Improved rapamycin activated synthetic receptor transgene constructs

Abstract

Provided herein are polynucleotide constructs for expression of a synthetic cytokine receptor complex and, in some cases co-expression with a chimeric antigen receptor system, as well as vectors, such as viral vectors, comprising same, cells comprising same, and methods of using same.

Description

260132004340IMPROVED RAPAMYCIN ACTIVATED SYNTHETIC RECEPTOR TRANSGENE CONSTRUCTSCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 738,510 filed on December 23, 2024, entitled “IMPROVED RAPAMYCIN ACTIVATED SYNTHETIC RECEPTOR TRANSGENE CONSTRUCTS,” the contents of which are incorporated by reference in its entirety.Reference to an Electronic Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 260132004340SeqList.xml, created December 22, 2025, which is 214,936 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure provides polynucleotide constructs for expression of a synthetic cytokine receptor complex and, in some cases co-expression with a chimeric antigen receptor system, as well as vectors, such as viral vectors comprising same, cells comprising same, and methods of using same.Background

[0004] Chimeric antigen receptor (CAR) T cell therapies have demonstrated limited efficacy against solid tumors, in part due to challenges overcoming solid tumor heterogeneity and CAR T cell exhaustion associated with the immunosuppressive tumor microenvironment (TME). In addition to this challenge, there are also challenges in methods of delivering CAR-expressing cells to subjects in a manner that can provide controllable persistence for therapeutic efficacy of the CAR in the treatment of various diseases, including cancer. Provided herein are embodiments that address such needs.Summary1MF-365319601260132004340

[0005] Provided herein is a polynucleotide construct encoding a fusion of a signal peptide and a heterologous protein of interest (POI). In some embodiments, the signal peptide is a modified CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7), and the heterologous POI comprises a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain.

[0006] In some of any embodiments, the FRB domain is the amino terminal protein of the chimeric protein.

[0007] In some of any embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor.

[0008] In some of any embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IE2RG).

[0009] In some of any embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0010] In some of any embodiments, a nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

[0011] In some of any embodiments, the IE2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IE2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

[0012] In some of any embodiments, a linker links the FRB domain and the signaling domain from the cytokine receptor. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21. In some embodiments, the linker comprises an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises the amino acid sequence of EAAAK (SEQ ID NO: 20) and the amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises2MF-365319601260132004340APAPAPAPAPAPAP (SEQ ID NO: 10). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the linker comprises EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19). In some embodiments, the comprises EAAAKEAAAKGGGGS (SEQ ID NO: 14). In some embodiments, the linker comprises DIGSGAPAPAPAP (SEQ ID NO: 15). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17). In some embodiments, the linker comprises DIGGSGGAP (SEQ ID NO: 18). In some embodiments, the linker comprises EAAAKEAAAK (SEQ ID NO: 145). In some embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO: 147).

[0013] In some aspects, provided herein is a polynucleotide construct encoding a fusion of a signal peptide and a heterologous protein of interest (POI). In some of any embodiments, the signal peptide is a modified CD4 signal peptide comprising amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24), and the heterologous POI comprises a chimeric protein comprising an FKBP12 domain.

[0014] In some of any embodiments, the FKBP12 domain is the amino terminal protein of the chimeric protein.

[0015] In some of any embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a cytokine receptor.

[0016] In some of any embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the beta chain signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

[0017] In some of any embodiments, the FKBP12 domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37.

[0018] In some of any embodiments, a nucleotide sequence encoding the FKBP12 domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NOS: 1 or 43.3MF-365319601260132004340

[0019] In some of any embodiments, the IL-2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148. In some embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

[0020] In some of any embodiments, a linker links the FKBP12 domain and the signaling domain from the cytokine receptor. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10-21, 145, or 147. In some embodiments, the linker comprises an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises the amino acid sequence of EAAAK (SEQ ID NO: 20) and the amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises APAPAPAPAPAPAP (SEQ ID NO: 10). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the linker comprises EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19). In some embodiments, the linker comprises EAAAKEAAAKGGGGS (SEQ ID NO: 14). In some embodiments, the linker comprises DIGSGAPAPAPAP (SEQ ID NO: 15). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17). In some embodiments, the linker comprises DIGGSGGAP (SEQ ID NO: 18). In some embodiments, the linker comprises EAAAKEAAAK (SEQ ID NO: 145). In some embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO: 147).

[0021] In some aspects, provided herein is a polynucleotide construct encoding a fusion of a signal peptide and a heterologous protein of interest (POI). In some embodiments, the heterologous POI comprises a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor. In some embodiments, a linker links the FRB domain and the signaling domain from the cytokine receptor.

[0022] In some of any embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10-21, 145, or 147. In some embodiments, the linker comprises an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises the amino acid sequence of EAAAK (SEQ ID NO: 20) and the amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the linker comprises4MF-365319601260132004340APAPAPAPAPAPAP (SEQ ID NO: 10). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the linker comprises EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the linker comprises EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19). In some embodiments, the linker comprises EAAAKEAAAKGGGGS (SEQ ID NO: 14). In some embodiments, the linker comprises DIGSGAPAPAPAP (SEQ ID NO: 15). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17). In some embodiments, the linker comprises DIGGSGGAP (SEQ ID NO: 18). In some embodiments, the linker comprises EAAAKEAAAK (SEQ ID NO: 145). In some embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO: 147).

[0023] In some of any embodiments, the polynucleotide construct further comprises a modified CD8 signal peptide comprising an amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7) and / or a modified CD4 signal peptide comprising an amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24).

[0024] In some of any embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain.

[0025] In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0026] In some of any embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0027] In some of any embodiments, a nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

[0028] In some of any embodiments, the IL2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.5MF-365319601260132004340

[0029] In some aspects, provided herein is a polynucleotide construct comprising a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI). In some embodiments, the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor. In some embodiments, a first linker links the FRB domain and the signaling domain from the cytokine receptor. In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI. In some embodiments, the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a cytokine receptor. In some embodiments, a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor. In some embodiments, the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide.

[0030] In some of any embodiments, the amino acid sequence of the first linker comprises a sequence selected from the SEQ ID NOS: 10-21, 145, or 147. In some of any embodiments, the amino acid sequence of the second linker comprises a sequence selected from the SEQ ID NOS: 10-21. In some embodiments, the first linker and / or the second linker comprise an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, the first linker and / or the second linker comprise an amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the first linker and / or the second linker comprise the amino acid sequence of EAAAK (SEQ ID NO: 20) and the amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, the first signal peptide encodes a modified CD8 signal peptide comprising an amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the second signal peptide encodes a modified CD4 signal peptide comprising an amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24). In some embodiments, the one or more linker comprises APAPAPAPAPAPAP (SEQ ID NO: 10). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19). In some embodiments, the one or more linker comprises EAAAKEAAAKGGGGS (SEQ ID NO: 14). In some embodiments, the one or more linker 6MF-365319601260132004340comprises DIGSGAPAPAPAP (SEQ ID NO: 15). In some embodiments, the one or more linker comprises GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17). In some embodiments, the one or more linker comprises DIGGSGGAP (SEQ ID NO: 18). In some embodiments, the one or more linker comprises EAAAKEAAAK (SEQ ID NO: 145). In some embodiments, the one or more linker comprises GGGGSGGGGS (SEQ ID NO: 147).

[0031] In some of any embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0032] In some of any embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0033] In some of any embodiments, a nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

[0034] In some of any embodiments, the IL2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

[0035] In some of any embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB).

[0036] In some embodiments, the IL-2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148. In some embodiments, the IL-2RB protein comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

[0037] In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 protein comprises the amino acid sequence of SEQ ID NO: 37.7MF-365319601260132004340

[0038] In some aspects, provided herein is a polynucleotide construct comprising a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI). In some embodiments, the first signal peptide encodes a modified CD8 signal peptide. In some embodiments, the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain; and a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI. In some embodiments, the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain. In some embodiments, the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide.

[0039] In some embodiments, the first signal peptide comprises a modified CD8 signal peptide comprising an amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

[0040] In some embodiments, the second signal peptide comprises a modified CD4 signal peptide comprising an amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24).

[0041] In some of any embodiments, the FRB domain is the amino terminal protein of the first chimeric protein.

[0042] In some of any embodiments, the FKBP12 domain is the amino terminal protein of the second chimeric protein.

[0043] In some of any embodiments, the first chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a first cytokine receptor.

[0044] In some of any embodiments, the first cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0045] In some embodiments, the IL2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

[0046] In some of any embodiments, the second chimeric protein comprises in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a second cytokine receptor.8MF-365319601260132004340

[0047] In some of any embodiments, the second cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB).

[0048] In some embodiments, the IL-2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148. In some embodiments, the IL-2RB protein comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

[0049] In some of any embodiments, the FKBP12 domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37.

[0050] In some of any embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a third heterologous POI comprising an FRB protein. In some embodiments, the nucleotide sequence encoding the FRB protein is of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB protein is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, an amino acid sequence encoding the FRB protein is of SEQ ID NOS: 29, 33, or 40. In some embodiments, the amino acid sequence encoding the FRB protein is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0051] In some of any embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises an anti-fluorescein isothiocyanate (FITC) E2. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0052] In some embodiments, the scFv VL comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence9MF-365319601260132004340of SEQ ID NO: 66. In some embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 66.

[0053] In some embodiments, the scFv VH comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 69.

[0054] In some embodiments, the scFv linker comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 72.

[0055] In some embodiments, the scFv comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 75.

[0056] In some of any embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a short hinge or a medium hinge domain. In some embodiments, the hinge domain comprises a CD8 or an IgG. In some embodiments, the CD8 hinge comprises CD8a hinge. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises the amino acid sequence of SEQ ID NOS: 78 or 79.10MF-365319601260132004340

[0057] In some of any embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises a CD8a transmembrane. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 81.

[0058] In some of any embodiments, the CAR comprises an endodomain. In some embodiments, the endodomain comprises a costimulatory molecule. In some embodiments, the endodomain comprises 4- IBB, CD3(^, and / or CD28. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises the amino acid sequence of SEQ ID NO: 89.

[0059] In some of any embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at 11MF-365319601260132004340least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes the amino acid sequence of SEQ ID NOS: 94, 141, 95, 96 or 142.

[0060] In some of any embodiments, the first fusion, the second fusion, and the third heterologous POI are separated by encoded cleavable signal peptides.

[0061] In some of any embodiments, the first fusion, the second fusion, the third heterologous POI, and the fourth heterologous POI are separated by encoded cleavable signal peptides.

[0062] In some of any embodiments, the first fusion and the second fusion are separated by encoded cleavable signal peptides.

[0063] In some embodiments, the one or more encoded cleavable signal peptides comprise a 2A cleavable linker sequence. In some embodiments, the one or more 2A cleavable linker sequences encoded in the polynucleotide construct are different from one another. In some embodiments, the 2A cleavable linker is independently a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the 2A cleavable linker is independently a P2A or a T2A. In some embodiments, at least one 2A cleavable linker is a P2A and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 50, 51, 52, or 53. In some embodiments, the nucleotide sequence encoding the P2A cleavable linker is set forth in SEQ ID NOS: 50, 51, 52, or 53. In some embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54. In some embodiments, the P2A cleavable linker comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, at least one 2A cleavable linker is a T2A and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO: 55. In some embodiments, the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56. In some embodiments, the T2A cleavable linker comprising the sequence set forth in SEQ ID NO: 56.

[0064] In some of any embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence. In some embodiments, the furin cleavage site sequence is located between the first fusion protein and the second fusion protein. In some embodiments, the12MF-365319601260132004340nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). In some embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the cleavage site sequence comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 60. In some of any embodiments, the first fusion and the second fusion are separated by a P2A, and the first fusion and the third heterologous POI are separated by a furin T2A.

[0065] In some of any embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 146. In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 146.

[0066] In some of any embodiments, the chimeric protein or the first chimeric protein comprises the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151. In some embodiments, the chimeric protein or the first chimeric protein comprises the amino acid sequence identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151.

[0067] In some of any embodiments, the chimeric protein or the second chimeric protein comprises the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 152 or 153. In some embodiments, the chimeric protein or the second chimeric protein comprises the amino acid sequence identical to the amino acid sequence of SEQ ID NOS: 152 or 153.13MF-365319601260132004340

[0068] In some aspects, provided herein is a polynucleotide construct of SEQ ID NO: 146. In some aspects, provided herein is a polynucleotide construct of sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 146.

[0069] In some of any embodiments, one or more amino acid sequences are codon optimized. In some of any embodiments, the polynucleotide construct is encoded in a bicistronic construct. In some of any embodiments, the polynucleotide construct is encoded in a polycistronic construct. In some aspects, provided herein is a viral vector comprising a polynucleotide construct of any of the preceding embodiments. In some embodiments, the viral vector is a lentiviral vector. In some aspects, provided herein is a cell comprising the viral vector of any of the preceding embodiments.

[0070] In some aspects, provided herein is a cell comprising the polynucleotide construct of any of the preceding embodiments.

[0071] In some aspects, provided herein is a cell comprising a rapamycin activated cytokine receptor (RACR) system. In some of any embodiments, the RACR system comprises (a) a first fusion protein comprising a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor; and (b) a second fusion protein comprising a second chimeric protein comprising an FKBP12 domain and a signaling domain from a cytokine receptor. In some of any embodiments, a first linker comprising the amino acid sequence EAAAK (SEQ ID NO: 21) links the FRB domain and the signaling domain from the cytokine receptor, and a second linker comprising the amino acid sequence EAAAK (SEQ ID NO: 21) links the FKBP12 domain and the signaling domain from the cytokine receptor.

