” refers to a cancer, an autoimmune disease, or an immunodeficiency. In one embodiment, immune disorders 15 encompass infectious disease. As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), 20 invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. 25         As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize. A “cancer cell” refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid cancers and liquid cancers. A “tumor” or “tumor cell” refers 30 generally to a swelling or lesion formed by an abnormal growth of cells, which may be 2024264639   14 Nov 2024 benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight 5 of the tumor. The term “relapse” refers to the diagnosis of return, or signs and symptoms of return, of a cancer after a period of improvement or remission. “Remission,” is also referred to as “clinical remission,” and includes both partial and complete remission. In partial remission, some, but not all, signs and symptoms of cancer have 10 disappeared. In complete remission, all signs and symptoms of cancer have disappeared, although cancer still may be in the body. “Refractory” refers to a cancer that is resistant to, or non-responsive to, therapy with a particular therapeutic agent. A cancer can be refractory from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to 15 the therapeutic agent, either over the course of a first treatment period or during a subsequent treatment period. “Antigen negative” refers to a cell that does not express antigen or expresses a negligible amount of antigen that is undetectable. In one embodiment, antigen negative cells do not bind receptors directed to the antigen. In one embodiment, antigen negative cells do not 20 substantially bind receptors directed to the antigen. An “autoimmune disease” refers to a disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as “self” and targets and attacks it as if it were foreign. Autoimmune diseases can be classified 25 into those in which predominantly one organ is affected (e.g., hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune 30 diseases are known in the art and include, for instance, Hashimoto’s thyroiditis, Grave’s 2024264639   14 Nov 2024 disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like. An “immunodeficiency” means the state of a patient whose immune system has been 5 compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, 10 chronic granulomatous disease, hyper-IgM syndrome, and diabetes. An “infectious disease” refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microbial or viral agent (e.g., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV / AIDS, diphtheria, 15 hepatitis B, hepatitis C, cholera, and influenza. As used herein, the terms “individual” and “subject” are often used interchangeably and refer to any animal that exhibits a symptom of cancer or other immune disorder that can be treated with the compositions and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm 20 animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. Typical subjects include human patients that have, have been diagnosed with, or are at risk or having, cancer or another immune disorder. As used herein, the term “patient” refers to a subject that has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods 25 disclosed elsewhere herein. As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction of the disease or condition, 30 or the delaying of the progression of the disease or condition, e.g., delaying tumor 2024264639   14 Nov 2024 outgrowth. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the 5 occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition. 10          As used herein, the phrase “ameliorating at least one symptom of” refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated. In particular embodiments, the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged 15 lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function). By “enhance” or “promote,” or “increase” or “expand” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater 20 physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, persistence, cytokine secretion, and / or an increase in cancer cell killing ability, among others apparent from the understanding in the art and the description herein. An “increased” or “enhanced” amount 25 is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition. By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to 30 the ability of composition contemplated herein to produce, elicit, or cause a lesser 2024264639   14 Nov 2024 physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all 5 integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage. By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to the ability of a composition 10 contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in a cell, as compared to the response caused by either vehicle, a control molecule / composition, or the response in a particular cell lineage. A comparable response is one that is not significantly different or measurable different from the reference response. 15          Additional definitions are set forth throughout this disclosure. C.   DARIC Immune Receptors In particular embodiments, one or more DARIC immune receptors that transduce an immunostimulatory signal upon exposure to a bridging factor is contemplated. As used herein, the term “DARIC immune receptor” refers to one or more non-naturally occurring 20 polypeptides that transduces an immunostimulatory signal in an immune effector cell upon exposure to a multimerizing agent or bridging factor, e.g., stimulating immune effector cell activity and function, increasing production and / or secretion of proinflammatory cytokines. In particular embodiments, the DARIC immune receptor is a polypeptide comprising a first multimerization domain, a transmembrane domain, an intracellular 25 signaling domain of an immune receptor, e.g., a human immune receptor, including, but not limited to a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an intracellular signaling domain of an immune 2024264639   14 Nov 2024 receptor, e.g., a human immune receptor, including, but not limited to a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor. In other particular embodiments, the DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a 5 transmembrane domain, and an intracellular signaling domain of an immune receptor including, but not limited to a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor; and a second polypeptide comprising a second multimerization domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor including, but not limited to a cytokine receptor, an 10 interleukin receptor, a pattern recognition receptor, and a toll-like receptor. As used herein, the term “immune receptor” refers to a receptor that is expressed on the surface of an immune cell that modulates an immune response upon binding its cognate ligand. Immune receptors suitable for use in particular embodiments include, but are not limited to: cytokine receptors, interleukin receptors, pattern recognition receptors, and toll- 15 like receptors, wherein signaling through the immune receptor stimulates an immune response. Illustrative examples of multimerization domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not limited to, an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP12-rapamycin binding (FRB) 20 polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYR1-like 1 (PYL1) polypeptide or variants thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, a GIB1 polypeptide or variants thereof, or a GAI polypeptide or variants thereof. 25         Illustrative examples of immune receptor transmembrane domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not limited to, the transmembrane region(s) of the alpha, beta, gamma, or delta chain of a T-cell receptor, CD3s, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, PD1, IL-12R02, IL- 2024264639   14 Nov 2024 7Ra, IL-2Ry, IL-2R0, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. Illustrative examples of immune receptor intracellular signaling domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not 5 limited to, intracellular signaling domains isolated from an IL-12 receptor, an IL-7 receptor, an IL-15 receptor, an IL-21 receptor, an IL-2 receptor, an IL-1 receptor, an IL-18 receptor, an IL-36 receptor, a type I IFN receptor, a TLR1 receptor, a TLR2 receptor, a TLR3 receptor, a TLR4 receptor, a TLR5 receptor, a TLR6 receptor, a TLR7 receptor, a TLR8 receptor, a TLR9 receptor, or a TLR10 receptor. 10          Illustrative examples of immune receptor intracellular signaling domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not limited to, IL-12R02, IL-7Ra, IL-2Ry, IL-2R0, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. 15          Illustrative examples of cytokine receptor intracellular signaling domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not limited to, IL-12R02, IL-7Ra, IL-2Ry, IL-2R0, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, and IL-1RL2. Illustrative examples of interleukin receptor intracellular signaling domains suitable 20 for use in particular DARIC immune receptors contemplated herein include, but are not limited to, IL-12R02, IL-7Ra, IL-2Ry, IL-2R0, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, and IL-1RL2. Illustrative examples of toll-like receptor intracellular signaling domains suitable for use in particular DARIC immune receptors contemplated herein include, but are not 25 limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. 1.   IL-18DARICImmuneReceptor Interleukin-18 (IL-18) is a cytokine that promotes T cell function and activity by, in part, increasing IFNy expression, increasing T cell proliferation, and protecting against 2024264639   14 Nov 2024 activation induced cell death (AICD). IL-18 binds interleukin 18 receptor 1, (IL-18R1, also known as CD218a) and interleukin 18 receptor accessory protein (IL-18RAP, CD218b). IL-18 signaling through IL-18R1 and IL-18RAP results in activation through the 5 MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase IFNy expression and increase sensitivity to IL-12. The transcriptional program induced by IL-18 also increases T cell proliferation and protects against AICD. 10         In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding an IL-18 DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding an IL-18 DARIC immune receptor and an 15 engineered antigen receptor. In particular embodiments, the IL-18 DARIC immune receptor transduces an IL-18-mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments an IL-18 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-18RAP intracellular 20 signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain. In particular embodiments an IL-18 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a 25 transmembrane domain, and an IL-18RAP intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a 2024264639   14 Nov 2024 Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. 5         In particular embodiments, the IL-18 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-18RAP intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-18R1 intracellular signaling domain. In particular embodiments, an IL- 10   18 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-18R1 intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-18RAP intracellular signaling domain. 15         In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PD1. In one embodiment, the first and second multimerization domains are the same. 20         In certain embodiments, an IL-18 DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain 25 comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-18R1 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-18RAP intracellular signaling domain. 2024264639   14 Nov 2024 In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-18R1 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a 5 CD4 or CD8a transmembrane domain, and an IL-18RAP intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP 10 polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 2.   IL-12DARICImmuneReceptor Interleukin-12 (IL-12) is a cytokine that promotes T cell function and activity by, in 15 part, increasing IFNy expression, increasing T cell proliferation, and potentiating IL-12 signaling. IL-12 binds interleukin 12 receptor, beta 1 (IL-12R^l, also known as CD212) and interleukin 12 receptor, beta 2 (IL-12R02). IL-12 signaling through IL-12R0l and IL-12R02 results in STAT3, STAT4, and STAT5 phosphorylation. Phosphorylated STAT3 / STAT4 translocates to the nucleus and 20 binds the IFNy promoter to increase IFNy expression. Phosphorylated STAT4 also recruits Jun oncogene (c-Jun) to IFNy promoter to increase IFNy expression and potentiates IL-12 signaling by increasing transcription of IL-12R02. STAT5 phosphorylation increases T cell proliferation. IL-12 signaling also increases expression of interleukin 2 receptor, alpha (IL-2R) by 25 recruiting STAT4 and c-Jun to the promoter of IL-2R, thereby enhancing T cell proliferation. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one 2024264639   14 Nov 2024 or more polynucleotides or vectors encoding an IL-12 DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding an IL-12 DARIC immune receptor and an engineered antigen receptor. 5         In particular embodiments, the IL-12 DARIC immune receptor transduces an IL- 12-mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments an IL-12 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-12R01 intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a 10 transmembrane domain, and an IL-12R02 intracellular signaling domain. In particular embodiments an IL-12 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-12R02 intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-12R01 intracellular signaling domain. 15          In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, 20 a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. In particular embodiments, the IL-12 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a 25 first transmembrane domain, and an IL-12R01 intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-12R02 intracellular signaling domain. In particular embodiments, an IL-12 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL- 2024264639   14 Nov 2024 12R02 intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-12R01 intracellular signaling domain. In particular embodiments, the first and second transmembrane domains are 5 selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PD1. In one embodiment, the first and second multimerization domains are the same. In certain embodiments, an IL-12 DARIC immune receptor comprises a first or 10 second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane 15 domain, and an IL-12R01 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-12R02 intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane 20 domain, and an IL-12R01 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-12R02 intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or 25 zotarolimus. In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 2024264639   14 Nov 2024 3.   IL-7DARICImmuneReceptor Interleukin-7 (IL-7) is a cytokine that promotes T cell function and activity by, in part, improving T cell precursor survival and proliferation. IL-7 binds interleukin 7 receptor alpha (IL-7Ra, also known as CD127) and interleukin 2 receptor, common gamma 5 chain (IL-2Ry, also known as CD132 and yc). IL-7 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and results in transcription of anti-apoptotic genes and genes that promote proliferation of T cell precursors. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one 10 or more polynucleotides or vectors encoding an IL-7 DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding an IL-7 DARIC immune receptor and an engineered antigen receptor. In particular embodiments, the IL-7 DARIC immune receptor transduces an IL-715 mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments an IL-7 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-7Ra intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular 20 embodiments an IL-7 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-7Ra intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving 25 polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one 2024264639   14 Nov 2024 embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. In particular embodiments, the IL-7 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a 5 first transmembrane domain, and an IL-7Ra intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular embodiments, an IL-7 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-2Ry intracellular 10 signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-7Ra intracellular signaling domain. In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 15  154, AMN, and PD1. In one embodiment, the first and second multimerization domains are the same. In certain embodiments, an IL-7 DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and 20 cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-7Ra intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or 25 CD8a transmembrane domain, and an IL-2Ry intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-7Raintracellular signaling domain; and a second polypeptide comprises 2024264639   14 Nov 2024 a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-2Ry intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or 5 zotarolimus. In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 10       4.   IL-15DARICImmuneReceptor Interleukin-15 (IL-15) is a cytokine that promotes T cell function and activity by, in part, improving T cell precursor survival and proliferation. IL-15 binds with high affinity to IL-15Ra (also known as CD215), which then associates with a complex comprising IL-2R0 (also known as IL-15RP and CD122) and IL-2Ry (also known as CD132 and yc), 15 expressed either on the same cell (cis-presentation) or on a different cell (transpresentation). IL-15 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and results in transcription of anti-apoptotic genes and genes that promote proliferation of T cell precursors. In various embodiments, one or more immune effector cells, including immune 20 effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding an IL-15 DARIC immune receptor, and optionally, a polynucleotide or vector encoding an IL-15Ra polypeptide. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding an IL-15 DARIC immune receptor and an engineered 25 antigen receptor, and optionally, a polynucleotide or vector encoding an IL-15Ra polypeptide. In particular embodiments, the IL-15 DARIC immune receptor transduces an IL-15-mediated immunostimulatory signal upon exposure to a bridging factor. In particular 2024264639   14 Nov 2024 embodiments an IL-15 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-2R0 intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular 5 embodiments an IL-15 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-2R0 intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving 10 polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one 15 embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. In particular embodiments, the IL-15 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-2R0 intracellular signaling domain; and a 20 polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular embodiments, an IL-15 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-2Ry intracellular signaling domain; and a polypeptide comprising a second multimerization 25 domain, a second transmembrane domain, and an IL-2R0 intracellular signaling domain. In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PD1. 2024264639   14 Nov 2024 In one embodiment, the first and second multimerization domains are the same. In certain embodiments, an IL-15 DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and 5 cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-2R0 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or 10 CD8a transmembrane domain, and an IL-2Ry intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-2R0 intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or 15 CD8a transmembrane domain, and an IL-2Ry intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP 20 polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 5.   IL-21DARICImmuneReceptor Interleukin-21 (IL-21) is a cytokine that promotes T cell function and activity by, in 25 part, improving T cell precursor survival and proliferation. IL-21 binds to interleukin 21 receptor (IL-21R, also known as CD360) and IL-2Ry (also known as CD132 and yc). IL-21 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and results in 2024264639   14 Nov 2024 transcription of anti-apoptotic genes and genes that promote proliferation of T cell precursors. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one 5 or more polynucleotides or vectors encoding an IL-21 DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding an IL-21 DARIC immune receptor and an engineered antigen receptor. In particular embodiments, the IL-21 DARIC immune receptor transduces an IL-10 21-mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments an IL-21 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-21R intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular 15 embodiments an IL-21 DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-21R intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving 20 polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one 25 embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. In particular embodiments, the IL-21 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-21R intracellular signaling domain; and a 2024264639   14 Nov 2024 polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular embodiments, an IL-21 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-2Ry 5 intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-21R intracellular signaling domain. In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 10  154, AMN, and PD1. In one embodiment, the first and second multimerization domains are the same. In certain embodiments, an IL-21 DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and 15 cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-21R intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or 20 CD8a transmembrane domain, and an IL-2Ry intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-21R intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or 25 CD8a transmembrane domain, and an IL-12Ry intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 2024264639   14 Nov 2024 In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 5       6.   IL-1DARICImmuneReceptor Interleukin-1 (IL-1) is a cytokine that promotes T cell function and activity by, in part, increasing IFNy expression, increasing T cell proliferation, and potentiating protecting against activation induced cell death (AICD). IL-1 binds interleukin 1 receptor 1, (IL-1R1, also known as CD121a) and interleukin 1 receptor accessory protein (IL-1RAP). 10          IL-1 signaling through IL-1R1 and IL-1RAP results in activation through the MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase IFNy expression and increase sensitivity to IL-12. The transcriptional program induced by IL-1 also increases T cell proliferation and protects 15 against AICD. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding an IL-1 DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more 20 polynucleotides or vectors encoding an IL-1 DARIC immune receptor and an engineered antigen receptor. In particular embodiments, the IL-1 DARIC immune receptor transduces an IL-1-mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments an IL-1 DARIC immune receptor contemplated herein comprises: a first 25 multimerization domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain. In particular embodiments an IL-1 DARIC immune receptor contemplated herein comprises: a first 2024264639   14 Nov 2024 multimerization domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving 5 polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one 10 embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. In particular embodiments, the IL-1 DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-1RAP intracellular signaling domain; and a 15 polypeptide comprising a second multimerization domain, a second transmembrane domain, and an IL-1R1 intracellular signaling domain. In particular embodiments, an IL-1 immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and an IL-1R1 intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a 20 second transmembrane domain, and an IL-1RAP intracellular signaling domain. In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PD1. 25         In one embodiment, the first and second multimerization domains are the same. In certain embodiments, an IL-1 DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 2024264639   14 Nov 2024 In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-1RAP intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a 5 CD4 or CD8a transmembrane domain, and an IL-1R1 intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and an IL-1RAP intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a 10 CD4 or CD8a transmembrane domain, and an IL-1R1 intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP 15 polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 7.   TLRDARICImmuneReceptor Toll like receptors (TLR1 through TLR10) are pattern recognition receptors that 20 detect invading pathogens and activate the innate and adaptive immune responses. Activation of TLRs by various ligands leads to induction of a pro-inflammatory transcriptional program and expression of multiple inflammatory cytokines. TLR signaling occurs via homodimerization of TLR signaling domains leading to activation through the MyD88 adaptor protein and IRAK4 phosphorylation. 25 Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase inflammatory cytokine production and induce proliferation. TLR activation can also lead to the activation of IRF3 and IRF7 transcription factors. 2024264639   14 Nov 2024 In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding a TLR DARIC immune receptor. In various embodiments, one or more immune effector cells are modified by introducing one or more 5 polynucleotides or vectors encoding a TLR DARIC immune receptor and an engineered antigen receptor. In particular embodiments, the TLR DARIC immune receptor transduces a TLR mediated immunostimulatory signal upon exposure to a bridging factor. In particular embodiments TLR DARIC immune receptor contemplated herein comprises: a first 10 multimerization domain, a transmembrane domain, and a TLR intracellular signaling domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an identical TLR intracellular signaling domain. In particular embodiments a TLR DARIC immune receptor contemplated herein comprises: a first multimerization domain, a transmembrane domain, and a TLR intracellular signaling 15 domain; a polypeptide cleavage signal; and a second multimerization domain, a transmembrane domain, and an identical TLR intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth 20 disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. 25         In particular embodiments, the TLR DARIC immune receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and a TLR intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an identical TLR intracellular signaling domain. In particular embodiments, a TLR immune 2024264639   14 Nov 2024 receptor is a complex of polypeptides comprising a first polypeptide comprising a first multimerization domain, a first transmembrane domain, and a TLR intracellular signaling domain; and a polypeptide comprising a second multimerization domain, a second transmembrane domain, and an identical TLR intracellular signaling domain. 5          In preferred embodiments, the TLR intracellular signaling domain is isolated from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. In particular embodiments, the first and second transmembrane domains are selected from the group consisting of: CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 10  154, AMN, and PD1. In one embodiment, the first and second multimerization domains are the same. In certain embodiments, a TLR DARIC immune receptor comprises a first or second multimerization domain selected from a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and 15 cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and a TLR intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a 20 transmembrane domain, and an identical TLR intracellular signaling domain. In certain embodiments, a first polypeptide comprises a multimerization domain comprising an FRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain, and a TLR intracellular signaling domain; and a second polypeptide comprises a multimerization domain comprising an FKBP polypeptide or variant thereof, a CD4 or 25 CD8a transmembrane domain, and an identical TLR intracellular signaling domain. In some embodiments, the bridging factor is AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 2024264639   14 Nov 2024 In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBP polypeptide is FKBP12; and the bridging factor is AP21967. In particular embodiments, the FRB polypeptide is FRB; the FKBP polypeptide is FKBP12; and the bridging factor is rapamycin, temsirolimus or everolimus. 5 D.    ENGINEERED ANTIGEN RECEPTORS In particular embodiments, a polypeptide comprises an engineered antigen receptor, a polypeptide cleavage signal and a DARIC immune receptor. In other particular embodiments, a polynucleotide or vector encoding a DARIC immune receptor is introduced into an immune effector cell that comprises an engineered antigen receptor. 10 Without wishing to be bound by any particular theory, it is contemplated in particular embodiments, that any mechanism known in the art may be used to introduce and coexpress an engineered antigen receptor and a DARIC immune receptor in the same immune effector cell or population of cells to increase the resistance of the immune effector cells to the TME and potentiate and increase the efficiency, potency, and durability of the immune 15 effector cell response. In particular embodiments, immune effector cells contemplated herein comprise an engineered antigen receptor and a DARIC immune receptor. In particular embodiments, the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), or a zetakine. 