[0072] In some aspects, provided herein is a cell comprising the polypeptide of SEQ ID NO: 144. In some embodiments, the polypeptide comprises the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 144.

[0073] In some aspects, provided is a cell comprising polynucleotide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set forth in SEQ ID NO: 146. In some embodiments, the cell comprises the polynucleotide set forth in SEQ ID NO: 146. In some aspects, the cell comprises a polypeptide encoded by a polynucleotide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set14MF-365319601260132004340forth in SEQ ID NO: 146. In some aspects, the cell comprises a polypeptide encoded by a polynucleotide set forth in SEQ ID NO: 146.

[0074] In some of any embodiments, the cell comprises a stem cell or a progenitor cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the peripheral blood mononuclear cell (PBMC) comprises lymphocytes. In some embodiments, the cell comprises a T cell. In some embodiments, the cell comprises a cytotoxic innate lymphocyte (CIL) cell. In some embodiments, the cell comprises a natural killer (NK) cell. In some embodiments, the cell comprises a white blood cell. In some embodiments, the white blood cell comprises a monocyte and / or a macrophage.

[0075] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with the polynucleotide constructs of any of the preceding embodiments.

[0076] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with the viral vector of any of the preceding embodiments.

[0077] In some of any embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the target cell comprises a progenitor cell. In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell.

[0078] In some aspects, provided herein is a method of increasing expression of a polynucleotide construct expressing a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of any of the preceding embodiments.

[0079] In some aspects, provided herein is a method of decreasing tonic signaling associated with a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of any of the preceding embodiments.

[0080] In some aspects, provided herein is a method of expressing a RACR system with decreased tonic signaling, the method comprising transducing a cell with a polynucleotide construct of any of the preceding embodiments.

[0081] Provided herein is a polynucleotide construct comprising: (1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein 15MF-365319601260132004340comprising a FK506-binding protein (FKBP12)-rapamycin-binding (FRB) domain and a signaling domain from a first cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and (2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a second cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor, wherein the encoded first fusion protein and the encoded second fusion protein are separated by an encoded cleavable peptide linker; and the first linker and / or the second linker each independently comprise an amino acid sequence set forth in SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158.

[0082] In some of any embodiments, the first linker and / or the second linker independently comprise an amino acid sequence selected from the group consisting of EAAAK (SEQ ID NO: 20), EAAAKEAAAK (SEQ ID NO: 145), EAAAKEAAAKEAAAK (SEQ ID NO: 12), GGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17), and EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19).

[0083] In some of any embodiments, the first linker is the amino acid sequence of EAAAKEAAAKEAAAK (SEQ ID NO: 12) and the second linker is the amino acid sequence of EAAAKEAAAKEAAAK (SEQ ID NO: 12).

[0084] Provided herein is a polynucleotide construct comprising: (1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein comprising a FK506-binding protein (FKBP12)-rapamycin-binding (FRB) domain and a signaling domain from a first cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and (2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a second cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor, wherein the encoded first fusion protein and the encoded second fusion protein are separated by a encoded cleavable peptide linker; and the first linker and the second linker independently16MF-365319601260132004340comprise an amino acid sequence selected from the sequence set forth in any of SEQ ID NOS: 10-21 and 145.

[0085] In some of any embodiments, the first linker and the second linker are the same amino acid sequence. In some of any embodiments, the first linker and the second linker are different amino acid sequences.

[0086] In some of any embodiments, the first signal peptide is a modified CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

[0087] In some of any embodiments, the second signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24) or a CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQG (SEQ ID NO: 23).

[0088] In some of any embodiments, the first cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain.

[0089] In some of any embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0090] In some of any embodiments, the FRB domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some of any embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some of any embodiments, the FRB domain encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some of any embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some of any embodiments, the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some of any embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

[0091] In some of any embodiments, the second cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some of any embodiments, the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB). In some of any embodiments, the IL-2RB comprises an amino acid sequence that is at least 80%, 85%, 90%,17MF-36531960126013200434095%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148. In some of any embodiments, the IL-2RB protein comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

[0092] In some of any embodiments, the FKBP12 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some of any embodiments, the FKBP12 protein comprises the amino acid sequence of SEQ ID NO: 37.

[0093] Provided herein is a polynucleotide construct comprising: (1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first signal peptide encodes a modified CD8 signal peptide, and wherein the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain; and (2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain, wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide.

[0094] In some of any embodiments, the first signal peptide comprises a modified CD8 signal peptide comprising an amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some of any embodiments, the second signal peptide comprises a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24).

[0095] In some of any embodiments, the FRB domain is the amino terminal protein of the first chimeric protein. In some of any embodiments, the FKBP12 domain is the amino terminal protein of the second chimeric protein. In some of any embodiments, the first chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a first cytokine receptor. In some of any embodiments, the first cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain.

[0096] In some of any embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG). In some of any embodiments, the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some of any embodiments, the IL2RG 18MF-365319601260132004340comprises the amino acid sequence of SEQ ID NOS: 34 or 149. In some of any embodiments, the FRB domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some of any embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0097] In some of any embodiments, the second chimeric protein comprises in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a second cytokine receptor. In some of any embodiments, the second cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some of any embodiments, the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB). In some of any embodiments, the IL-2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148. In some of any embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

[0098] In some of any embodiments, the FKBP12 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some of any embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37.

[0099] In some of any embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a third heterologous POI comprising an FRB protein. In some of any embodiments, the FRB protein is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some of any embodiments, the FRB protein is encoded by the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some of any embodiments, the FRB protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some of any embodiments, the FRB protein comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0100] In some of any embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR). In some of any embodiments, the CAR comprises an scFv domain. In some of any embodiments, the scFv domain comprises an anti-fluorescein isothiocyanate (FITC) E2.19MF-365319601260132004340

[0101] In some of any embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH). In some of any embodiments, the scFv VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some of any embodiments, the scFv VL is encoded by the nucleotide sequence of SEQ ID NOS: 64 or 65. In some of any embodiments, the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66. In some of any embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 66. In some of any embodiments, the scFv VH is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some of any embodiments, the scFv VH is encoded by the nucleotide sequence of SEQ ID NOS: 67 or 68. In some of any embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 69. In some of any embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 69.

[0102] In some of any embodiments, the scFv linker is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some of any embodiments, the scFv linker is encoded by the nucleotide sequence of SEQ ID NOS: 70 or 71. In some of any embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some of any embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 72. In some of any embodiments, the scFv is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some of any embodiments, the scFv is encoded by the nucleotide sequence of SEQ ID NOS: 73 or 74. In some of any embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some of any embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 75.

[0103] In some of any embodiments, the hinge domain comprises a short hinge or a medium hinge domain. In some of any embodiments, the hinge domain comprises a CD8 or an IgG. In some of any embodiments, the CD8 hinge comprises CD8a hinge. In some of any embodiments,20MF-365319601260132004340the CD8a hinge is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some of any embodiments, the CD8a hinge is encoded by the nucleotide sequence of SEQ ID NOS: 76 or 77. In some of any embodiments, the CD8a hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some of any embodiments, the CD8a hinge comprises the amino acid sequence of SEQ ID NOS: 78 or 79.

[0104] In some of any embodiments, the CAR comprises a transmembrane domain. In some of any embodiments, the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain. In some of any embodiments, the CD8 transmembrane domain comprises a CD8a transmembrane domain. In some of any embodiments, the transmembrane domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 80. In some of any embodiments, the transmembrane domain is encoded by the nucleotide sequence of SEQ ID NO: 80. In some of any embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some of any embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 81.

[0105] In some of any embodiments, the CAR comprises an endodomain. In some of any embodiments, the endodomain comprises a costimulatory molecule. In some of any embodiments, the endodomain comprises 4- IBB, CD3(^, and / or CD28. In some of any embodiments, the 4- IBB endodomain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some of any embodiments, the 4- IBB endodomain is encoded by the nucleotide sequence of SEQ ID NOS: 82 or 83. In some of any embodiments, the 4- IBB endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some of any embodiments, the 4- IBB endodomain comprises the amino acid sequence of SEQ ID NOS: 84 or 113. In some of any embodiments, the CD3(^ endodomain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some of any embodiments, the CD3(^ endodomain is encoded by the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some 21MF-365319601260132004340of any embodiments, the CD3(^ endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89. In some of any embodiments, the CD3(^ endodomain comprises the amino acid sequence of SEQ ID NO: 89.

[0106] In some of any embodiments, the fourth heterologous POI is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some of any embodiments, the fourth heterologous POI is encoded by the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some of any embodiments, the fourth heterologous POI comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some of any embodiments, the fourth heterologous POI comprises the amino acid sequence of SEQ ID NOS: 94, 141, 95, 96 or 142.

[0107] In some of any embodiments, the each of the first fusion, the second fusion, and the third heterologous POI is operably linked in series, and each adjacent pair of (i) the first fusion, (ii) the second fusion, and (iii) the third heterologous POI is separated by a cleavable linker. In some of any embodiments, each of the first fusion, the second fusion, the third heterologous POI, and the fourth heterologous POI is operably linked in series, and each adjacent pair of (i) the first fusion, (ii) the second fusion, (iii) the third heterologous POI, and (iv) the fourth heterologous POI is separated by a cleavable linker. In some of any embodiments, the cleavable linker comprises a 2A cleavable linker sequence. In some of any embodiments, the 2A cleavable linker sequences in the polynucleotide construct are different from one another when there are two or more cleavable linker sequences. In some of any embodiments, the 2 A cleavable linker sequence is independently a T2A, P2A, E2A or F2A cleavable linker sequence. In some of any embodiments, at least one of the 2 A cleavable linkers is a P2A cleavable linker and the P2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 50, 51, 52, or 53. In some of any embodiments, the P2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NOS: 50, 51, 52, or 53. In some of any embodiments, the P2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54. In some of any embodiments, the P2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 54. In some of any embodiments, at least one 22MF-365319601260132004340of the 2A cleavable linkers is a T2A cleavable linker and the T2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55. In some of any embodiments, the T2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 55. In some of any embodiments, the T2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56. In some of any embodiments, the T2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 56. In some of any embodiments, at least one of the 2 A cleavable linkers comprises a furin cleavage site sequence.

[0108] In some of any embodiments, the furin cleavage site sequence is located between the first fusion protein and the second fusion protein. In some of any embodiments, the furin cleavage site sequence comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57. In some of any embodiments, the furin cleavage site sequence comprises the nucleotide sequence set forth in SEQ ID NO: 57. In some of any embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58. In some of any embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 58. In some of any embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavable linker (furinT2A).

[0109] In some of any embodiments, the cleavage site sequence is comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some of any embodiments, the cleavage site sequence is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 59.

[0110] In some of any embodiments, the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some of any embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 60.

[0111] In some of any embodiments, the first fusion and the second fusion are separated by a P2A, and the first fusion and the third heterologous POI are separated by a furin T2A.

[0112] In some of any embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the23MF-365319601260132004340nucleotide sequence of SEQ ID NO: 146. In some of any embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 146.

[0113] In some of any embodiments, the chimeric protein or the first chimeric protein comprises the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151. In some of any embodiments, the chimeric protein or the first chimeric protein comprises the amino acid sequence identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151.

[0114] Provided herein is a polynucleotide construct of SEQ ID NO: 146. In some of any embodiments, one or more nucleotide sequences are codon optimized. In some of any embodiments, the polynucleotide construct is encoded in a bicistronic construct. In some of any embodiments, the polynucleotide construct is encoded in a polycistronic construct.

[0115] Provided herein is a viral vector particle comprising a polynucleotide construct of any one of the embodiments provided herein. In some of any embodiments, the viral vector is a lentiviral vector. In some of any embodiments, the lentiviral vector is a self-inactivating and replication-incompetent lentiviral vector particle. In some of any embodiments, the viral vector comprises a pseudotyped viral envelope protein, optionally wherein the pseudotyped viral envelope protein is cocal virus glycoprotein (G) (Cocal G), optionally wherein the Cocal G is a Cocal envelope variant containing a R354Q mutation or a K47Q mutation. In some of any embodiments, the vector particle comprises a membrane-bound multidomain fusion (MDF) protein comprising a T cell activating domain and at least one costimulatory molecule on the surface of the lentiviral particle. In some of any embodiments, the MDF protein comprises a transmembrane domain that anchors the MDF protein to the lentiviral envelope membrane. In some of any embodiments, the MDF protein comprises: (a) a first costimulatory ligand comprising an extracellular domain portion of CD58 that binds to CD2; (b) a T-cell activating domain comprising a single-chain variable fragment (scFv) that specifically binds to CD3e; and (c) a second costimulatory ligand comprising CD80.

[0116] Provided herein is a cell comprising the polynucleotide construct of any one of the embodiments provided herein. Provided herein is a cell transduced by the viral vector particle of any one of the embodiments provided herein. Provided herein is a cell comprising a rapamycin activated cytokine receptor (RACR) system, wherein the RACR system comprises: (a) a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein comprising a 24MF-365319601260132004340FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and (b) a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor, wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide and the first linker and / or the second linker comprise an amino acid sequence represented by SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158. Provided herein is a cell comprising a rapamycin activated cytokine receptor (RACR) system, wherein the RACR system comprises: (a) a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first signal peptide encodes a modified CD8 signal peptide, and wherein the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain; and (b) a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, w, and wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain, wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide. Provided herein is a cell comprising a polypeptide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set forth in SEQ ID NO: 144 or that is encoded by a polynucleotide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set forth in SEQ ID NO: 146.