20       1.   Engineered TCRs In particular embodiments, immune effector cells contemplated herein comprise an engineered TCR and a DARIC immune receptor. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and a DARIC immune receptor separated by one or more polypeptide cleavage signals. In one 25 embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and a polynucleotide or vector encoding a DARIC immune receptor. In 2024264639   14 Nov 2024 one embodiment, T cells are engineered to express an engineered TCR are further engineered by introducing a polynucleotide or vector encoding a DARIC immune receptor. Naturally occurring T cell receptors comprise two subunits, an alpha chain and a beta chain subunit, each of which is a unique protein produced by recombination event in 5 each T cell’s genome. Libraries of TCRs may be screened for their selectivity to particular target antigens. In this manner, natural TCRs, which have a high-avidity and reactivity toward target antigens may be selected, cloned, and subsequently introduced into a population of T cells used for adoptive immunotherapy. In one embodiment, T cells are modified by introducing a TCR subunit has the 10 ability to form TCRs that confer specificity to T cells for tumor cells expressing a target antigen. In particular embodiments, the subunits have one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunits retain the ability to form TCRs and confer upon transfected T cells the ability to home to target cells and participate in immunologically-relevant 15 cytokine signaling. The engineered TCRs preferably also bind target cells displaying the relevant tumor-associated peptide with high avidity, and optionally mediate efficient killing of target cells presenting the relevant peptide in vivo. The nucleic acids encoding engineered TCRs are preferably isolated from their natural context in a (naturally-occurring) chromosome of a T cell and can be incorporated 20 into suitable vectors as described elsewhere herein. Both the nucleic acids and the vectors comprising them can be transferred into a cell, preferably a T cell in particular embodiments. The modified T cells are then able to express one or more chains of a TCR encoded by the transduced nucleic acid or nucleic acids. In preferred embodiments, the engineered TCR is an exogenous TCR because it is introduced into T cells that do not 25 normally express the particular TCR. The essential aspect of the engineered TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component. In contrast to engineered TCRs, CARs are engineered to bind target antigens in an MHC independent manner. 2024264639   14 Nov 2024 The TCR can be expressed with additional polypeptides attached to the aminoterminal or carboxyl-terminal portion of the alpha chain or beta chain of a TCR so long as the attached additional polypeptide does not interfere with the ability of the alpha chain or beta chain to form a functional T cell receptor and the MHC dependent antigen recognition. 5         Antigens that are recognized by the engineered TCRs contemplated in particular embodiments include, but are not limited to cancer antigens, including antigens on both hematological cancers and solid tumors. Illustrative antigens include, but are not limited to alpha folate receptor, alpha folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, 10 CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D 15 Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1. 2.    ChimericAntigenReceptors In various embodiments, immune effector cells express CARs that redirect cytotoxicity toward tumor cells. CARs are molecules that combine antibody-based 20 specificity for a target antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity. As used herein, the term, “chimeric,” describes being composed of parts of different proteins or DNAs from different origins. In particular embodiments, immune effector cells contemplated herein comprise 25 CAR and a DARIC immune receptor. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding a CAR and a DARIC immune receptor separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding a CAR and a polynucleotide 2024264639   14 Nov 2024 or vector encoding a DARIC immune receptor. In one embodiment, T cells are engineered to express a CAR are further engineered by introducing a polynucleotide or vector encoding a DARIC immune receptor. In various embodiments, a CAR comprises an extracellular domain that binds to a 5 specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and an intracellular signaling domain. The main characteristic of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility 10 (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors. In particular embodiments, CARs comprise an extracellular binding domain that specifically binds to a target polypeptide. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a 15 biological molecule of interest. In particular embodiments, the extracellular binding domain comprises an antibody or antigen binding fragment thereof. In one preferred embodiment, the binding domain is an scFv. In another preferred embodiment, the binding domain is a camelid antibody. 20         In particular embodiments, the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, 25 FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1. 2024264639   14 Nov 2024 In particular embodiments, the CARs comprise an extracellular binding domain, e.g., antibody or antigen binding fragment thereof that binds an antigen, wherein the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex. 5         In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be 10 altered. In another embodiment, the hinge domain comprises a CD8a hinge region. In one embodiment, the hinge is a PD-1 hinge or CD152 hinge. The transmembrane domain (TM) of the CAR fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain may be derived either from a natural, 15 synthetic, semi-synthetic, or recombinant source. Illustrative TM domains may be derived from (i.e., comprise at least the transmembrane region(s) of the alpha, beta, gamma, or delta chain of the T-cell receptor, CD38, CD3s, CD3y, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1. 20        In one embodiment, a CAR comprises a TM domain derived from CD8a. In another embodiment, a CAR contemplated herein comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TM domain and the intracellular signaling domain of the CAR. A glycine-serine linker provides a particularly suitable linker. 25         In preferred embodiments, a CAR comprises an intracellular signaling domain that comprises one or more “co-stimulatory signaling domains” and a “primary signaling domain.” Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. 2024264639   14 Nov 2024 Illustrative examples of ITAM containing primary signaling domains suitable for use in CARs contemplated in particular embodiments include those derived from FcRy, FcRp, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. In particular preferred embodiments, a CAR comprises a CD3Z primary signaling domain and one or 5 more co-stimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain. In particular embodiments, a CAR comprises one or more co-stimulatory signaling domains to enhance the efficacy and expansion of T cells expressing CAR receptors. 10          Illustrative examples of such co-stimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, a CAR comprises one or more co- 15 stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3Z primary signaling domain. In various embodiments, the CAR comprises: an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from a polypeptide selected from the group 20 consisting of: CD4, CD8a, CD154, and PD-1; one or more intracellular co-stimulatory signaling domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcR0, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. 25        3.    Zetakines In various embodiments, immune effector cells comprise chimeric cytokine receptor that redirect cytotoxicity toward tumor cells. Zetakines are chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a 2024264639   14 Nov 2024 soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain. Zetakines, when expressed on the surface of T lymphocytes, direct T cell activity to those cells expressing a receptor for which the soluble receptor ligand is specific. Zetakine 5 chimeric immunoreceptors redirect the antigen specificity of T cells, with application to treatment of a variety of cancers, particularly via the autocrine / paracrine cytokine systems utilized by human malignancy. In particular embodiments, immune effector cells contemplated herein comprise one or more chains of a zetakine receptor and a DARIC immune receptor. In one 10 embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and a DARIC immune receptor separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and a polynucleotide or vector encoding a DARIC immune receptor. In one embodiment, 15 T cells are engineered to express one or more chains of a zetakine receptor are further engineered by introducing a polynucleotide or vector encoding a DARIC immune receptor. In particular embodiments, the zetakine comprises an immunosuppressive cytokine or cytokine receptor binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain. 20          In particular embodiments, the cytokine or cytokine receptor binding variant thereof is selected from the group consisting of: interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and interleukin-13 (IL-13). In certain embodiments, the linker comprises a CH2CH3 domain, hinge domain, or the like. In one embodiment, a linker comprises the CH2 and CH3 domains of IgG1, IgG4, 25 or IgD. In one embodiment, a linker comprises a CD8a or CD4 hinge domain. In particular embodiments, the transmembrane domain is selected from the group consisting of: the alpha or beta chain of the T-cell receptor, CD38, CD3s, CD3y, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1. 2024264639   14 Nov 2024 In particular embodiments, the intracellular signaling domain is selected from the group consisting of: an ITAM containing primary signaling domain and / or a costimulatory domain. In particular embodiments, the intracellular signaling domain is selected from the 5 group consisting of: FcRy, FcR0, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. In particular embodiments, the intracellular signaling domain is selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 10  (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, a chimeric cytokine receptor comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3Z primary signaling domain. 15 E.    POLYPEPTIDES Various polypeptides are contemplated herein, including, but not limited to, DARIC immune receptors, engineered TCRs, CARs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof. In preferred embodiments, a polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-6. 20 “Polypeptide,” “peptide” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. In one embodiment, a “polypeptide” includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may 25 comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In particular 2024264639   14 Nov 2024 preferred embodiments, fusion polypeptides, polypeptides, fragments and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides. An “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from a cellular 5 environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances. In particular embodiments, an isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source. Polypeptides include “polypeptide variants.” Polypeptide variants may differ from 10 a naturally occurring polypeptide in one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide by introducing one or more substitutions, deletions, 15 additions and / or insertions the polypeptide. In particular embodiments, polypeptides include polypeptides having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant 20 maintains at least one biological activity of the reference sequence. In particular embodiments, the biological activity is binding affinity. In particular embodiments, the biological activity is enzymatic activity. In certain embodiments, a polypeptide complex comprises (i) a first polypeptide, e.g., first fusion polypeptide, having a first multimerization domain and (ii) second polypeptide, e.g., 25 second fusion polypeptide, having a second multimerization domain that is not the same as the first multimerization domain, wherein the first and second multimerization domains substantially contribute to or efficiently promote formation of the polypeptide complex in the presence of a bridging factor. The interaction(s) between the first and second multimerization domains substantially contributes to or efficiently promotes the multimerization of the first and 30 second fusion polypeptides if there is a statistically significant reduction in the association 2024264639   14 Nov 2024 between the first and second fusion polypeptides in the absence of the first multimerization domain, the second multimerization domain, or the bridging factor. In certain embodiments, when the first and second fusion polypeptides are co-expressed, at least about 60%, for instance, at least about 60% to about 70%, at least about 70% to about 80%, at least about 80% 5 to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second single chain polypeptides form multimers with each other in the presence of a bridging factor. Polypeptides variants include biologically active “polypeptide fragments.” Illustrative examples of biologically active polypeptide fragments include binding domains, signaling 10 domains, and the like. As used herein, the term “biologically active fragment” or “minimal biologically active fragment” refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to 15 about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long. 20          In particular embodiments, the polypeptides set forth herein may comprise one or more amino acids denoted as “X.” “X” if present in an amino acid SEQ ID NO, refers to any one or more amino acids. In particular embodiments, SEQ ID NOs denoting a fusion protein comprise a sequence of continuous X residues that cumulatively represent any scFv. As noted above, polypeptides may be altered in various ways including amino acid 25 substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367 30   382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, Fourth 2024264639   14 Nov 2024 Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.). 5          In certain embodiments, a polypeptide variant comprises one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the 10 polynucleotides and polypeptides contemplated in particular embodiments and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence, e.g., 15 according to Table 1. TABLE 1- Amino Acid Codons Amino Acids One letter code Three letter code Codons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Aspartic acid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine F Phe UUC UUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU Isoleucine I Iso AUA AUC AUU Lysine K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC   CUG   CUU Methionine M Met AUG Asparagine N Asn AAC AAU 2024264639   14 Nov 2024 Proline P Pro CCA CCC CCG CCU Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGU Serine S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACU Valine V Val GUA GUC GUG GUU Tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. Preferably, amino acid changes in the protein variants disclosed herein are 5 conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, 10 methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those 15 of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). In one embodiment, where expression of two or more polypeptides is desired, the 20 polynucleotide sequences encoding them can be separated by an IRES sequence as disclosed elsewhere herein. Polypeptides contemplated in particular embodiments include fusion polypeptides. In particular embodiments, fusion polypeptides and polynucleotides encoding fusion 2024264639   14 Nov 2024 polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments. In preferred embodiments, a fusion polypeptide is a DARIC immune receptor. In other preferred embodiments, the fusion polypeptide comprises one or more DARIC immune 5 receptors. In particular preferred embodiments, fusion polypeptides comprise one or more segments, fragments, or domains of one or more human polypeptides. In another embodiment, two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein. 10         Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), binding domains, signaling domains, etc., epitope tags (e.g., maltose binding protein (“MBP”), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus 15 to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. In particular embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. 20 Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein. Fusion polypeptides may optionally comprise a linker that can be used to link the one 25 or more polypeptides or domains within a polypeptide. A peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art. Suitable 30 peptide linker sequences may be chosen based on the following factors: (1) their ability to 2024264639   14 Nov 2024 adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral 5 amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions 10 that can be used to separate the functional domains and prevent steric interference. Preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein. Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between. 15          Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26). Suitable protease cleavages sites and self-cleaving peptides are known to the skilled 20   person (see, e.g., in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC proteases, potyvirus P1 (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 25 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO: 18), for example, ENLYFQG (SEQ ID NO: 19) and ENLYFQS (SEQ ID NO: 20), 30 wherein X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S). 2024264639   14 Nov 2024 In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or ribosomal skipping sequence. Illustrative examples of ribosomal skipping sequences include but are not limited to: a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). 5 In a particular embodiment, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In one embodiment, the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) 10  (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. Illustrative examples of 2A sites are provided in Table 2. TABLE 2: SEQ ID NO: 21 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 22 ATNFSLLKQAGDVEENPGP SEQ ID NO: 23 LLKQAGDVEENPGP SEQ ID NO: 24 GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 25 EGRGSLLTCGDVEENPGP SEQ ID NO: 26 LLTCGDVEENPGP SEQ ID NO: 27 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 28 QCTNYALLKLAGDVESNPGP SEQ ID NO: 29 LLKLAGDVESNPGP SEQ ID NO: 30 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 31 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 32 LLKLAGDVESNPGP SEQ ID NO: 33 LLNFDLLKLAGDVESNPGP SEQ ID NO: 34 TLNFDLLKLAGDVESNPGP SEQ ID NO: 35 LLKLAGDVESNPGP SEQ ID NO: 36 NFDLLKLAGDVESNPGP SEQ ID NO: 37 QLLNFDLLKLAGDVESNPGP 2024264639   14 Nov 2024 SEQ ID NO: 38 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 39 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 40 LNFDLLKLAGDVESNPGP SEQ ID NO: 41 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 42 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP In preferred embodiments, a polypeptide or fusion polypeptide comprises a DARIC immune receptor polypeptide. F.    Polynucleotides In particular embodiments, polynucleotides encoding DARIC immune receptors, 5 engineered TCRs, CARs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof are provided. As used herein, the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), 10 messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to 15 a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that “intermediate lengths,” in 20 this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In particular embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 2024264639   14 Nov 2024 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence. In particular embodiments, polynucleotides may be codon-optimized. As used herein, the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a 5 polypeptide in order to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to 10 their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, 15   (x) systematic variation of codon sets for each amino acid, and / or (xi) isolated removal of spurious translation initiation sites. As used herein the term “nucleotide” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at 20 the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose. Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). 25 The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. Nucleotides are usually mono, di- or triphosphates. The nucleotides can be unmodified or modified at the sugar, phosphate and / or base moiety, (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see for example, WO 92 / 07065 and WO 93 / 15187). Examples of modified 2024264639   14 Nov 2024 nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 21832196). A nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the 5 term “nucleoside” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and / or base moiety, (also referred to interchangeably as 10 nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As also noted above, examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196). Illustrative examples of polynucleotides include but are not limited to polynucleotides encoding SEQ ID NOs: 1-6. 15          In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to polynucleotides encoding DARIC immune receptors, engineered antigen receptors, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein. As used herein, the terms “polynucleotide variant” and “variant” and the like refer 20 to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” 25 and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. 2024264639   14 Nov 2024 In one embodiment, a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. To hybridize under “stringent conditions” describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized. Generally, stringent conditions are 5 selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are 10 occupied at equilibrium. The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally 15 aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window 20 of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the 25 reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity”. A 30 “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer 2024264639   14 Nov 2024 units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons 5 between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous 10 positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, 15 FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids 20  Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 19941998, Chapter 15. As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA 25 fragment that has been removed from the sequences that are normally adjacent to the fragment. An “isolated polynucleotide” also refers to a complementary DNA (cDNA), a recombinant DNA, or other polynucleotide that does not exist in nature and that has been made by the hand of man. In particular embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or 30 derived from a recombinant source. 2024264639   14 Nov 2024 In various embodiments, a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail. Terms that describe the orientation of polynucleotides include: 5' (normally the end 5 of the polynucleotide having a free phosphate group) and 3' (normally the end of the polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the premessenger (premRNA) [except for uracil (U) in RNA, 10 instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as “template,” “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse orientation” refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation. 15          The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'. The latter sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'. A sequence that is equal to its reverse 20 complement is said to be a palindromic sequence. Complementarity can be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there can be “complete” or “total” complementarity between the nucleic acids. Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a 25 polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and / or primate codon selection. In particular embodiments, the polynucleotides 30 are codon optimized for expression and / or stability. Further, alleles of the genes 2024264639   14 Nov 2024 comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The term “nucleic acid cassette” or “expression cassette” as used herein refers to 5 genetic sequences within the vector which can express an RNA, and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. Vectors may comprise 1, 2, 3, 4, 5, 6, 10   7, 8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate 15 intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3 ' and 5 ' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit. 20          Polynucleotides include polynucleotide(s)-of-interest. As used herein, the term “polynucleotide-of-interest” refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein. The polynucleotides contemplated herein, regardless of the length of the coding 25 sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides 30 encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as 2024264639   14 Nov 2024 known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. 5          Polynucleotides can be prepared, manipulated, expressed and / or delivered using any of a variety of well-established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into appropriate vector. Illustrative examples of vectors include, but are not limited to plasmid, autonomously 10 replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac. Additional Illustrative examples of vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. 15           Illustrative examples of viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Illustrative examples of expression vectors include but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-20 DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells. In particular embodiments, the vector is an episomal vector or a vector that is 25 maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. “Expression control sequences,” “control elements,” or “regulatory sequences” present 30 in an expression vector are those non-translated regions of the vector including but not limited 2024264639   14 Nov 2024 to the origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions, all of which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. 5 Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used. In particular embodiments, a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors. A vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and / or 10 enhancers. An “endogenous control sequence” is one which is naturally linked with a given gene in the genome. An “exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being 15 genetically manipulated. A “synthetic” control sequence may comprise elements of one more endogenous and / or exogenous sequences, and / or sequences determined in vitro or in silico that provide optimal promoter and / or enhancer activity for the particular therapy. The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and 20 transcribes polynucleotides operably linked to the promoter. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide. 25         The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “promoter / enhancer” refers to a segment of DNA which contains sequences capable of 30 providing both promoter and enhancer functions. 2024264639   14 Nov 2024 The term “operably linked”, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and / or enhancer) and a second polynucleotide 5 sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence 10 may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively. Illustrative ubiquitous expression control sequences suitable for use in particular 15 embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H 20 (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), p-kinesm (P-KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 - 1482 (2007)), a Ubiquitin C promoter (UBC), a phosphoglycerate 25 kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken p-actin (CAG) promoter, a p-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) U3 promoter (Haas et al. Journal of Virology. 2003;77(17): 9439-9450). In one embodiment, a vector comprises an MNDU3 promoter. 2024264639   14 Nov 2024 In one embodiment, a vector comprises an EF1a promoter comprising the first intron of the human EF1a gene. In one embodiment, a vector comprises an EF1a promoter that lacks the first intron of the human EF1a gene. 5          In a particular embodiment, it may be desirable to use a cell, cell type, cell lineage or tissue specific expression control sequence to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development). 