[0117] In some of any embodiments, the cell comprises a stem cell or a progenitor cell. In some of any embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some of any embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some of any embodiments, the peripheral blood mononuclear cell (PBMC) comprises lymphocytes. In some of any embodiments, the cell comprises a T cell. In some of any embodiments, the cell comprises a cytotoxic innate lymphocyte (CIL) cell. In some of any embodiments, the cell comprises a natural killer (NK) cell. In some of any embodiments, the cell comprises a white blood cell. In some of any embodiments, the white blood cell comprises a monocyte and / or a macrophage.25MF-365319601260132004340

[0118] Provided herein is a method of transducing a cell comprising contacting a target cell with the polynucleotide constructs of any one of the embodiments provided herein. Provided herein is a method of transducing a cell comprising contacting a target cell with the vector particle of any one of the embodiments provided herein. In some of any embodiments, the target cell comprises a stem cell. In some of any embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some of any embodiments, the target cell comprises a progenitor cell. In some of any embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some of any embodiments, the target cell comprises a T cell. In some of any embodiments, the T cell comprises a CD4+ or CD8+ T cell.

[0119] Provided herein is a method of increasing expression of a polynucleotide construct expressing a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of any one of the embodiments provided herein or the vector particle of any one of the embodiments provided herein. Provided herein is a method of decreasing tonic signaling associated with a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of any one of the embodiments provided herein or the vector particle of any one of the embodiments provided herein. Provided herein is a method of expressing a RACR system with decreased tonic signaling, the method comprising transducing a cell with a polynucleotide construct of any one of the embodiments provided herein or the vector particle of any one of the embodiments provided herein.Brief Description of the Drawings

[0120] FIG. 1 shows an exemplary RACR polynucleotide construct schematic. The polynucleotide construct encodes a free FKBP12-rapamycin-binding (FRB), a first fusion of an FRB domain and a cytokine gamma chain polypeptide, a second fusion of a FKBP12 domain and a cytokine beta chain polypeptide, and a chimeric antigen receptor (CAR) that binds a tumor tag (TagCAR). The components of the polynucleotide constructs are separated by cleavable linkers.

[0121] FIG. 2 shows the measured STAT5 activation in the presence or absence of rapamycin for cells expressing a RACR construct. The RACR constructs include linker sequence modifications and signal peptide sequence modifications.26MF-365319601260132004340

[0122] FIG. 3 shows the modal distribution of Rapa-AF647 positive cells. Cells transduced with RACR (“Reference RACR” and “Variant 3”) and cells without RACR (“Parent”) were exposed to rapamycin and stained with Rapa-AF647 for two hours. Cells expressing higher levels of Rapa-AF647 positive cells have a higher MFI (mean fluorescence intensity).

[0123] FIG. 4 shows an exemplary RACR complex schematic of “Variant 3”, which has a higher rapamycin binding capacity than the reference RACR complex.Detailed Description

[0124] Provided herein are embodiments relating to engineered polynucleotide constructs encoding chimeric proteins (also referred to herein interchangeably as “fusion proteins”) that comprise an FKBP12 domain and / or an FKBP12-rapamycin-binding (FRB) domain, which together mediate ligand-dependent dimerization or signaling. In some embodiments, the FKBP12 or FRB domain is part of a chimeric protein by operable linkage to a signaling domain derived from a cytokine receptor (e.g., IL2Ry or IL2RP). In certain embodiments, the engineered polynucleotide constructs include a signal peptide that enhances secretion or surface localization of an FRB- or FKBP12-containing polypeptide, and / or a linker sequence that improves the signaling efficiency between domains of such chimeric protein. In certain embodiments, an FRB -containing polypeptide includes a particular variant CD4 or CD8 signal peptide that is modified compared to the natural signal peptide and shown to substantially improve expression of the encoded protein. In some examples, the constructs are employed in synthetic receptor systems, such as a rapamycin-activated cytokine receptor (RACR), although the optimized signal peptides and linker configurations described herein are applicable to other chimeric proteins that utilize FKBP12 or FRB fusions.

[0125] In some embodiments, the polynucleotide construct is a polycistronic construct encoding a heterologous protein comprising variant signal peptides provided herein and variant linkers provided herein. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a heterologous protein comprising variant signal peptides provided herein. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a heterologous protein comprising variant linkers provided herein.

[0126] In some aspects, provided herein are polynucleotide constructs encoding polypeptide sequences with increased expression levels. In some embodiments, the increased expression27MF-365319601260132004340levels are reflected in the RNA expression levels and protein expression levels of the encoded polypeptide sequences.

[0127] In some aspects, the disclosure herein relates to the use of variant signal peptides (e.g., CD4 and CD8) in polynucleotide constructs for improving cell surface expression of the encoded polypeptides. In some embodiments, improving cell surface expression improves engineered cell functionality.

[0128] The disclosure relates generally to a polynucleotide construct encoding at least two polypeptide chains of a synthetic receptor and methods for uses thereof. In some embodiments, the synthetic receptors comprise a rapamycin activated cytokine receptor (RACR) complex. In certain embodiments, the polynucleotide constructs are polycistronic, such that a single promoter drives transcription of a single mRNA encoding two or more distinct polypeptides. In some examples, the polycistronic construct encodes the two polypeptide chains of a synthetic cytokine receptor, such as an FKBP12-containing chain and an FRB-containing chain, separated by a self-cleaving peptide sequence (e.g., a 2A peptide). The synthetic cytokine receptor components encoded within these polycistronic constructs may include the optimized features described herein, including modified signal peptides that enhance expression and variant linkers that reduce tonic signaling and improve ligand responsiveness. Accordingly, the polycistronic architecture enables coordinated expression of multiple receptor or signaling modules under a single promoter, facilitating precise regulation of receptor composition and functionality within the same engineered cell.

[0129] In additional embodiments, the polycistronic construct further encodes a chimeric antigen receptor (CAR) or a soluble FRB polypeptide that can be expressed in the same cell as the synthetic cytokine receptor. In such embodiments, the FRB polypeptide may be freely diffusible within the cell or secreted to the extracellular space to modulate ligand-dependent dimerization and signaling. In some embodiments, the polynucleotide construct encodes a chimeric protein encoding a synthetic cytokine receptor, a chimeric antigen receptor (CAR), and a freely diffusible FRB, in which the synthetic cytokine receptor is responsive to rapamycin binding. Advantageously, FRB reduces the inhibitory effects of rapamycin on mTOR in cells engineered to express the polynucleotide constructs provided herein. Expression of the freely diffusible FRB can promote consistent activation and proliferation of engineered cells.

[0130] The disclosure relates generally to a polynucleotide construct encoding a chimeric protein encoding a synthetic cytokine receptor, a synthetic chimeric antigen receptor (CAR), and 28MF-365319601260132004340a freely diffusible FRB and methods for uses thereof. In some aspects, provided herein is a polynucleotide construct encoding one or more heterologous proteins (e.g., protein of interest (POI)). In some embodiments, the one or more heterologous proteins are separated by cleavage site sequences. In some embodiments, the polynucleotide construct comprises a sequence, in 5’ to 3’ order, encoding a first heterologous protein comprising an FRB domain, a second heterologous protein comprising a first synthetic cytokine receptor, a third heterologous protein comprising a second synthetic cytokine receptor, and a fourth heterologous protein comprising a CAR. In some embodiments, the first synthetic cytokine receptor comprises a FKBP12-rapamycin-binding (FRB) domain and a cytokine gamma chain polypeptide. In some embodiments, the second synthetic cytokine receptor comprises a FKBP12 domain and a cytokine beta chain polypeptide. In some embodiments, the first synthetic cytokine receptor comprises a FKBP12 domain and a cytokine gamma chain polypeptide. In some embodiments, the second synthetic cytokine receptor comprises an FRB domain and a cytokine beta chain polypeptide.

[0131] In any of the preceding embodiments, the synthetic cytokine receptor comprises one or more modified signal peptides. In any of the preceding embodiments, a linker links the 1) FRB domain or 2) FKBP12 domain with 3) a transmembrane domain and a signaling domain from a cytokine receptor. In any of the preceding embodiments, the chimeric protein comprises a variant linker sequence. In any of the preceding embodiments, the chimeric protein comprises a variant linker sequence and one or more modified signal peptides.

[0132] In some embodiments, the polynucleotide construct encodes a synthetic cytokine receptor comprising either an N-terminal wild-type CD8 signal peptide (e.g., SEQ ID NO: 6) or a variant CD8 signal peptide (e.g., SEQ ID NO: 7). In some embodiments, the polynucleotide construct encodes a synthetic cytokine receptor comprising either an N-terminal wild-type CD4 signal peptide (e.g., SEQ ID NO: 23) or a variant CD4 signal peptide (e.g., SEQ ID NO: 24). In some embodiments, the polynucleotide construct encodes a synthetic cytokine receptor comprising either an N-terminal wild-type CD8 signal peptide (e.g., SEQ ID NO: 6) or a variant CD8 signal peptide (e.g., SEQ ID NO: 7) and a synthetic cytokine receptor comprising either an N-terminal wild-type CD4 signal peptide (e.g., SEQ ID NO: 23) or a variant CD4 signal peptide (e.g., SEQ ID NO: 24).

[0133] In some embodiments, the polynucleotide construct encodes a synthetic cytokine receptor comprising two polypeptide chains, one including an FKBP12 domain and the other 29MF-365319601260132004340including an FKBP12-rapamycin-binding (FRB) domain. Either or both polypeptide chains may comprise an N-terminal signal peptide selected from a wild-type or variant CD4 or CD8 signal peptide sequence. For example, in certain embodiments, the FRB -containing chain comprises a wild-type CD8 signal peptide (e.g., SEQ ID NO: 6) or a variant CD8 signal peptide (e.g., SEQ ID NO: 7) that includes a substitution of alanine for proline at the terminal position (P21A), which enhances predicted signal peptide cleavage and improves expression.

[0134] In certain embodiments, the FKBP12-containing chain comprises a wild- type CD4 signal peptide (e.g., SEQ ID NO: 23) or a variant CD4 signal peptide (e.g., SEQ ID NO: 24) that includes a substitution of alanine for glycine at the terminal position (G25A).

[0135] In some embodiments, at least one of the two chains includes a variant CD4 or a variant CD8 signal peptide. In some embodiments, both chains include variant signal peptides, for example, one comprising a variant CD4 signal peptide and the other comprising a variant CD8 signal peptide.. A representative construct, designated Variant 3, includes the FRB-containing chain with the CD8 P21A signal peptide (SEQ ID NO: 7) and the FKBP12-containing chain with the CD4 G25A signal peptide (SEQ ID NO: 24). Such engineered polynucleotide constructs as provided encode synthetic cytokine receptors that exhibit enhanced signal peptide cleavage efficiency, improved expression and improved activity relative to a reference construct employing wild- type signal peptides.

[0136] In some embodiments, one or both of the FRB- or FKBP12-containing chains of a synthetic cytokine receptor are linked to their respective signaling domains through linker sequence, including those that contain rigid or semi-rigid motifs. The results demonstrate that particular linker sequences engineered between the extracellular ligand-binding domain (e.g., FRB or FKBP12) and the intracellular cytokine receptor signaling domain substantially influence receptor assembly and improve signaling behavior. For instance, constructs incorporating linkers composed of rigid or semi-rigid motif sequences, containing EAAAK (SEQ ID NO: 20) sequences or a hybrid sequence containing EAAAK (SEQ ID NO: 20) and GGGGS (SEQ ID NO: 21_, exhibit reduced ligand- independent (“tonic”) signaling while maintaining or enhancing ligand-dependent STAT5 activation compared to a reference construct. These results indicate that linker architecture modulates the spatial orientation and flexibility between domains, thereby improving the balance between receptor stability and activation responsiveness. In particular, a construct incorporating an (EAAAK)s linker (e.g. Variant 3 RACR) achieved enhanced rapamycin binding sensitivity, reduced background 30MF-365319601260132004340activation, and robust inducible signaling, supporting that optimized linker length and composition can improve both the dynamic range and specificity of synthetic receptor function.

[0137] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a cytokine gamma chain polypeptide, wherein a linker links the FRB domain and the cytokine gamma chain polypeptide. In some embodiments, the amino acid sequence of the linker comprises an amino acid sequence of any one of SEQ ID NOS: 156, 157 or 158. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, or 147. In some embodiments, the amino acid sequence of the linker is (EAAAK)3 (SEQ ID NO: 12).

[0138] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a cytokine beta chain polypeptide, wherein a linker links the FRB domain and the cytokine beta chain polypeptide. In some embodiments, the amino acid sequence of the linker comprises an amino acid sequence of any one of SEQ ID NOS: 156, 157 or 158. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, or 147. In some embodiments, the amino acid sequence of the linker is (EAAAK)3 (SEQ ID NO: 12).

[0139] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12 domain and a cytokine gamma chain polypeptide, wherein a linker links the FKBP12 domain and the cytokine gamma chain polypeptide. In some embodiments, the amino acid sequence of the linker comprises an amino acid sequence of any one of SEQ ID NOS: 156, 157 or 158. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, or 147. In some embodiments, the amino acid sequence of the linker is (EAAAK)3 (SEQ ID NO: 12).