10          In a particular embodiment, it may be desirable to express a polynucleotide a T cell specific promoter. As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, 15 etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest. 20          Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system 25 (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducer agents include, but are not limited to glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof. Conditional expression can also be achieved by using a site specific DNA 30 recombinase. According to certain embodiments the vector comprises at least one 2024264639   14 Nov 2024 (typically two) site(s) for recombination mediated by a site specific recombinase. As used herein, the terms “recombinase” or “site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, 5 five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, 10 Gin, OC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA. The polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms “recombination 15 sequence,” “recombination site,” or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds. For example, one recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current 20 Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include but are not limited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995). Suitable recognition sites for the FLP recombinase include, but are not limited to: 25 FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988). Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme X Integrase, e.g., phi-c31. The y C31 SSR mediates recombination only between the heterotypic sites attB (34 bp in 30 length) and attP (39 bp in length) (Groth et al., 2000). attB and attP, named for the 2024264639   14 Nov 2024 attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by y C31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert to further yC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. For 5 catalyzing insertions, it has been found that attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, typical strategies position by homologous recombination an attP-bearing “docking site” into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion. 10          As used herein, an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art 15 include those described in U.S. Pat. No. 6,692,736. Further examples of “IRES” known in the art include but are not limited to IRES obtainable from picornavirus (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), and insulin- 20 like growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4, the encephelomycarditis virus (EMCV) which is commercially available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRES have also been reported in viral genomes of Picornaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia 25 virus (FrMLV) and Moloney murine leukemia virus (MoMLV). In one embodiment, the IRES used in polynucleotides contemplated herein is an EMCV IRES. In particular embodiments, the polynucleotides comprise polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide. As used herein, the term 30 “Kozak sequence” refers to a short nucleotide sequence that greatly facilitates the initial 2024264639   14 Nov 2024 binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:43), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). Elements directing the efficient termination and polyadenylation of the heterologous 5 nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors comprise a polyadenylation sequence 3’ of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent 10 RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3‘ end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant 15 AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product. In particular embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is an SV40 polyA 20 sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit p-globin polyA sequence (r^gpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In particular embodiments, the poly(A) sequence is synthetic. In some embodiments, a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and / or 25 uncontrolled proliferation. In specific embodiments, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID). In certain embodiments, polynucleotides comprise gene segments that cause the 30 immune effector cells, e.g., T cells, to be susceptible to negative selection in vivo. By 2024264639   14 Nov 2024 “negative selection” is meant that the infused cell can be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes are known in the art, and include, inter alia the 5 following: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)). 10          In some embodiments, genetically modified immune effector cells, such as T cells, comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene which, upon being introduced into the host cell expresses a dominant phenotype permitting positive selection of cells carrying the gene. Genes of this type are known in the 15 art, and include, inter alia, hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene. 20          In one embodiment, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker. In a particular embodiment, the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other. An example of a fused polynucleotide that yields as an expression product a polypeptide that 25 confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT US91 / 08442 and PCT / US94 / 05601, by S. D. Lupton, describing the use of bifunctional 2024264639   14 Nov 2024 selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers. Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred negative selectable markers are derived from 5 genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker. 10          In particular embodiments, polynucleotides encoding one or more polypeptides, or fusion polypeptides may be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods. In particular embodiments, delivery of one or more polynucleotides may be provided by the same method or by different methods, and / or by the same vector or by different vectors. 15          The term “vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. In particular embodiments, non-viral vectors are used to deliver one or 20 more polynucleotides contemplated herein to a T cell. Illustrative examples of non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes. Illustrative methods of non-viral delivery of polynucleotides contemplated in 25 particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock. Illustrative examples of polynucleotide delivery systems suitable for use in particular 30 embodiments contemplated in particular embodiments include, but are not limited to those 2024264639   14 Nov 2024 provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See 5   e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments. Viral vectors comprising polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, typically by systemic 10   administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient. 15          In one embodiment, viral vectors comprising polynucleotides contemplated herein are administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of 20 administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Illustrative examples of viral vector systems suitable for use in particular embodiments contemplated in particular embodiments include but are not limited to adeno- 25   associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors. In various embodiments, one or more polynucleotides are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides. 2024264639   14 Nov 2024 AAV is a small (~26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Recombinant AAV (rAAV) are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal 5 repeats (ITRs). The ITR sequences are about 145 bp in length. In particular embodiments, the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype. For 10 example, a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In particular embodiments, the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences 15 derived from AAV6. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2. In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest. Construction of rAAV vectors, production, and purification thereof have been 20 disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety. In various embodiments, one or more polynucleotides are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising the one or more polynucleotides. 25          As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus 30 (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), 2024264639   14 Nov 2024 feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus. As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to: HIV (human 5 immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. 10          In various embodiments, a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT / FLAP, a Psi (T) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein. 15          In particular embodiments, lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus. As used herein, the term “integration defective lentivirus” or “IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is herein 20 incorporated by reference in its entirety. Illustrative mutations in the HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, 25  R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H. The term “long terminal repeat (LTR)” refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain 30  U3, R and U5 regions. 2024264639   14 Nov 2024 As used herein, the term “FLAP element” or “cPPT / FLAP” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. 5          As used herein, the term “packaging signal” or “packaging sequence” refers to psi [ T] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. The term “export element” refers to a cis-acting post-transcriptional regulatory element 10 which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE). 15          In particular embodiments, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; 20 Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766). Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs. “Self-inactivating” (SIN) vectors refers to replication-defective 25 vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self-inactivation is preferably achieved through in the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to produce the vector RNA. Thus, during reverse 30 transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In particular 2024264639   14 Nov 2024 embodiments, it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells. In the case of HIV based lentivectors, it has been discovered that such vectors tolerate significant U3 5 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers. An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, 10 viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. The terms “pseudotype” or “pseudotyping” as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing 15 preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ presenting cells. In certain embodiments, lentiviral vectors are produced according to known methods. 20 See e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22. According to certain specific embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it is to be understood that many different sources of retroviral and / or lentiviral sequences can be 25 used or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein. Moreover, a variety of lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a 30 viral vector or transfer plasmid contemplated herein. 2024264639   14 Nov 2024 In various embodiments, one or more polynucleotides are introduced into an immune effector cell, by transducing the cell with an adenovirus comprising the one or more polynucleotides. Adenoviral based vectors are capable of very high transduction efficiency in many 5 cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and / or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce 10 multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. Generation and propagation of the current adenovirus vectors, which are replication deficient, may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 15 proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions (Graham & Prevec, 1991). Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & 20 Prevec, 1992). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with 25 intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). In various embodiments, one or more polynucleotides are introduced into an immune effector cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides. The mature HSV virion consists of an enveloped icosahedral capsid with a viral 30 genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one 2024264639   14 Nov 2024 embodiment, the HSV based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be deficient 5 in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof. Advantages of the HSV vector are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent 10 Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which are incorporated by reference herein in its entirety. G.   Genetically Modified Cells In various embodiments, cells are modified to express DARIC immune receptors, engineered TCRs, CARs, zetakines, and / or fusion proteins contemplated herein, for use in 15 the treatment of cancer. Cells may be non-genetically modified to express the polypeptides contemplated herein, or in particular preferred embodiments, cells may be genetically modified to express the polypeptides contemplated herein. As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms, 20 “genetically modified cells,” “modified cells,” and “redirected cells,” are used interchangeably in particular embodiments. In particular embodiments, the DARIC immune receptor polypeptides contemplated herein are introduced and expressed in immune effector cells to improve the resistance of the cells to the immunosuppressive signals in the TME. In particular embodiments, 25 DARIC immune receptor polypeptides are introduced and expressed in immune effector cells that have been redirected to a target cell by virtue of co-expressing an engineered antigen receptor in the cell. An “immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of 2024264639   14 Nov 2024 ADCC and / or CDC). The illustrative immune effector cells contemplated herein are T lymphocytes, in particular cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells. In one embodiment, immune effector cells include natural killer (NK) cells. In one embodiment, immune effector cells include natural killer T (NKT) cells. Immune 5 effector cells can be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous,” as used herein, refers to cells from the same subject. “Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different subject that are 10 genetically identical to the cell in comparison. “Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are autologous. Illustrative immune effector cells suitable for introducing the DARIC immune receptor polypeptides contemplated herein include T lymphocytes. The terms “T cell” or 15 “T lymphocyte” are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other subset of T 20 cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. As would be understood by the skilled person, other cells may also be used as immune effector cells with DARIC immune receptor polypeptides contemplated herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and 25 macrophages. Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in particular embodiments, immune effector cell includes progenitors of immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from cord blood, bone marrow or mobilized peripheral 2024264639   14 Nov 2024 blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells. As used herein, immune effector cells genetically engineered to contain a specific chimeric receptor may be referred to as, “antigen specific redirected immune effector 5 cells.” The term, “CD34+ cell,” as used herein refers to a cell expressing the CD34 protein on its cell surface. “CD34,” as used herein refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entrance into lymph nodes. The CD34+ cell population contains hematopoietic stem cells 10 (HSC), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils and cells of the monocyte / macrophage lineage. Methods for making the immune effector cells which express one or more DARIC immune receptor polypeptides contemplated herein are provided in particular 15 embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells with one or more nucleic acids and / or vectors or combination thereof comprising one or more DARIC immune receptor polypeptides as contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an 20 individual such that the immune effector cells express one or more DARIC immune receptor polypeptides and engineered antigen receptors contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered into the individual. In further embodiments, the immune effector cells are 25 first activated and stimulated to proliferate in vitro prior to being genetically modified. In this regard, the immune effector cells may be cultured before and / or after being genetically modified. In particular embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. 30 In particular embodiments, the modified immune effector cells comprise T cells. 2024264639   14 Nov 2024 T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject 5 using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation. In other embodiments, an isolated or purified population of T cells is used. In some embodiments, after isolation of PBMC, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, 10 expansion, and / or genetic modification. In one embodiment, an isolated or purified population of T cells expresses one or more of the markers including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof In certain embodiments, the T cells are isolated from an individual and first activated and stimulated to proliferate in vitro prior to being modified to express one or more DARIC 15 immune receptor polypeptides. In order to achieve sufficient therapeutic doses of T cell compositions, T cells are often subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 20   7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety. In particular embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of vectors or polynucleotides encoding one or more DARIC immune receptor polypeptides. Optionally in combination with an engineered antigen 25 receptor contemplated herein. In one embodiment, T cells are activated at the same time that they are modified. In various embodiments, a method of generating an immune effector cell comprises activating a population of cells comprising T cells and expanding the population of T cells. T cell activation can be accomplished by providing a primary stimulation signal through the T 2024264639   14 Nov 2024 cell TCR / CD3 complex and by providing a secondary co-stimulation signal through an accessory molecule, e.g., CD28. The TCR / CD3 complex may be stimulated by contacting the T cell with a suitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonal antibody. Illustrative 5 examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1. In addition to the primary stimulation signal provided through the TCR / CD3 complex, induction of T cell responses requires a second, co-stimulatory signal. In particular embodiments, a CD28 binding agent can be used to provide a co-stimulatory signal. Illustrative examples of CD28 binding agents include but are not limited to: natural CD 28 10 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7 family of proteins, such as B7-1(CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibody or fragment thereof capable of crosslinking the CD28 molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10. In one embodiment, the molecule providing the primary stimulation signal, for example 15 a molecule which provides stimulation through the TCR / CD3 complex and the co-stimulatory molecule are coupled to the same surface. In certain embodiments, binding agents that provide stimulatory and co-stimulatory signals are localized on the surface of a cell. This can be accomplished by transfecting or transducing a cell with a nucleic acid encoding the binding agent in a form suitable for its 20 expression on the cell surface or alternatively by coupling a binding agent to the cell surface. In another embodiment, the molecule providing the primary stimulation signal, for example a molecule which provides stimulation through the TCR / CD3 complex and the costimulatory molecule are displayed on antigen presenting cells. In one embodiment, the molecule providing the primary stimulation signal, for example 25 a molecule which provides stimulation through the TCR / CD3 complex and the co-stimulatory molecule are provided on separate surfaces. In a certain embodiment, one of the binding agents that provides stimulatory and costimulatory signals is soluble (provided in solution) and the other agent(s) is provided on one or more surfaces. 2024264639   14 Nov 2024 In a particular embodiment, the binding agents that provide stimulatory and costimulatory signals are both provided in a soluble form (provided in solution). In various embodiments, the methods for making T cells contemplated herein comprise activating T cells with anti-CD3 and anti-CD28 antibodies. 5          In one embodiment, expanding T cells activated by the methods contemplated herein further comprises culturing a population of cells comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or any hourly integer value in between. In another embodiment, the T cell composition may be cultured for 14 days. In a particular embodiment, T cells are cultured for about 21 days. In another embodiment, the T cell compositions are 10 cultured for about 2-3 days. Several cycles of stimulation / activation / expansion may also be desired such that culture time of T cells can be 60 days or more. In particular embodiments, conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not 15 limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGF0, and TNF-a or any other additives suitable for the growth of cells known to the skilled artisan. Further illustrative examples of cell culture media include, but are not limited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, 20 with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are 25 maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02). In particular embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and co-stimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, 30 and / or IL-15. 2024264639   14 Nov 2024 In other embodiments, artificial APC (aAPC) made by engineering K562, U937, 721.221, T2, and C1R cells to direct the stable expression and secretion, of a variety of costimulatory molecules and cytokines. In a particular embodiment K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the AAPC cell 5 surface. Populations of T cells can be expanded by aAPCs expressing a variety of costimulatory molecules including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8a T cells. aAPCs provided in WO 03 / 057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety. 10         In a particular embodiment, polynucleotide encoding one or more DARIC immune receptor polypeptides and an engineered antigen receptor are introduced into the population of T cells. In a particular embodiment, polynucleotide encoding one or more DARIC immune receptor polypeptides is introduced into a population of T cells that express an engineered antigen receptor. The polynucleotides may be introduced into the T cells by microinjection, 15 transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and the like. In a preferred embodiment, polynucleotides are introduced into a T cell by viral transduction. Illustrative examples of viral vector systems suitable for introducing a polynucleotide 20 into an immune effector cell or CD34+ cell include but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer. In one embodiment, polynucleotides are introduced into a T cell by AAV transduction. In one embodiment, polynucleotides are introduced into a T cell by retroviral transduction. 25          In one embodiment, polynucleotides are introduced into a T cell by lentiviral transduction. In one embodiment, polynucleotides are introduced into a T cell by adenovirus transduction. In one embodiment, polynucleotides are introduced into a T cell by herpes simplex 30 virus transduction. 2024264639   14 Nov 2024 In one embodiment, polynucleotides are introduced into a T cell by vaccinia virus transduction. H.   Compositions and Formulations The compositions contemplated herein may comprise one or more polypeptides, 5 polynucleotides, vectors comprising same, genetically modified immune effector cells, bridging factors, etc. Compositions include but are not limited to pharmaceutical compositions. A “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of 10 therapy. It will also be understood that, if desired, the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not 15 adversely affect the ability of the composition to deliver the intended therapy. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or 20 complication, commensurate with a reasonable benefit / risk ratio. The term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the bridging factors, polypeptides, polynucleotides, vectors comprising same, or genetically modified immune effector cells are administered. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, 25 including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in particular embodiments, include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium 2024264639   14 Nov 2024 chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 5         In one embodiment, a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In particular embodiments, a composition comprising a carrier is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In particular embodiments, a composition comprising a pharmaceutically acceptable 10 carrier is suitable for intraventricular, intraspinal, or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the bridging factors, polypeptides, polynucleotides, vectors comprising 15 same, or genetically modified immune effector cells, use thereof in the pharmaceutical compositions is contemplated. In particular embodiments, compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier. A composition comprising a cell-based composition contemplated herein can be administered separately by enteral or 20 parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals. In particular embodiments, compositions contemplated herein comprise a bridging factor and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier must be of sufficiently high purity and of 25 sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition. For example, the pharmaceutically 30 acceptable carrier can be, without limitation, a binding agent (e.g., pregelatinized maize 2024264639   14 Nov 2024 starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated 5 vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic 10 acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like. Such carrier solutions also can contain buffers, diluents and other suitable additives. The term “buffer” as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered 15 saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal / physiologic saline (0.9% NaCl). The pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7. Alternatively, the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still another 20 embodiment, the composition has a pH of about 7.4. Compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium. The compositions may be a suspension. The term “suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are 25 not adhered to a support, such as a culture dish. In particular embodiments, compositions contemplated herein are formulated in a suspension, where the modified T cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like. Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, 2024264639   14 Nov 2024 isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium. In certain embodiments, a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition 5 comprising a population of modified T cells. The therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum. In some embodiments, compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to 10 human subjects. In particular embodiments, the pharmaceutically acceptable cell culture medium is a serum free medium. Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost. In various 15 embodiments, the serum-free medium is animal-free, and may optionally be protein-free. Optionally, the medium may contain biopharmaceutically acceptable recombinant proteins. “Animal-free” medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources. 20 “Protein-free” medium, in contrast, is defined as substantially free of protein. Illustrative examples of serum-free media used in particular compositions includes but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10. In a preferred embodiment, the compositions comprising modified T cells are 25 formulated in PlasmaLyte. In various embodiments, compositions comprising modified T cells are formulated in a cryopreservation medium. For example, cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw. Illustrative examples of cryopreservation media used in particular compositions includes, 30 but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2. 