[0140] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12 domain and a cytokine beta chain polypeptide, wherein a linker links the FKBP12 domain and the cytokine beta chain polypeptide. In some embodiments, the amino acid sequence of the linker comprises an amino acid sequence of any one of SEQ ID NOS: 156, 157 or 158. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, or 147. In some embodiments, the amino acid sequence of the linker is (EAAAK)3 (SEQ ID NO: 12).

[0141] In some aspects, provided herein is a viral vector comprising any one of the polynucleotide constructs disclosed herein. In some embodiments, references to “viral vector”31MF-365319601260132004340in the context of administration, transduction, dosing / titers, or particle- surface components are intended to refer to viral vector particles. In the present disclosure, viral vector is sometimes used as a shorthand for viral vector particles; for example, a lentiviral vector particle is commonly referred to as an LVV. For clarity, a “viral vector” may refer to the engineered viral nucleic acid (DNA or RNA) comprising the transgene cassette and one or more vector elements, whereas a “viral vector particle” refers to a viral particle / virion comprising structural components (e.g., capsid and optionally an envelope) that packages a viral vector genome and is capable of delivering the viral vector to a cell (e.g., transducing a cell).

[0142] In some aspects, provided herein is a cell comprising any of the viral vectors disclosed herein.

[0143] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with any one of the viral vectors disclosed herein. In some embodiments, viral vectors disclosed herein improve the tonic signaling associated with RACR in transduced cells. In some embodiments, viral vectors disclosed herein improve the rapamycin sensitivity. In a therapeutic context, increased sensitivity to rapamycin would, for example, be expected to decrease the rapamycin dose patients need to receive.

[0144] In some aspects, provided herein is a method of expressing a chimeric protein encoding a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell comprising contacting the target cell with any one of the viral vectors disclosed herein.

[0145] In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

[0146] In some aspects, provided herein is a method of administering to a subject any of the cells disclosed herein. In some aspects, provided herein is a method of administering to a subject any of the viral vectors disclosed herein.

[0147] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.32MF-365319601260132004340

[0148] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. POLYNUCLEOTIDE CONSTRUCTS

[0149] Provided herein are polynucleotide constructs encoding one or more separate proteins.

[0150] In some embodiments, the polynucleotide constructs comprise one, two, three, or four nucleotide sequence open reading frames each encoding a separate protein. In some embodiments, the polynucleotide constructs comprise four nucleotide sequence open reading frames each encoding a separate protein. In some embodiments, the nucleotide sequences are separated by cleavable linkers.

[0151] In some embodiments, the polynucleotide constructs provided herein encode a fusion of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD8 signal peptide comprises the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the heterologous POI comprises a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain. In some embodiments, the heterologous POI is a chimeric protein of a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the signaling domain from a cytokine receptor is a cytokine gamma chain polypeptide or a cytokine beta chain polypeptide. In some embodiments, the cytokine gamma chain comprises interleukin 2 receptor subunit y (IL2RG). In some embodiments, the cytokine beta chain comprises interleukin 2 receptor subunit P (IL2RB).

[0152] In some embodiments, the polynucleotide constructs provided herein encode a fusion of a signal peptide and a heterologous protein of interest, wherein the signal peptide is a wildtype CD4 signal peptide (MNRGVPFRHLLLVLQLALLPAATQG; SEQ ID NO:23) or a modified CD4 signal peptide comprises the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24). In some embodiments, the heterologous POI comprises a chimeric protein comprising an FKBP12 domain. In some embodiments, the chimeric protein is a chimeric protein of a synthetic cytokine receptor comprising in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the signaling domain from a cytokine receptor is cytokine gamma chain polypeptide or a cytokine beta chain polypeptide. In33MF-365319601260132004340some embodiments, the cytokine gamma chain comprises interleukin 2 receptor subunit y (IL2RG). In some embodiments, the cytokine beta chain comprises interleukin 2 receptor subunit P (IL2RB).

[0153] In some embodiments, the polynucleotide construct encodes a synthetic cytokine receptor polypeptide that is encoded as the fusion proteins described above, where one is a fusion of a CD8 signal peptide with a FRB polypeptide and a signaling domain from a cytokine receptor (e.g., signaling domain of IL2RG or signaling domain of IL2RB) and the other is a fusion of a signal peptide such as a CD4 signal peptide or modified CD4 signal peptide as described with a FKBP12 polypeptide and the other of the signaling domain from the cytokine receptor (e.g. the other of the signaling domain of IL2RG or signaling domain of IL2RB).

[0154] In some embodiments, the polynucleotide constructs are polycistronic constructs that encode one or more additional proteins, such as a third heterologous POI and in some cases also a fourth heterologous POI. In some embodiments, the additional protein or proteins is an FRB (such that a freely diffusible FRB may be produced by a cell in which the polynucleotide construct is expressed) or is a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide constructs provided herein comprise a nucleotide sequence encoding an FRB. In some embodiments, the polynucleotide constructs provided herein further comprise a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a third heterologous POI comprising an FRB protein. In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR). In some embodiments, the first fusion, the second fusion, and the third heterologous POI are separated by encoded cleavable signal peptides. In some embodiments, the first fusion, the second fusion, the third heterologous POI, and the fourth heterologous POI are separated by encoded cleavable signal peptides.

[0155] In some embodiments, the polynucleotide construct provided herein comprises nucleotide sequences encoding an FRB, a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor, a chimeric protein comprising a FKBP12 domain and a signaling domain from a cytokine receptor, and a CAR.

[0156] In some embodiments, the polynucleotide construct comprises in 5’ to 3’ order a nucleotide sequence encoding FRB, a nucleotide sequence encoding a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a 34MF-365319601260132004340cytokine receptor, a chimeric protein comprising a FKBP12 domain and a signaling domain from a cytokine receptor, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the chimeric protein comprises in 5’ to 3’ order a first nucleotide sequence encoding FRB:IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the chimeric protein polypeptide comprises in 5’ to 3’ order a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding FRB:IL2RB.

[0157] Features of the nucleotide sequences and encoded components of provided polynucleotide constructs are described in the following subsections.A. Rapamycin Activated Cytokine Receptor (RACR) System

[0158] Disclosed herein are polynucleotide constructs encoding one or more of the polypeptide chains of a rapamycin activated cytokine receptor (RACR) complex. The rapamycin activated cytokine receptor (RACR) system is a synthetic cytokine receptor composed of two chimeric polypeptides that mimics the JAK / STAT signal downstream to induce STAT5 activation (which is associated with cytokine signaling in T cells) in the presence of rapamycin or an analog and induce cell survival and growth. Each polypeptide chain is a chimeric protein composed of an extracellular head domain of FKBP12 or FRB and either an intracellular gamma chain or beta chain signaling domain of a cytokine receptor, in which binding of rapamycin or an analog to FKBP12 and FRB forms a complex to engage the JAK / STAT intracellular signaling from the gamma and beta signaling domains. In some embodiments, the FKBP12 and FRB domains may be replaced with or modified to comprise functionally equivalent variants, derivatives, homologs, or engineered versions thereof that retain the ability to bind one another in the presence of rapamycin or an analog, thereby forming a dimerization complex capable of initiating cytokine receptor signaling.

[0159] In particular embodiments, provided polynucleotide constructs encoding the synthetic cytokine receptor of a RACR system incorporate sequences encoding specific signal peptides and inter-domain linkers that facilitate enhanced expression, membrane localization, and receptor assembly, and signaling performance of the synthetic cytokine receptor.

[0160] In some embodiments, the polynucleotide construct encodes a chimeric protein of a synthetic cytokine receptor. The chimeric polypeptides of synthetic cytokine receptors of the present disclosure comprise a signaling domain from a cytokine receptor, wherein the cytokine35MF-365319601260132004340receptor is a cytokine gamma chain polypeptide (e.g., comprising a gamma chain signaling domain) or the cytokine receptor is a cytokine beta chain polypeptide (e.g., comprising a beta chain signaling domain), in which each signaling domain is associated with a sequence comprising a dimerization domain. The dimerization domains may be heterodimerization domains, including but not limited to FK506-Binding Protein of size 12 kD (FKBP) and a FKBP12-rapamycin binding (FRB) domain. The dimerization domains controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling by the synthetic cytokine receptor.

[0161] In particular embodiments, the synthetic cytokine receptor is a two chain polypeptide, in which one chain includes the FKBP 12 dimerization domain and the other chain includes the FRB dimerization domain. In some embodiments, the polynucleotide construct encoding the synthetic cytokine receptor encodes the two distinct polypeptide chains, each encoded by a single polycistronic polynucleotide construct. The polycistronic construct may comprise at least two open reading frames (ORFs), each encoding one of the two polypeptide chains, optionally separated by a self-cleaving peptide sequence (e.g., a 2A peptide) or other cleavable linker sequence, thereby allowing co-expression of both chains from a single transcript.

[0162] The chimeric polypeptides of the synthetic cytokine receptor can include transmembrane receptor proteins that include a chimeric cytokine gamma chain polypeptide and a chimeric cytokine beta chain polypeptide, such as provided as a first transmembrane receptor and a second transmembrane receptor. The cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2G intracellular domain). The cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain). In some embodiments, the polynucleotide construct provided herein comprises a first fusion 36MF-365319601260132004340encoding IL2RG chain of the synthetic cytokine receptor and a second fusion encoding IL2RB chain of the synthetic cytokine receptor. In particular embodiments, the dimerization domain of the cytokine gamma chain polypeptide and the cytokine beta chain polypeptide are extracellular (N-terminal to the transmembrane domain).

[0163] In some embodiments, each polypeptide chain comprises a chimeric protein that includes, in order from the N- to C-terminus, (i) the dimerization domain, (ii) a transmembrane domain, and (iii) a signaling domain derived from either the gamma chain or the beta chain of a cytokine receptor. In some embodiments, the transmembrane domains of the two chains are derived from the same cytokine receptor.

[0164] The polynucleotide constructs disclosed herein comprise sequence modifications, including modified signal peptides and variant linkers. In some embodiments, the cytokine gamma chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide. In some embodiments, the cytokine beta chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide. A skilled artisan is readily familiar with signal peptides that can provide a signal to transport a nascent protein in the cells. In some embodiments, the signal peptide is modified. In some embodiments, the signal peptide is a modified CD8 signal peptide. In some embodiments, the modified CD8 signal peptide comprises the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). In some embodiments, the modified signal peptide is a modified CD4 signal peptide. In some embodiments, the modified CD4 signal peptide comprises the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24).

[0165] In some embodiments, an inter-domain linker is provided between the dimerization domain and the transmembrane domain in one or both of the chains. The linker may serve to spatially separate or orient the dimerization domains for optimal interaction. In improved embodiments, the linker is a rigid or semi-rigid peptide linker, such as a proline-rich linker, an a-helical linker (e.g., (EAAAK)n), or a structured synthetic linker that maintains the dimerization domains in a fixed relative geometry. The linker thereby connects the dimerization domain to the transmembrane domain, and by extension, to the signaling domain of each chain.

[0166] In some embodiments, one or more linkers comprises an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, one or more linkers comprises an amino acid sequence of GGGGS (SEQ ID NO: 21). In some embodiments, one or more linkers comprises the amino acid sequence of EAAAK (SEQ ID NO: 20) and the amino acid sequence of GGGGS 37MF-365319601260132004340(SEQ ID NO: 21). In some embodiments, the one or more linker comprises APAPAPAPAPAPAP (SEQ ID NO: 10). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the one or more linker comprises EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19). In some embodiments, the one or more linker comprises EAAAKEAAAKGGGGS (SEQ ID NO: 14). In some embodiments, the one or more linker comprises DIGSGAPAPAPAP (SEQ ID NO: 15). In some embodiments, the one or more linker comprises GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17). In some embodiments, the one or more linker comprises DIGGSGGAP (SEQ ID NO: 18). In some embodiments, the one or more linker comprises EAAAKEAAAK (SEQ ID NO: 145). In some embodiments, the one or more linker comprises GGGGSGGGGS (SEQ ID NO: 147).

[0167] In particular embodiments, each linker is EAAAKEAAAKEAAAK (SEQ ID NO: 12).

[0168] Upon dimerization of the dimerization domains (e.g., FKBP12-FRB association), the two chimeric chains assemble into a functional receptor complex, bringing the respective signaling domains into proximity and activating the downstream signaling cascade. In some embodiments, the receptor is constitutively active or inducible by a dimerizing agent.

[0169] In some embodiments, the synthetic cytokine receptor is able to be bound by the non-physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to the non-physiological ligand rapamycin or a rapamycin analog, in which binding of the non-physiological ligand to the dimerization domains of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, such as via the JAK / STAT pathway.1. Dimerization Domain

[0170] The dimerization domains may be heterodimerization domains, including but not limited to FK506-Binding Protein of size 12 kD (FKBP12) and a FKBP12-rapamycin binding (FRB) domain, which are known in the art to dimerize in the presence of rapamycin or a rapalog. In some embodiments, a two-chain synthetic cytokine receptor (e.g., RACR) as provided herein includes a first polypeptide chain that comprises the FKBP12 dimerization domain and a second polypeptide chain that comprises the FRB dimerization domain. Each of38MF-365319601260132004340these chains may further include a signal peptide (e.g., a modified CD8 or modified CD4 signal peptide), an inter-domain linker positioned between the dimerization domain and a transmembrane domain, and an intracellular cytokine receptor signaling domain (e.g., a y-chain or P-chain signaling domain).