2024264639   14 Nov 2024 In one embodiment, the compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10. In particular embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contamination. By “substantially free” with respect to endotoxin 5 is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU / kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells. In particular embodiments, compositions contemplated herein contain about 0.5 EU / mL to about 5.0 EU / mL, or about 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 10 EU / mL, or 5.0 EU / mL. In particular embodiments, formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., enteral and parenteral, e.g., intravascular, 15 intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It would be understood by the skilled artisan that particular embodiments contemplated herein may comprise other formulations, such as those that are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 20 volume I and volume II. 22nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated by reference herein, in its entirety. In particular embodiments, compositions comprise an amount of immune effector cells, including CAR T cells, that express one or more DARIC immune receptor polypeptides contemplated herein. As used herein, the term “amount” refers to “an amount effective” or 25 “an effective amount” of cells comprising one or more DARIC immune receptor polypeptides contemplated herein, etc., to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. A “prophylactically effective amount” refers to an amount of cells comprising one or more DARIC immune receptor polypeptides contemplated herein, etc., effective to 30 achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic 2024264639   14 Nov 2024 dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount. A “therapeutically effective amount” refers to an amount of cells comprising one or more DARIC immune receptor polypeptides contemplated herein that is effective to “treat” a 5 subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells described herein may be administered at 10 a dosage of 102 to 1010 cells / kg body weight, preferably 105 to 106 cells / kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs or less. Hence the density of the desired cells is 15 typically greater than 106 cells / ml and generally is greater than 107 cells / ml, generally 108 cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some embodiments, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106 / kilogram (106-1011 20 per patient) may be administered. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction of the immune response. Generally, compositions comprising the cells activated and expanded as described 25 herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions contemplated herein are used in the treatment of cancer. In particular embodiments, the immune effector cells may be administered either alone, or as a pharmaceutical compositions in combination with carriers, diluents, excipients, and / or with other components such as IL-2 or other cytokines 30 or cell populations. 2024264639   14 Nov 2024 In particular embodiments, pharmaceutical compositions comprise an amount of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. In a particular embodiment, compositions comprise an effective amount of immune 5 effector cells comprising one or more DARIC immune receptor polypeptides contemplated herein, alone or in combination with one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular 10 disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, antiinflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents. In certain embodiments, compositions comprising immune effector cells 15 comprising one or more DARIC immune receptor polypeptides contemplated herein may be administered in conjunction with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; 20 ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; 25 nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic 30 acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, 2024264639   14 Nov 2024 rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, 5 azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; 10 demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; 15 mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin 20 C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin) ; ONTAK™ (denileukin diftitox) ; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the 25 above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically 30 acceptable salts, acids or derivatives of any of the above. 2024264639   14 Nov 2024 A variety of other therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, the composition comprising immune effector cells comprising one or more DARIC immune receptor polypeptides contemplated herein is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs 5 include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal antiinflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. 10         Other exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids are chosen from the group consisting of cortisone, 15 dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers 20 include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline. Illustrative examples of therapeutic antibodies suitable for combination with the 25 modified T cells comprising one or more DARIC immune receptor polypeptides contemplated herein, include but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, 2024264639   14 Nov 2024 lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab. In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. By “cytokine” as used herein is meant a generic term for 5 proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as 10 follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth 15 factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, 20  IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF- beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. I.    Therapeutic Methods 25         The immune effector cells, including CAR T cells, comprising a DARIC immune receptor contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of, or for preventing, treating, or ameliorating at least one symptom associated with, a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. 2024264639   14 Nov 2024 The immune effector cells that comprise an engineered receptor and a DARIC immune receptor contemplated herein provide improved drug products for use in the prevention, treatment, or amelioration of at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. As used herein, 5 the term “drug product” refers to modified cells produced using the compositions and methods contemplated herein. In particular embodiments, the drug product comprises genetically modified immune effector cells, T cells comprising an engineered receptor, or CAR T cells further modified to express a DARIC immune receptor. Moreover, the modified T cells contemplated in particular embodiments provide safer and more efficacious 10 adoptive cell therapies because they are resistant to T cell exhaustion and display increased durability and persistence in the tumor microenvironment that can lead to sustained therapy. In particular embodiments, an effective amount of modified immune effector cells or T cells comprising an engineered receptor and a DARIC immune receptor are administered to a subject to prevent, treat, or ameliorate at least one symptom of a cancer, 15 GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. In particular embodiments, a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of modified immune effector cells or T cells comprising a DARIC immune receptor and an engineered 20 TCR, CAR, or other therapeutic transgene to redirect the cells to a tumor or cancer. The genetically modified cells are a more durable and persistent drug product because the cells are more resistant to immunosuppressive signals from the tumor microenvironment by virtue of transducing a chemically regulatable immunostimulatory signal. In particular embodiments, the modified immune effector cells contemplated herein are 25 used in the treatment of solid tumors or cancers. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, 30 brain / CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, 2024264639   14 Nov 2024 cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, 5 fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, 10 malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell 15 tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, 20 small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms Tumor. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, 25 pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung. 2024264639   14 Nov 2024 In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of liquid cancers or hematological cancers. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, 5 lymphomas, and multiple myeloma. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell 10 leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone 15 lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma. Preferred cells for use in the methods contemplated herein include 20 autologous / autogeneic (“self”) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells. In particular embodiments, methods comprising administering a therapeutically effective amount of modified immune effector cells contemplated herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more 25 therapeutic agents, are provided. In certain embodiments, the cells are used in the treatment of patients at risk for developing a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. Thus, particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising 2024264639   14 Nov 2024 administering to a subject in need thereof, a therapeutically effective amount of the modified immune effector cells contemplated herein. In one embodiment, a method of treating a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency in a subject in need 5 thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising modified immune effector cells contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. 10          In one illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is at least 2 x 106 cells / kg, at least 3 x 106 cells / kg, at least 4 x 106 cells / kg, at least 5 x 106 cells / kg, at least 6 x 106 cells / kg, at least 7 x 106 cells / kg, at least 8 x 106 cells / kg, at least 9 x 106 cells / kg, or at least 10 x 106 cells / kg, or more cells / kg, including all intervening doses of cells. 15          In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2 x 106 cells / kg, about 3 x 106 cells / kg, about 4 x 106 cells / kg, about 5 x 106 cells / kg, about 6 x 106 cells / kg, about 7 x 106 cells / kg, about 8 x 106 cells / kg, about 9 x 106 cells / kg, or about 10 x 106 cells / kg, or more cells / kg, including all intervening doses of cells. 20          In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is from about 2 x 106 cells / kg to about 10 x 106 cells / kg, about 3 x 106 cells / kg to about 10 x 106 cells / kg, about 4 x 106 cells / kg to about 10 x 106 cells / kg, about 5 x 106 cells / kg to about 10 x 106 cells / kg, 2 x 106 cells / kg to about 6 x 106 cells / kg, 2 x 106 cells / kg to about 7 x 106 cells / kg, 2 x 106 cells / kg to about 8 x 106 cells / kg, 25   3 x 106 cells / kg to about 6 x 106 cells / kg, 3 x 106 cells / kg to about 7 x 106 cells / kg, 3 x 106 cells / kg to about 8 x 106 cells / kg, 4 x 106 cells / kg to about 6 x 106 cells / kg, 4 x 106 cells / kg to about 7 x 106 cells / kg, 4 x 106 cells / kg to about 8 x 106 cells / kg, 5 x 106 cells / kg to about 6 x 106 cells / kg, 5 x 106 cells / kg to about 7 x 106 cells / kg, 5 x 106 cells / kg to about 8 x 106 cells / kg, or 6 x 106 cells / kg to about 8 x 106 cells / kg, including all intervening doses of 30 cells. 2024264639   14 Nov 2024 One of ordinary skill in the art would recognize that multiple administrations of the compositions contemplated in particular embodiments may be required to effect the desired therapy. For example, a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more. In certain embodiments, it may be desirable to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be 10 activated from blood draws of from 10cc to 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Not to be bound by theory, using this multiple blood draw / multiple reinfusion protocol may serve to select out certain populations of T cells. In one embodiment, a method of treating a subject diagnosed with a cancer, 15 comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding an engineered antigen receptor and one or more DARIC immune receptors and producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells. 20          The methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo modified immune effector cells or on reintroduction of the modified progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells. One method comprises modifying peripheral blood T cells ex vivo by introducing 25 one or more vectors encoding an engineered antigen receptor and one or more DARIC immune receptors and returning the transduced cells into the subject. 2024264639   14 Nov 2024 All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference. Although the foregoing embodiments have been described in some detail by way of 5 illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical 10 parameters that could be changed or modified in particular embodiments to yield essentially similar results. 2024264639   14 Nov 2024 EXAMPLES EXAMPLE 1 Rapamycin Induces IFNy Expression in IL-18 Receptor DARIC T Cells IL-18 receptor (IL-18R) DARIC binding and signaling components were designed, 5 constructed, and verified. An IL-18R DARIC lentiviral vector was constructed comprising an MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8a-signal peptide, an FRB variant (T82L), and an IL-18R1 transmembrane and signaling domain); a P2A sequence; and a DARIC binding component (an IgK-signal peptide, an FKBp12 domain, and a IL-18RAp transmembrane and signaling domain). An 10  IL-18R DARIC-GFp lentiviral vector was constructed by appending another polynucleotide encoding a p2A sequence and GFp to the to the polynucleotide encoding the IL-18R1 signaling domain in aforementioned vector. T cells transduced with the IL-18R DARIC and IL-18R DARIC-GFp lentiviral vectors express the polypeptides shown in Figure 1A. See, e.g., sEQ ID Nos: 1-4. 15        GFp expression was measured in donor pBMCs transduced with LVV encoding IL- 18R-DARIC-GFp to assess transduction efficiency. Figure 1B. Cell growth was measured in donor pBMCs transduced with LVV encoding IL-18R-DARIC-GFp. The transduced cells showed similar growth kinetics to Untransduced control pBMCs. Figure 1C. 20        CD62L and CD45Ra expression were measure in donor pBMCs transduced with LVV encoding IL-18R-DARIC-GFp. T cell subset development as assessed by CD62L and CD45Ra expression was similar among IL-18R-DARIC-GFp transduced cells and untransduced controls. Figure 1D. Donor pBMCs were transduced with LVV encoding IL-18R-DARIC-GFp. 25 Transduced pBMCs and untransduced control pBMCs were pre-treated with 50 ng / mL human recombinant IL-12 for 24 hours. The treated cells were washed and cultured in medium only, medium with 100 ng / mL human recombinant IL-18 or medium with 1 nM rapamycin. T cells cultured in IL-18 showed increased IFNy production in both 2024264639   14 Nov 2024 untransduced controls and cells transduced with IL-18R-DARIC-GFP. In contrast, only cells transduced with IL-18R-DARIC-GFP showed increased IFNy production when treated with rapamycin. Figure 2. 5          In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 10 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge 15 in the field of endeavour to which this specification relates.