[0171] In some embodiments, the FRB domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises or consists of a variant, derivative, or fragment of SEQ ID NO: 29, 33, or 40 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains the ability to heterodimerize with an FKBP12 domain in the presence of rapamycin or an analog thereof.

[0172] In some embodiments, a nucleotide sequence encoding the FRB domain has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain comprises or consists of a variant, homolog, derivative, or codon-optimized sequence of SEQ ID NO: 4, 32, or 39 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that encodes a polypeptide having at least 80% sequence identity to, and retaining the dimerization function of, an FRB domain as set forth in SEQ ID NO: 29, 33, or 40. In some embodiments, the nucleotide sequence has a sequence in which degenerate codons are used to encode the same amino acid sequence as SEQ ID NO: 4, 32, or 39.

[0173] In some embodiments, the FRB domain comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOS: 33. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 33. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NOS: 33. In some embodiments, the FRB domain comprises a variant, derivative, or fragment of SEQ ID NO: 33 (e.g., having at least 80%, 85%,39MF-36531960126013200434090%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains the ability to heterodimerize with an FKBP12 domain in the presence of rapamycin or an analog thereof.

[0174] In some embodiments, a nucleotide sequence encoding the FRB domain has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain comprises or consists of a variant, homolog, derivative, or codon-optimized sequence of SEQ ID NO: 32 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that encodes a polypeptide having at least 80% sequence identity to, and retaining the dimerization function of, an FRB domain as set forth in SEQ ID NO: 33. In some embodiments, the nucleotide sequence has a sequence in which degenerate codons are used to encode the same amino acid sequence as SEQ ID NO: 32.

[0175] In some embodiments, the FKBP12 domain comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NOS: 37. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NOS: 37. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NOS: 37. In some embodiments, the FKBP12 domain comprises a variant, derivative, or fragment of SEQ ID NO: 37 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains the ability to heterodimerize with an FRB domain in the presence of rapamycin or an analog thereof.

[0176] In some embodiments, a nucleotide sequence encoding the FKBP12 domain has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises or consists of a variant, homolog, derivative, or codon-optimized sequence of SEQ ID NO: 1 or 43 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that encodes a polypeptide having at least 80% sequence identity to, and retaining the dimerization function of, an FKBP12 domain as set forth in SEQ ID NO:40MF-36531960126013200434037. In some embodiments, the nucleotide sequence has a sequence in which degenerate codons are used to encode the same amino acid sequence as SEQ ID NO: 37.

[0177] In some embodiments, other ligand-dependent dimerization systems may be used in place of FKBP12 and FRB. These include both heterodimeric and homodimeric domain pairs that permit small-molecule-regulated or constitutive receptor assembly.

[0178] In some embodiments, the first and second dimerization domains may be domains that interact indirectly through a small-molecule ligand. For example, the first dimerization domain and the second dimerization domain may be a FK506-Binding Protein of size 12 kD (FKBP12) and a calcineurin domain, which are known in the art to dimerize in the presence of FK506 or an analogue thereof.

[0179] In some embodiments, the dimerization domains involve homodimerization systems that allow ligand-dependent association of two polypeptide chains with an identical dimerization domain. In some embodiments the dimerization domains are homodimerization domains selected from:i) FK506-Binding Protein of size 12 kD (FKBP);ii) ii) cyclophiliA (CypA); oriii) iii) gyrase B (CyrB);with the corresponding non-physiological ligands being, respectively.i) FK1012, AP1510, AP1903, or AP20187;ii) ii) cyclosporin-A (CsA); oriii) iii) coumermycin or analogs thereof.

[0180] In some embodiments, the dimerization domains involve alternative heterodimerization systems that allow ligand-dependent association of two polypeptide chains with different dimerization domains.

[0181] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a cyclophilin domain.

[0182] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0183] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a calcineurin domain and a cyclophilin domain.41MF-365319601260132004340

[0184] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).2. Transmembrane Domain

[0185] The transmembrane domain (TM) is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0186] In the context of the rapamycin-activated cytokine receptor (RACR) system, each of the two polypeptide chains includes a transmembrane domain positioned between the dimerization domain and the intracellular signaling domain. In provided embodiments, the transmembrane domain anchors the chimeric receptor within the plasma membrane when expressed in a cell, thereby orienting the dimerization domain extracellularly and positioning the signaling domain intracellularly. In some embodiments, an inter-domain linker is positioned between the dimerization domain and the transmembrane domain, as described in a separate subsection below.

[0187] In some embodiments, the transmembrane domain may be heterologous or homologous to the intracellular signaling domain of the same receptor chain. For example, the transmembrane domain may be derived from a cytokine receptor different from the source of the intracellular domain (heterologous), or it may be derived from the same cytokine receptor subunit (homologous). In some embodiments, such substitution permits modulation of receptor stability, expression, or signaling properties while maintaining the overall membrane topology of the RACR system.

[0188] In some embodiments, a linker sequence may be present between the transmembrane domain and the intracellular signaling domain, such as to provide structural flexibility or spatial separation of the cytoplasmic signaling motifs. In other embodiments, the transmembrane domain and intracellular domain are directly contiguous (i.e., the C-terminus of the transmembrane segment is directly fused to the N-terminus of the intracellular signaling domain) without an intervening linker sequence.

[0189] In some embodiments, the transmembrane domain is contiguous with, or maps directly to, the corresponding cytokine receptor intracellular domain sequence. In some embodiments, the TM domain and the corresponding intracellular signaling domain are encoded42MF-365319601260132004340within a single continuous open reading frame, thereby providing a structural and functional linkage between the membrane-spanning segment and the cytoplasmic signaling motifs. In some embodiments, the TM and intracellular domains correspond to the same cytokine receptor subunit, thereby preserving native membrane topology and signal transduction characteristics within the synthetic receptor design.

[0190] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain.

[0191] In some embodiments, one or more additional contiguous amino acids of the ectodomain directly adjacent to the TM domain of the cytokine receptor also can be included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor. In some embodiments, 1-20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor is included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor. The portion of the ectodomain may be a contiguous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent (e.g., N-terminal to) the TM sequence. In some embodiments, such inclusion of a portion of the ectodomain contributes to the correct membrane insertion, folding, or surface presentation of the receptor chain, and maintains appropriate spacing between the dimerization domain and the membrane surface. In some embodiments, such ectodomain residues may be present along with a separate inter-domain linker as described.3. Intracellular Domain

[0192] In some embodiments, the intracellular domain is a signaling domain from a cytokine receptor. In some embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In the context of the rapamycin-activated cytokine receptor (RACR) system, each of the two polypeptide chains of the synthetic cytokine receptor functions as a transmembrane receptor protein that includes the43MF-365319601260132004340dimerization domain and transmembrane domain and also includes an intracellular signaling domain that functions cooperatively upon dimerization of the FKBP12- and FRB -containing receptor chains. Dimerization of the extracellular dimerization domains by rapamycin or a rapamycin analog brings the two intracellular domains into proximity at the cytoplasmic face of the membrane, thereby initiating JAK / STAT-mediated signaling.

[0193] In some embodiments, the intracellular signaling domains suitable for use in the RACR system are derived from, or functionally analogous to, intracellular domains of cytokine receptors that activate members of the Janus kinase (JAK) family (e.g., JAK1 or JAK3) and signal through downstream STAT transcription factors (e.g., STAT3 or STAT5). In particular embodiments, the intracellular domains are derived from the common y-chain (IL-2RG) receptor and from one of the P-chain family receptors, such as the interleukin-2 receptor subunit P (IL-2RB), interleukin-7 receptor subunit P (IL-7RB), or interleukin- 21 receptor subunit P (IL-21RB), which together mediate ligand- inducible JAK / STAT activation in the RACR system.

[0194] In some embodiments, the intracellular signaling domain from a cytokine receptor comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. The IL-2RG domain provides the y-chain signaling function that participates in, for example, JAK3 recruitment and activation following receptor dimerization.

[0195] In some embodiments, the intracellular signaling domain from a cytokine receptor comprises interleukin-2 receptor subunit beta (IL2RB) domain. In some embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL2RB can also mean IL15RB. That is, the terms are used interchangeably in the present disclosure. The IL-2RB intracellular domain cooperates with the IL-2RG domain of the paired receptor chain to mediate, for example, JAK1 / JAK3 activation and downstream STAT5 signaling in the RACR system. In addition to IL-2RB, the P-chain intracellular domain may alternatively derive from other cytokine receptor subunits that use related signaling mechanisms, such as the interleukin-7 receptor subunit beta (IL-7RB) or interleukin-21 receptor subunit beta (IL-21RB).

[0196] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. In some embodiments, the intracellular signaling domain of the second transmembrane receptor protein 44MF-365319601260132004340comprises an interleukin-2 receptor subunit beta (IL2RB) domain. These paired intracellular domains represent a functional y-chain / p-chain receptor configuration that mimics native cytokine receptor signaling and exemplifies the two-chain RACR design.

[0197] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain (e.g., FRB), a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain (e.g., FKBP12). Upon rapamycin-dependent FKBP12-FRB dimerization, the intracellular IL-2RG and IL-2RB domains are brought together at the inner membrane surface, resulting in cytokine-receptor-like activation of the JAK / STAT pathway.

[0198] In some embodiments, the intracellular signaling domain of one of the transmembrane receptor proteins is an IL-2RG intracellular domain. In some embodiments, the intracellular domain sequence may include the transmembrane region contiguous with its cytoplasmic signaling motifs. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 149, which includes the transmembrane region contiguous with the intracellular signaling motifs of IL-2RG. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises an amino-acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 149. In some embodiments, the IL-2RG transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 149. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 149 (e.g., having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains IL-2RG signaling function, including JAK1 / JAK3 activation and downstream STAT3 and / or STAT5 signaling, upon rapamycin-induced heterodimerization with a P-chain cytokine receptor intracellular domain (e.g., IL-2RB, IL-7RB, or IL-21RB).

[0199] In some embodiments, a IL-2RG derived sequence that comprises the transmembrane and intracellular domain may further include a contiguous portion of the IL-2RG ectodomain. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 34, which includes the transmembrane region contiguous with the intracellular signaling motifs of IL-2RG. In some embodiments, the IL-2RG45MF-365319601260132004340transmembrane and intracellular domain comprises an amino-acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 34. In some embodiments, the IL-2RG transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 34. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 34 (e.g., having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains IL-2RG signaling function, including JAK1 / JAK3 activation and downstream STAT3 and / or STAT5 signaling, upon rapamycin-induced heterodimerization with a P-chain cytokine receptor intracellular domain (e.g., IL-2RB, IL-7RB, orIL-21RB).

[0200] In some embodiments, a nucleotide sequence encoding the IL-2RG portion of the polypeptide transmembrane receptor encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 149 or SEQ ID NO: 34, or a variant, derivative, or fragment thereof as described herein. In some embodiments, the nucleotide sequence encodes a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino-acid sequence identity to SEQ ID NO: 149 or SEQ ID NO: 34, and retains IL-2RG signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization. In some embodiments, the nucleotide sequence is codon-optimized for expression in a mammalian or human cell and / or comprises degenerate codons encoding the same amino-acid sequence as SEQ ID NO: 149 or SEQ ID NO: 34. In some embodiments, the nucleotide sequence comprises or consists of SEQ ID NO: 2, which encodes the IL2RG portion of the polypeptide transmembrane receptor. In some embodiments, the nucleotide sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 2, and encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 34 or a variant, derivative, or fragment thereof retaining IL-2RG signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization.

[0201] In some embodiments, the intracellular signaling domain of one of the transmembrane receptor proteins is an IL-2RB intracellular domain. In some embodiments, the intracellular domain sequence may include the transmembrane region contiguous with its cytoplasmic signaling motifs. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 148, which includes the 46MF-365319601260132004340transmembrane region contiguous with the intracellular signaling motifs of IL-2RB. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises an amino-acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 148. In some embodiments, the IL-2RB transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 148. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 148 (e.g., having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains IL-2RB signaling function, including JAK1 / JAK3 activation and downstream STAT3 and / or STAT5 signaling, upon rapamycin-induced heterodimerization with a Y-chain cytokine receptor intracellular domain (e.g., IL-2RG).

[0202] In some embodiments, a IL-2RB derived sequence that comprises the transmembrane and intracellular domain may further include a contiguous portion of the IL-2RB ectodomain. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the aminoacid sequence of SEQ ID NOS: 38 or 49, which includes the transmembrane region contiguous with the intracellular signaling motifs of IL-2RB. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises an amino-acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS: 38 or 49. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL-2RB transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NOS: 38 or 49 (e.g., having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto) that retains IL-2RB signaling function, including JAK1 / JAK3 activation and downstream STAT3 and / or STAT5 signaling, upon rapamycin-induced heterodimerization with a Y-chain cytokine receptor intracellular domain (e.g., IL-2RG).

[0203] In some embodiments, a nucleotide sequence encoding the IL2RB portion of the polypeptide transmembrane receptor encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 148, SEQ ID NO: 38, or SEQ ID NO: 49 or a variant, derivative, or fragment thereof as described herein. In some embodiments, the nucleotide sequence encodes a47MF-365319601260132004340polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino-acid sequence identity to SEQ ID NO: 148, SEQ ID NO: 38, or SEQ ID NO: 49, and retains IL-2RB signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization. In some embodiments, the nucleotide sequence is codon-optimized for expression in a mammalian or human cell and / or comprises degenerate codons encoding the same aminoacid sequence as SEQ ID NO: 148, SEQ ID NO: 38, or SEQ ID NO: 49. In some embodiments, the nucleotide sequence comprises or consists of SEQ ID NO: 3 or 48, which encodes the IL2RB portion of the polypeptide transmembrane receptor. In some embodiments, the nucleotide sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 3 or 48, and encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 38 or SEQ ID NO: 49 or a variant, derivative, or fragment thereof retaining IL-2RB signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization.

[0204] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain (e.g., FKBP12), a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain (e.g., FRB). In these embodiments, the heterologous pairing of an IL-2RG intracellular domain with an IL-7RB intracellular domain provides an P-chain signaling configuration that, upon rapamycin-induced dimerization of the receptor chains, promotes recruitment and activation of JAK1 and JAK3 and subsequent phosphorylation of downstream STAT transcription factors (e.g., STAT3 and / or STAT5), thereby eliciting cytokine-type signaling within the engineered cell.4. Linkers

[0205] The polynucleotide constructs encoding one or more of the transmembrane receptor proteins of a rapamycin-activated cytokine receptor (RACR) complex can include inter-domain linkers positioned between the dimerization domain (e.g., FKBP12 or FRB) and the transmembrane domain of each receptor chain. The inclusion of such linkers can improve rapamycin sensitivity, enhance JAK / STAT pathway activation (e.g., STAT5 phosphorylation) in the presence of rapamycin or a rapamycin analog, and minimize baseline signaling in its absence.48MF-365319601260132004340

[0206] In some embodiments, each transmembrane receptor polypeptide chain includes a linker connecting the FRB domain and the transmembrane domain, and / or a linker connecting the FKBP12 domain and the transmembrane domain. These linkers serve to orient the dimerization domains relative to the cell surface, thereby promoting correct receptor folding, membrane insertion, and ligand-dependent dimerization. In particular embodiments, the interdomain linkers are located immediately C-terminal to the dimerization domain and immediately N-terminal to the transmembrane domain of each transmembrane receptor protein.

[0207] In some embodiments, the linker connecting the FKBP12 domain and the transmembrane domain is referred to as a first linker, and the linker connecting the FRB domain and the transmembrane domain is referred to as a second linker. In some embodiments, the first linker and the second linker are the same amino acid sequence. In other embodiments, the first linker and the second linker are different amino acid sequences.

[0208] In certain embodiments, each linker is a peptide linker, such as a contiguous amino acid sequence connecting the dimerization domain and the transmembrane domain of a transmembrane receptor protein. In nucleic acid embodiments, the polynucleotide encoding the receptor protein encodes the peptide linker sequence positioned between the dimerization-domain-encoding region and the transmembrane-domain-encoding region.

[0209] In some embodiments, the inter-domain linker is heterologous to the cytokine receptor sequence. In particular embodiments, a provided inter-domain linker is included in receptor constructs that lack ectodomain sequences of a naturally occurring cytokine receptor. In such embodiments, the heterologous peptide linker replaces the natural extracellular-to-transmembrane junction and provides controlled spacing and orientation between the dimerization domain (e.g., FKBP12 or FRB) and the transmembrane domain.

[0210] In some embodiments, each linker is a peptide of about 2 to 50 amino acids in length, such as about 2 to 20 amino acids or about 5 to 15 amino acids. The linkers may be designed to be rigid, semi-rigid, or flexible depending on the desired receptor spacing and mechanical stability. In some embodiments, rigid or semi-rigid linkers are particularly desirable to maintain a defined distance between the dimerization domain and the plasma membrane, improving the geometry of receptor pairing and signaling efficiency in the RACR system. In some embodiments, a flexible linker comprises small, uncharged residues such as glycine and serine, which confer rotational freedom and minimize structural constraints. Exemplary flexible linkers include glycine-serine repeats such as (GGGGS)n (SEQ ID NO: 156), (GGS)n, or short GS 49MF-365319601260132004340dipeptides that provide high mobility between the connected domains. In other embodiments, a semi-rigid linker includes sequences that adopt partial a-helical structure or contain alternating rigid and flexible residues; examples include EAAAKEAAAK (SEQ ID NO: 145), EAAAK (SEQ ID NO:20), or combinations of EAAAK (SEQ ID NO: 20) and GGGGS (SEQ ID NO: 147) motifs that balance rigidity and flexibility. In some embodiments, the amino acid sequence of the linker comprises an amino acid sequence of any one of SEQ ID NOS: 156, 157 or 158. In further embodiments, a rigid linker forms a stable a-helical spacer that maintains a fixed distance and orientation between domains; exemplary rigid linkers include multiple tandem EAAAK (SEQ ID NO: 20) repeats, proline-rich sequences such as APAPAPAPAPAPAP (SEQ ID NO: 10), or structured a-helical motifs such as EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11).

[0211] In some embodiments, rigid or semi-rigid linkers are particularly desirable to maintain a defined distance between the dimerization domain and the plasma membrane, which may improve the geometry of receptor pairing and signaling efficiency in the RACR system.

[0212] Exemplary linkers include, but are not limited to, the following peptide sequences: EAAAK (SEQ ID NO: 20), GGGGS (SEQ ID NO: 21), combinations thereof, or repeats thereof. Additional exemplary linkers include APAPAPAPAPAPAP (SEQ ID NO: 10), EAAAKEAAAKEAAAKGGGGSGGGGSGGGGS (SEQ ID NO: 11), EAAAKEAAAKEAAAK (SEQ ID NO: 12), EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19), EAAAKEAAAKGGGGS (SEQ ID NO: 14), DIGSGAPAPAPAP (SEQ ID NO: 15), GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17), DIGGSGGAP (SEQ ID NO: 18), EAAAKEAAAK (SEQ ID NO: 145), and GGGGSGGGGS (SEQ ID NO: 147).

[0213] The linkers described herein may be employed individually, in tandem, or in combination, and may be repeated or concatenated to achieve the desired inter-domain spacing and rigidity. In certain embodiments, the linkers form a-helical or proline-rich structures that maintain a fixed geometry between the dimerization and transmembrane domains.

[0214] In particular embodiments, each peptide linker comprises the amino acid sequence EAAAKEAAAKEAAAK (SEQ ID NO: 12).5. Signal Peptide Modifications

[0215] The transmembrane receptor proteins of a synthetic cytokine receptor, such as a RACR complex, each include an N-terminal signal peptide that directs the nascent receptor50MF-365319601260132004340chain into the secretory pathway for processing and membrane insertion. The signal peptides may be derived from a variety of known leaders suitable for mammalian expression, including but not limited to wild-type CD8 (SEQ ID NO: 6), wild-type CD4 (SEQ ID NO: 23), and IL-2Ra (CD25; SEQ ID NO: 5) signal peptides, as well as modified versions thereof. Each receptor chain necessarily contains a signal peptide; however, the particular sequence used can vary depending on the desired level and uniformity of expression.

[0216] In some embodiments, at least one of the receptor chains comprises a modified signal peptide, and in certain embodiments both receptor chains comprise modified signal peptides. The inclusion of a modified signal peptide can improve signal-peptide cleavage efficiency, protein trafficking, and cell-surface expression of the chimeric receptor components in mammalian cells. Such modifications may be especially useful for the polynucleotide constructs that include FKBP12 and FRB dimerization domains, where balanced surface expression of both chains is important for controlled rapamycin-dependent signaling.

[0217] In some embodiments, the modified signal peptide is a modified CD8 signal peptide, which differs from the wild-type human CD8 sequence (SEQ ID NO: 6) by a single amino-acid substitution at the C-terminus, where the terminal proline (P) residue is replaced with an alanine (A) residue to yield MALPVTALLLPLALLLHAARA (SEQ ID NO: 7). This modification is contemplated to improve signal-peptide cleavage and translocation efficiency, resulting in more consistent membrane expression of chimeric receptor proteins.

[0218] In some embodiments, the modified signal peptide is a modified CD4 signal peptide, which differs from the wild-type human CD4 sequence (SEQ ID NO: 23) by a single amino-acid substitution at the C-terminus, where the terminal glycine (G) residue is replaced with an alanine (A) residue to yield MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24). This substitution is also contemplated to enhance cleavage accuracy and promote uniform processing of chimeric receptor constructs.

[0219] Any of the signal peptides described herein, wild-type or modified, can be used interchangeably with either of the synthetic cytokine receptor (e.g., RACR) chains. In some embodiments, one receptor chain (e.g., containing an FKBP12 dimerization domain) includes the modified CD8 signal peptide, and the other receptor chain (e.g., containing an FRB dimerization domain) includes the modified CD4 signal peptide. In other embodiments, both chains include the same modified signal peptide, or one chain includes a modified signal peptide and the other a wild-type signal peptide. At least one chain, however, includes a modified signal 51MF-365319601260132004340peptide in many embodiments, thereby enhancing reliable co-expression and balanced surface display of both transmembrane receptor proteins.

[0220] In particular embodiments, the first transmembrane receptor protein of the synthetic cytokine receptor (e.g., containing an FRB dimerization domain) includes the modified CD8 signal peptide (SEQ ID NO: 7), and the second transmembrane receptor protein (e.g., containing an FKBP12dimerization domain) includes the modified CD4 signal peptide (SEQ ID NO: 24).

[0221] In particular embodiments, the first transmembrane receptor protein of the synthetic cytokine receptor (e.g., containing an FKBP12 dimerization domain) includes the modified CD8 signal peptide (SEQ ID NO: 7), and the second transmembrane receptor protein (e.g., containing an FRB dimerization domain) includes the modified CD4 signal peptide (SEQ ID NO: 24).

[0222] In some embodiments, the use of the modified CD8 or CD4 signal peptides described above promotes consistent N-terminal processing and cell- surface localization of these receptor chains, supporting rapamycin-dependent dimerization of the FKBP12- and FRB -containing transmembrane receptor proteins and downstream activation of cytokine-type signaling pathways in the RACR system.6. Exemplary Synthetic Cytokine Receptors

[0223] The synthetic cytokine receptors of the present disclosure comprise a cytokine gamma chain and a cytokine beta chain, each comprising a dimerization domain (e.g., FRB and FKPB12). The dimerization domains controllable dimerize in the present of a non-physiological ligand, thereby activating signaling the synthetic cytokine receptor.

[0224] The cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IE-2RG) intracellular domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IE-2G intracellular domain. In some embodiments, the cytokine gamma chain polypeptide comprises an FRB:IE2RG fusion protein. In some embodiments, the synthetic gamma chain polypeptide comprises an FKBP12:IE2RG fusion protein.

[0225] The cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IE-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IE-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IE-21RB) intracellular52MF-365319601260132004340domain. The dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain). In some embodiments, the cytokine beta chain polypeptide comprises an FKBP12:IL2RB fusion protein. In some embodiments, the cytokine beta chain polypeptide comprises an FRB:IL2RB fusion protein.

[0226] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain, and a transmembrane domain and signaling domain from a cytokine receptor (e.g., IL2RG or IL-2RB). In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12 domain, and a transmembrane domain and signaling domain from a cytokine receptor (e.g., IL2RG or IL-2RB). In any of the preceding embodiments, a linker links the 1) FRB domain orFKBP12 domain with 2) a transmembrane domain and a signaling domain from the cytokine receptor. In any of the preceding embodiments, a linker links the 1) FRB domain or FKBP12 domain with 2) a signaling domain from a cytokine receptor.

[0227] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a cytokine gamma chain polypeptide, wherein a linker links the FRB domain and the cytokine gamma chain polypeptide. In provided embodiments, the cytokine gamma chain polypeptide is a transmembrane domain and signaling domain portion of IL2RG. In some embodiments, the linker links the FRB domain and the cytokine gamma chain polypeptide. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, and 147. In particular embodiments, each peptide linker comprises the amino acid sequence EAAAKEAAAKEAAAK (SEQ ID NO: 12)

[0228] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a cytokine beta chain polypeptide, wherein a linker links the FRB domain and the cytokine beta chain polypeptide. In provided embodiments, the cytokine beta chain polypeptide is a transmembrane domain and signaling domain portion of IL2RB. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, and 147. In particular embodiments, each peptide linker comprises the amino acid sequence EAAAKEAAAKEAAAK (SEQ ID NO: 12)53MF-365319601260132004340

[0229] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12 domain and a cytokine gamma chain polypeptide, wherein a linker links the FKBP12 domain and the cytokine gamma chain polypeptide. In provided embodiments, the cytokine gamma chain polypeptide is a transmembrane domain and signaling domain portion of IL2RG. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, and 147. In particular embodiments, each peptide linker comprises the amino acid sequence EAAAKEAAAKEAAAK (SEQ ID NO: 12)

[0230] In some embodiments, the polynucleotide construct encodes a chimeric protein comprising a FKBP12 domain and a cytokine beta chain polypeptide, wherein a linker links the FKBP12 domain and the cytokine beta chain polypeptide. In provided embodiments, the cytokine beta chain polypeptide is a transmembrane domain and signaling domain portion of IL2RB. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10 - 21, 145, and 147. In particular embodiments, each peptide linker comprises the amino acid sequence EAAAKEAAAKEAAAK (SEQ ID NO: 12). In some embodiments, the polynucleotide construct includes a signal peptide to control expression of the chimeric transmembrane receptor protein in a cell. Exemplary signal peptides include those described in Section I.A.5.