Claims

1. A fusion polypeptide comprising:(a) a first polypeptide comprising:(i) a first multimerization domain;(ii) a first transmembrane domain; and(iii) an IL-2Rp or an IL-2Ry immune receptor intracellular signaling domain;(b) a ribosomal skipping sequence; and(c) a second polypeptide comprising:(i) a second multimerization domain;(ii) a second transmembrane domain; and(iii) an IL-2Ry or an IL-2RP immune receptor intracellular signaling domain, wherein the first and second multimerization domains are selected from FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) T2098L; andwherein the first polypeptide and the second polypeptide, when expressed in an immune effector cell, are capable of increasing cell proliferation in the presence of a bridging factor.

2. The fusion polypeptide of claim 1, wherein the first multimerization domainand the second multimerization domain associate with the bridging factor when the first polypeptide and second polypeptide are expressed,wherein the bridging factor is selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

3. The fusion polypeptide of claim 1 or claim 2, wherein the bridging factor isAP21967.2024264639   14 Sep 20264.     The fusion polypeptide of any one of claims 1 to 3, wherein the firsttransmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, a CD154 transmembrane domain, and a CD71 transmembrane domain.

5. The fusion polypeptide of any one of claims 1 to 4, wherein the firsttransmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.

6. The fusion polypeptide of any one of claims 1 to 5, wherein the firsttransmembrane domain and the second transmembrane domain are the same or different.

7. The fusion polypeptide of any one of claims 1 to 6, wherein the ribosomalskipping sequence is a viral polypeptide.

8. The fusion polypeptide of any one of claims 1 to 7, wherein the ribosomalskipping sequence is a viral 2A polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

9. The fusion polypeptide of any one of claims 1 to 8, wherein the firstmultimerization domain localizes extracellularly when the first polypeptide is expressed and the second multimerization domain localizes extracellularly when the second polypeptide is expressed.

10. A composition comprising the fusion polypeptide of any one of claims 1 to 9.

11. A composition comprising:(a) a first polypeptide comprising:2024264639   14 Sep 2026(i) a first multimerization domain;(ii) a first transmembrane domain; and(iii) an IL-2RP or an IL-2Ry immune receptor intracellular signaling domain; and(b) a second polypeptide comprising:(i) a second multimerization domain;(ii) a second transmembrane domain; and(iii) an IL-2Ry or an IL-2RP immune receptor intracellular signaling domain, wherein the first and second multimerization domains are selected from FRB T2098L and FKBP12; andwherein the first polypeptide and the second polypeptide, when expressed in an immune effector cell, are capable of increasing cell proliferation in the presence of a bridging factor.

12. The composition of claim 10 or claim 11, wherein the first multimerization domain and the second multimerization domain associate with the bridging factor when the first polypeptide and the second polypeptide are expressed,wherein the bridging factor is selected from the group consisting of: rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FK506 / cyclosporin A (FKCsA) or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof, AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

13. The composition of any one of claims 10-12, wherein the bridging factor is AP21967.

14. The composition of any one of claims 10-13, wherein the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, an2024264639   14 Sep 2026amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, a CD154 transmembrane domain, and a CD71 transmembrane domain.

15. The composition of any one of claims 10-14, wherein the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.

16. The composition of any one of claims 10-15, wherein the first transmembrane domain and the second transmembrane domain are the same or different.

17. The composition of any one of claims 10-16, further comprising a cell.

18. A polypeptide complex comprising the first and second polypeptides as defined in any one of claims 1 to 17.

19. A polynucleotide encoding the fusion polypeptide of any one of claims 1-9 or the first and second polypeptides of the composition of any one of claims 10-17.

20. A cDNA and / or RNA encoding the fusion polypeptide of any one of claims 19 or the first and second polypeptides of the composition of any one of claims 10-17.

21. A vector comprising the polynucleotide of claim 19 or claim 20.

22. A cell comprising the fusion polypeptide of any one of claims 1 to 9, the firstand second polypeptides as defined in any one of claims 1 to 17, the polypeptide complex of claim 18, the polynucleotide of claim 19 or claim 20, or the vector of claim 21.

23. The cell of claim 22, wherein the cell is:a hematopoietic cell;a T cell;a CD3+, CD4+, and / or CD8+ cell;an immune effector cell;2024264639   14 Sep 2026a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell; and / ora natural killer (NK) cell or natural killer T (NKT) cell.

24. The cell of claim 22 or claim 23, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.

25. The cell of any one of claims 22 to 24, further comprising an engineered antigen receptor.

26. The cell of claim 25, wherein the engineered antigen receptor is selected from the group consisting of: an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a DARIC receptor or components thereof, and a chimeric cytokine receptor.

27. A composition comprising the fusion polypeptide of any one of claims 1 to 9, the first and second polypeptides as defined in any one of claims 1 to 17, the polypeptide complex of claim 18, the polynucleotide of claim 19 or claim 20, the vector of claim 21, or the cell of any one of claims 22 to 26.

28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion polypeptide of any one of claims 1 to 9, the first and second polypeptides as defined in any one of claims 1 to 17, the polypeptide complex of claim 18, the polynucleotide of claim 19 or claim 20, the vector of claim 21, or the cell of any one of claims 22 to 26.

29. A method of treating a solid cancer or hematological malignancy comprising administering to the subject an effective amount of the composition of claim 28, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer, or wherein the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.2024264639   14 Sep 202630. Use of the composition of claim 28 in the manufacture of a medicament for treating a solid cancer or hematological malignancy, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer, or wherein the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.

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