[0231] In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the synthetic cytokine receptor. In some embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the synthetic cytokine receptor is an FRB:IL2RG fusion protein. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 2. In some54MF-365319601260132004340embodiments, the nucleotide encoding the gamma chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain consists of the nucleotide sequence of SEQ ID NO: 2.

[0232] In some embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG). In some embodiments, the IL2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some55MF-365319601260132004340embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG consists of the amino acid sequence of SEQ ID NOS: 34 or 149.

[0233] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 148.

[0234] In some embodiments, polynucleotide construct encoding the chimeric protein comprises a FKBP12-rapamycin-binding (FRB) domain. In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the FRB domain. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 32. In some56MF-365319601260132004340embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NO: 32.

[0235] In some embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NO: 33.

[0236] In some embodiments, the polynucleotide construct is codon optimized.57MF-365319601260132004340

[0237] In some embodiments, a chimeric transmembrane protein of a synthetic cytokine receptor contains a FRB dimerization domain and an IL2RG transmembrane and intracellular domain (FRB:IL2RG). An exemplary FRB:IL2RG sequence and its components is set forth in Table 1 with respective SEQ ID Nos listed.

[0238] In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 30.58MF-365319601260132004340

[0239] In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 30.

[0240] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 31, 150, or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence consists of the sequence of SEQ ID NO: 31, 150, or 151.

[0241] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90%59MF-365319601260132004340identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 150 or 151. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NO: 150 or 151.

[0242] In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the synthetic cytokine receptor. In some embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the synthetic cytokine receptor is an FKBP12:IL2RG fusion protein. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NO:2. In some60MF-365319601260132004340embodiments, the nucleotide encoding t the gamma chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain comprises the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide encoding the gamma chain signaling domain consists of the nucleotide sequence of SEQ ID NO:2.

[0243] In some embodiments, the cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG). In some embodiments, the IL2RG comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149. In some embodiments, the IL2RG consists of the amino acid sequence of SEQ ID NOS: 34 or 149.61MF-365319601260132004340

[0244] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 148.

[0245] In some embodiments, polynucleotide construct encoding the chimeric protein comprises a FKBP12 domain. In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the FKBP12 domain. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the62MF-365319601260132004340nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NOS: 1 or 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NOS: 1 or 43.

[0246] In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NO: 37.

[0247] In some embodiments, a chimeric transmembrane protein of a synthetic cytokine receptor contains a FKBP12 dimerization domain and an IL2RG transmembrane and63MF-365319601260132004340intracellular domain (FKBP12:IL2RG). Exemplary FKBP12:IL2RG sequences and their components are set forth in Table 2 with respective SEQ ID Nos listed.

[0248] In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct comprises a nucleotide sequence64MF-365319601260132004340comprising the nucleotide sequence of SEQ ID NOS: 41 or 44. In some embodiments, the polynucleotide construct consists of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NOS: 41 or 44.

[0249] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting of the amino acid sequence of SEQ ID NOS: 27, 42, 154 or 45.

[0250] In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the synthetic cytokine receptor. In some embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the synthetic cytokine receptor is an FKBP12:IL2RB fusion protein. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta 65MF-365319601260132004340chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain comprises the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain consists of the nucleotide sequence of SEQ ID NOS: 3 or 48.

[0251] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit P (IL2RB).

[0252] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an 66MF-365319601260132004340amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 38 or 49.

[0253] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 148.

[0254] In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding FKBP12.

[0255] In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide 67MF-365319601260132004340sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NO: 1.

[0256] In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the 68MF-365319601260132004340nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NO: 43.

[0257] In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NO: 37.

[0258] In some embodiments, the polynucleotide construct is codon optimized.

[0259] In some embodiments, a chimeric transmembrane protein of a synthetic cytokine receptor contains a FKBP12 dimerization domain and an IL2RB transmembrane and intracellular domain (FKBP12:IL2RB). Exemplary FKBP12:IL2RB sequences and their components are set forth in Table 3 with respective SEQ ID Nos listed.69MF-365319601260132004340

[0260] In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding FKBP12:IL2RB with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 35.

[0261] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID70MF-365319601260132004340NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 152 or 153. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NO: 152 or 153.

[0262] In some embodiments, the chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the polynucleotide construct comprises a nucleotide sequence encoding the synthetic cytokine receptor. In some embodiments, the cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the synthetic cytokine receptor is an FRB:IL2RB fusion protein. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS:3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NOS:3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NOS:3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling 71MF-365319601260132004340domain is at least 95% identical to the nucleotide sequence of SEQ ID NOS:3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NOS:3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain comprises the nucleotide sequence of SEQ ID NOS: 3 or 48. In some embodiments, the nucleotide encoding the beta chain signaling domain consists of the nucleotide sequence of SEQ ID NOS: 3 or 48.

[0263] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit P (IL2RB).

[0264] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 38 or 49. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ 72MF-365319601260132004340ID NOS: 38 or 49. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 38 or 49.

[0265] In some embodiments, the IL2RB comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NOS: 148. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NOS: 148.

[0266] In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding FRB domain. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence73MF-365319601260132004340encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NO:32. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NO:32.

[0267] In some embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NO: 33.

[0268] In some embodiments, a chimeric transmembrane protein of a synthetic cytokine receptor contains a FRB dimerization domain and an IL2RB transmembrane and intracellular domain (FRB:IL2RB). Exemplary FRB:IL2RB sequences and their components are set forth in Table 4 with respective SEQ ID Nos listed.74MF-365319601260132004340

[0269] In some embodiments, the polynucleotide construct comprises a nucleotide encoding a chimeric protein comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a cytokine receptor. In some embodiments, the cytokine receptor polypeptide is a cytokine beta chain polypeptide comprising a beta chain signaling domain. In some embodiments, the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB). In some embodiments, the chimeric protein is an FRB:IL2RB fusion protein. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID 75MF-365319601260132004340NO: 46. In some embodiments, the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 46.

[0270] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NOS: 47, 155 or 28. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NOS: 47, 155 or 28.

[0271] In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino 76MF-365319601260132004340acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NOS: 47 or 155. In some embodiments, the polynucleotide construct encodes an amino acid sequence consisting the sequence of SEQ ID NOS: 47 or 155.

[0272] In some embodiments, the RACR system includes features of the FRB-IL-2RG fusion protein in Table 1 and features of the FKBP12-IL-2RB in Table 3. In some embodiments, the RACR system comprises a first transmembrane protein (e.g., containing the FKBP12 dimerization domain) comprising the amino acid sequence set forth in SEQ ID NO: 152 and a second transmembrane protein (e.g., containing the FRB dimerization domain) comprising the amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, the RACR system may be encoded by a polynucleotide construct in which is present in 5’ to 3 order the nucleotide sequence encoding the FRB-IL-2RG (e.g., SEQ ID NO: 150), a nucleotide sequence encoding a cleavable linker (e.g., 2A sequence), and the nucleotide sequence encoding FKBP12-IL-2RB (e.g., SEQ ID NO: 152). In some embodiments, the RACR system may be encoded by a polynucleotide construct in which is present in 5’ to 3 order the nucleotide sequence encoding FKBP12-IL-2RB (e.g., SEQ ID NO: 152), a nucleotide sequence encoding a cleavable linker (e.g., 2A sequence), and the nucleotide sequence encoding FRB-IL-2RG (e.g., SEQ ID NO: 150). In some embodiments, the cleavable linker is a 2A linker, such as a P2A (e.g., set forth in SEQ ID NO:54). The polynucleotide constructs can be inserted into a nucleic acid vector, such as a viral vector, for preparing a engineered retroviral particle, such as described in Section II. In some embodiments, the RACR system (including the 2A cleavable linker) comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some77MF-365319601260132004340embodiments, the RACR system (including the 2 A cleavable linker) comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some embodiments, the RACR system (including the 2A cleavable linker) comprises an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some embodiments, the RACR system (including the 2A cleavable linker) comprises the amino acid sequence of SEQ ID NO: 22.

[0273] In some embodiments, the 2A cleavable linker results in ribosome skipping during translation to produce two separate proteins, the FKBP12-IL-2RB fusion protein (e.g., SEQ ID NO: 152) and the FRB-IL-2RG fusion protein (e.g., SEQ ID NO: 150) that are separately processed to remove the signal peptide to result in expression on the surface of a lipid bilayer, such as a cell. In some embodiments, a cell surface expressed RACR system comprises an FRB-IL-2RG fusion protein set forth in SEQ ID NO: 151 and a FKBP12-IL-2RB fusion protein set forth in SEQ ID NO: 153. Also provided herein is a cell comprising a RACR system comprises an FRB-IL-2RG fusion protein set forth in SEQ ID NO: 151 and a FKBP12-IL-2RB fusion protein set forth in SEQ ID NO: 153. The cell may be any as described herein.B. Cytosolic FRB

[0274] In some embodiments, an expression cassette of the polynucleotide construct further encodes an FRB domain. The FRB domain is an approximately 270 base pair (bp) domain derived from the mTOR protein kinase. It may be expressed in the cytosol as a freely diffusible soluble protein.

[0275] In some embodiments, the first expression cassette in the polynucleotide construct comprises a nucleotide sequence encoding an FRB. In some embodiments, when the FRB domain is expressed, it is a freely diffusible soluble protein.

[0276] In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB78MF-365319601260132004340domain is at least 80% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39. In some embodiments, the nucleotide sequence encoding the FRB consists of the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

[0277] In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the 79MF-365319601260132004340nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the FRB consists of the nucleotide sequence of SEQ ID NO: 4.

[0278] In some embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

[0279] In some embodiments, the FRB domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 29. In some80MF-365319601260132004340embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NO: 29.

[0280] In some embodiments, the cytosolic FRB polypeptide binds to the same ligand as the synthetic cytokine receptor complex. In some embodiments, the ligand is a non-physiological ligand (e.g., rapamycin or rapalog).

[0281] Advantageously, the cytosolic FRB confers resistance to the immunosuppressive effect of the non-physiological ligand (e.g., rapamycin or rapalog).C. Chimeric Antigen Receptor

[0282] In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR).

[0283] In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain contains a costimulatory signaling domain and / or an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising a costimulatory signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding portion, a transmembrane domain and an intracellular signaling domain comprising a costimulatory signaling domain and an activation signaling domain.81MF-365319601260132004340

[0284] In any embodiments described herein, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv) of an antibody, a Fab, Fv, Fc, or (Fab’)2 fragment, and the like.

[0285] In some embodiments, a costimulatory signaling domain serves to enhance the proliferation and survival of the lymphocytes upon binding of the CAR to a targeted moiety. The identity of the costimulatory signaling domain is limited only in that it has the ability to enhance cellular proliferation and survival activation upon binding of the targeted moiety by the CAR. Suitable costimulatory signaling domains include, but are not limited to: CD28 (see, e.g., Alvarez- Vallina, L. et al., Eur J Immunol. 1996. 26(10):2304-9); CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family (see, e.g., Imai, C. et al., Leukemia. 2004.18:676-84); and CD134 (0X40), a member of the TNFR-superfamily of receptors (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994. 24:677). A skilled artisan will understand that sequence variants of these costimulatory signaling domains can be used, where the variants have the same or similar activity as the domain on which they are modeled. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0286] In some embodiments of the invention, the CAR constructs comprise two costimulatory signaling domains. While the particular combinations include all possible variations of the four noted domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (0X40).

[0287] In some embodiments, the activation signaling domain serves to activate cells upon binding of the CAR to a targeted moiety. The identity of the activation signaling domain is limited only in that it has the ability to induce activation of the selected cell upon binding of the targeted moiety by the CAR. Suitable activation signaling domains include the CD3(^ chain and Fc receptor y. In some embodiments, the signaling domain is a signaling domain of NKG2C or NKp44. The skilled artisan will understand that sequence variants of these noted activation signaling domains can be used without adversely impacting the invention, where the variants have the same or similar activity as the domain on which they are modeled. Such variants may have at least about 80%, at least about 90%, at least about 95%. at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.82MF-365319601260132004340

[0288] In some embodiments, the CARs may include additional elements, such a signal peptide to ensure proper export of the fusion protein to the cells surface, a transmembrane domain to ensure the fusion protein is maintained as an integral membrane protein, and a hinge domain that imparts flexibility to the recognition region and allows strong binding to the targeted moiety.

[0289] In some embodiments, nucleotide sequence that encodes a CAR comprising an extracellular domain, optionally a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain and an activation signaling domain. In some embodiments, the costimulatory and activation signaling domains are a single domain, for example a single intracellular domain that provides both costimulation and activation signals to a cell. In other embodiments, the intracellular signaling domain comprises either a costimulatory domain or an activation signaling domain. In some embodiments, the CAR comprises an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, a nucleotide sequence encodes an extracellular domain, an CD28 hinge domain, a CD28 transmembrane domain, a CD28 co- stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the nucleotide sequence encodes a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4- IBB costimulatory domain, and a CD3zeta signaling domain.

[0290] Illustrative CAR constructs suitable for the provided polynucleotide constructs are provided below:(1) SCFV-CD8TM-4-1BBIC-CD3^S (see, e.g., Liu E, Tong Y, Doth G, et al., Leukemia. 2018; 32: 520-531);(2) SCFV-CD28TM+IC-CD3^S (see, e.g., Han J, Chu J, Keung CW et al., Sci Rep. 2015; 5:11483; Kruschinski A, Moosmann A, Poschke I et al., Proc Natl Acad Sci USA. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927);(3) SCFV-DAP12TM+IC (see, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015; 38: 197-210);(4) SCFV-CD8TM-2B4IC-CD3^S (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49);83MF-365319601260132004340(5) SCFV-2B4TM+IC-CD3^S (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866);(6) SCFV-CD28TM+IC-4-1BBIC-CD3^S (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913);(7) SCFV-CD16TM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(8) SCFV-NKP44TM-DAP10IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(9) SCFV-NKP46TM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(10) SCFV-NKG2DTM-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(11) SCFV-NKG2DTM-4-1BBIC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(12) SCFV-NKG2DTM-2B4IC-DAP12IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(13) SCFV-NKG2DTM-2B4IC-DAP10IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192);(14) SCFV-NKG2DTM-4-1BBIC-2B4IC-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and(15) SCFV-NKG2DTM-CD3^S (see, e.g., Li Y, Hermanson DL, Moriarity BS Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).1. Extracellular Domain

[0291] In some embodiments, the binding portion of the CAR can be directed to any antigen that is desired to be targeted, such as due to its overexpression on cells or association with a disease or conditions like cancer.

[0292] In some embodiments, the binding portion of the CAR is specific to a tumor antigen. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma- associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, P-human chorionic gonadotropin,84MF-365319601260132004340alphafetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NAU, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD 171, CD 179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, R0R1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor- specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29VBCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET. In some embodiments, the CAR comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA 85MF-365319601260132004340and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD19 and CD22, BCMA and GPRC5D, or CD20 and CD22. In some embodiments, the CAR comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA.

[0293] A skilled artisan is readily familiar with CARs against diverse tumor antigens. Any one of such CARs can be employed as the CAR. Numerous CARs have been incorporated into products approved by the FDA and include, but are not limited to, anti-CD19 and anti-BCMA CAR T cells such as tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta), brexucabtagene autoleucel (Tecartus), lisocabtagene maraleucel (Breyanzi), idecabtagene vicleucel (Abecma), or ciltacabtagene autoleucel (Carvykti). It is within the level of a skilled artisan to generate similar constructs for specific targeting of a desired tumor antigen.

[0294] In some embodiments, the binding portion of the CAR can be directed to a universal antigen to target a wide variety of tumors without the need to prepare separate CAR constructs. The targeted moiety recognized by the CAR may also remain constant. In some embodiments, a ligand may be administered to the subject to allow interaction with target cells and interaction with the binding portion of the CAR. It is only the ligand portion of the small conjugate molecule that needs to be altered to allow the system to target cancer cells of different identity. Exemplary CAR systems are described in the section below.

[0295] In some embodiments, the CAR is an anti-CD19 CAR and the extracellular binding domain of the CD 19 CAR is specific to CD 19, for example, human CD 19. In some embodiments, the extracellular domain of the CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16- 17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. An exemplary anti-CD19 CAR is shown in Table 6 with its different portions including the extracellular domain.

[0296] In some embodiments, the CAR is an anti-CD20 CAR and the extracellular binding domain of the CD20 CAR is specific to CD20, for example, human CD20. In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific to CD20, including, for example, Leul6, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and86MF-365319601260132004340ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies. An exemplary anti-CD20 CAR is shown in Table 7 and Table 8 with its different portions including its extracellular domain.a. Universal CARs

[0297] Conventionally, CARs are generated by fusing a polynucleotide encoding a VL, VH, or scFv to the 5' end of a polynucleotide encoding transmembrane and intracellular domains, and transducing cells with that polynucleotide as well as with the corresponding VH or VL, if needed. Numerous variations on CARs well known in the art and the disclosure contemplates using any of the known variations. Additionally, VL / VH pairs and scFvs for innumerable haptens are known in the art or can be generated by conventional methods routinely.Accordingly, the present disclosure contemplates using any known hapten-binding domain.

[0298] In some embodiments, the CAR is an anti-FITC CAR and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) moiety conjugated to an agent that binds to a desired target cell (such as a cancer cell). Exemplary ligands are described below. In some embodiments, the ligand is FITC-folate.

[0299] An exemplary anti-FITC CAR is shown in Table 5 and its different portions.

[0300] In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises anti-fluorescein isothiocyanate (FITC) E2. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0301] In some embodiments, the scFv VL comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of 87MF-365319601260132004340SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID NOS: 64 or 65. In some embodiments, the scFv VL consists of the nucleotide sequence of SEQ ID NOS: 64 or 65.

[0302] In some embodiments, the scFv VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 66.1n some embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv VL consists of the amino acid sequence of SEQ ID NO: 66.88MF-365319601260132004340

[0303] In some embodiments, the scFv VH comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID NOS: 67 or 68. In some embodiments, the scFv VH consists of the nucleotide sequence of SEQ ID NOS: 67 or 68.

[0304] In some embodiments, the scFv VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH 89MF-365319601260132004340comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the scFv VH consists of the amino acid sequence of SEQ ID NO: 69.

[0305] In some embodiments, the scFv linker comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker comprises the nucleotide sequence of SEQ ID NOS: 70 or 71. In some embodiments, the scFv linker consists of the nucleotide sequence of SEQ ID NOS: 70 or 71.

[0306] In some embodiments, the scFv linker comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 95% identical to the amino acid sequence 90MF-365319601260132004340of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the scFv linker consists of the amino acid sequence of SEQ ID NO: 72.

[0307] In some embodiments, the scFv comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv comprises the nucleotide sequence of SEQ ID NOS: 73 or 74. In some embodiments, the scFv consists of the nucleotide sequence of SEQ ID NOS: 73 or 74.

[0308] In some embodiments, the scFv comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv91MF-365319601260132004340comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 75.

[0309] Various methods to target CARs and CAR-expressing cells have been described in the art, including, for example in US 2020 / 0123224, the disclosure of which is incorporated by reference herein. For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety may be conjugated to an agent that binds to a desired target cell (such as a cancer cell), and thereby a CAR expressing an anti-fluorescein / FITC chimeric antigen receptor may be selectively targeted to the target cell labeled by the conjugate. In variations, other haptens recognized by CARs may be used in place of fluorescein / FITC. The CAR may be generated using various scFv sequences known in the art, or scFv sequences generated by conventional and routine methods. Further illustrative scFv sequences for fluorescein / FITC and for other haptens are provided in, for example, WO 2021 / 076788, the disclosure of which is incorporated by reference herein.

[0310] In one embodiment, the disclosure provides an illustration of this conjugate molecule / CAR system.

[0311] In some embodiments, the CAR system of the disclosure utilizes conjugate molecules as the bridge between CAR-expressing cells and targeted cancer cells. The conjugate molecules are conjugates comprising a hapten and a cell-targeting moiety, such as any suitable tumor cell-specific ligand. Illustrative haptens that can be recognized and bound by CARs, include small molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, along with fluorescein and derivatives thereof, including 92MF-365319601260132004340FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, a knottin, a centyrin, and a DARPin. Suitable celltargeting moiety that may themselves act as a hapten for a CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrins, and DARPins (see Reichert, J.M. AZAto 2009. 1(3): 190-209).

[0312] In some embodiments, the cell-targeting moiety is DUPA (DUPA-(99m) Tc), a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (KD = 14 nM; see Kularatne, S.A. et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, a DUPA derivative can be the ligand of the small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, incorporated herein by reference.

[0313] In some embodiments, the cell-targeting moiety is CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0314] In some embodiments, the cell-targeting moiety is folate, folic acid, or an analogue thereof, a ligand bound by the folate receptor on cells of cancers that include cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, E.I. et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0315] In some embodiments, the cell-targeting moiety is an NK-1R ligand. Receptors for NK-1R the ligand are found, for example, on cancers of the colon and pancreas. In some embodiments, the NK-1R ligand may be synthesized according to the method disclosed in Int’l Patent Appl. No. PCT / US2015 / 044229, incorporated herein by reference.

[0316] In some embodiments, the cell-targeting moiety may be a peptide ligand, for example, the ligand may be a peptide ligand that is the endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand may be a regulatory peptide that belongs to the family of tachykinins which target tachykinin receptors. Such regulatory peptides include Substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K), (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0317] In some embodiments, the cell-targeting moiety is a CAIX ligand. Receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers. The CAIX ligand may also be referred to herein as CA9.93MF-365319601260132004340

[0318] In some embodiments, the cell-targeting moiety is a ligand of gamma glutamyl transpeptidase. The transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytic gliomas, melanomas, and leukemias.

[0319] In some embodiments, the cell-targeting moiety is a CCK2R ligand. Receptors for the CCK2R ligand found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, among others.

[0320] In some embodiments, the cell-targeting moiety is a PSMA ligand.

[0321] In some embodiments, the cell-targeting moiety is a FAP ligand.

[0322] In one embodiment, the cell-targeting moiety may have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about 4000 Daltons, about 1 to about 3500 Daltons, about 1 to about 3000 Daltons, about 1 to about 2500 Daltons, about 1 to about 2000 Daltons, about 1 to about 1500 Daltons, about 1 to about 1000 Daltons, or about 1 to about 500 Daltons.

[0323] In one illustrative embodiment, the linkage in a conjugate described herein can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and a free amine group of a small molecule ligand) or the linkage can be through an intermediary linker. In one embodiment, if present, an intermediary linker can be any biocompatible linker known in the art, such as a divalent linker. In one illustrative embodiment, the divalent linker can comprise about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can comprise about 2 to about 20 carbon atoms. In other embodiments, lower molecular weight divalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300 Da) are employed. In another embodiment, linkers lengths that are suitable include, but are not limited to, linkers having 2, 3, 4, 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 or 40, or more atoms.94MF-365319601260132004340

[0324] In some embodiments, the hapten and the cell-targeting moiety can be directly conjugated through such means as reaction between the isothiocyanate group of FITC and free amine group of small ligands (e.g., folate, DUPA, and CCK2R ligand). However, the use of a linking domain to connect the two molecules may be helpful as it can provide flexibility and stability. Examples of suitable linking domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) sugars; 5) unnatural peptidoglycans; 6) polyvinylpyrrolidone; 7) Pluronic F-127. Linker lengths that are suitable include, but are not limited to, linkers having 2, 3, 4, 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 or 40, or more atoms.

[0325] In some embodiments, the linker may be a divalent linker that may include one or more spacers.

[0326] An illustrative conjugate of the disclosure is FITC-Folateillustrative conjugate of the disclosure is FITC-CA9

[0327] Illustrative conjugates of the disclosure include the following molecules: FITC-(PEG)i2-Folate, FITC-(PEG)2o-Folate, FITC-(PEG)io8-Folate, FITC-DUPA, FITC-(PEG)I2-DUPA, FITC-CCK2R ligand, FITC-(PEG)I2-CCK2R ligand, FITC-(PEG)n-NKlR ligand, and FITC-(PEG)2-CA9.

[0328] While the affinity at which the ligands and cancer cell receptors bind can vary, and in some cases low affinity binding may be preferable (such as about 1 pM), the binding affinity of the ligands and cancer cell receptors will generally be at least about 100 pM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, even more preferably at least about 100 pM.95MF-365319601260132004340

[0329] Examples of conjugates and methods of making them are provided in U.S. patent applications US 2017 / 0290900, US 2019 / 0091308, and US 2020 / 0023009, all of which are incorporated herein by reference.b. Spacer (e.g., hinge domain)

[0330] In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a short hinge or a medium hinge domain. In some embodiments, the hinge domain comprises a CD8 or an IgG. In some embodiments, the CD8 hinge comprises CD8a hinge. In some embodiments, the IgG hinge comprises an IgG4 hinge. In some embodiments, the IgG4 hinge is modified. In some embodiments, the IgG hinge comprises an IgGl hinge. In some embodiments, the hinge domain comprises a PD1 hinge. In some embodiments, the hinge domain comprises a CD28 hinge.

[0331] In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge comprises the nucleotide sequence of SEQ ID NOS: 76 or 77. In some embodiments, the CD8a hinge consists of the nucleotide sequence of SEQ ID NOS: 76 or 77.96MF-365319601260132004340

[0332] In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge comprises the amino acid sequence of SEQ ID NOS: 78 or 79. In some embodiments, the CD8a hinge consists of the amino acid sequence of SEQ ID NOS: 78 or 79.

[0333] In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD897MF-365319601260132004340hinge comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge comprises the nucleotide sequence of SEQ ID NOS: 61 or 79. In some embodiments, the CD8 hinge consists of the nucleotide sequence of SEQ ID NOS: 61 or 79.

[0334] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 62.

[0335] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 85% identical 98MF-365319601260132004340to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 63.

[0336] In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the99MF-365319601260132004340IgGl hinge comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IgGl hinge consists of the amino acid sequence of SEQ ID NO: 97.

[0337] In some embodiments, the PD1 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the PD1 hinge consists of the amino acid sequence of SEQ ID NO: 98.

[0338] In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises 100MF-365319601260132004340an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 99.c. Transmembrane Domain

[0339] In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 or a CD28. In some embodiments, the transmembrane domain comprises a CD8 domain. In some embodiments, the transmembrane domain comprises a CD28 domain. In some embodiments, the CD8 transmembrane domain comprises CD8a transmembrane domain.

[0340] In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain101MF-365319601260132004340comprises the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists of the nucleotide sequence of SEQ ID NO: 80.

[0341] In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 81.d. Intracellular domain (i.e., endodomain)

[0342] In some embodiments, the CAR comprises an endodomain. In some embodiments, the endodomain comprises a costimulatory molecule. In some embodiments, the endodomain comprises 4- IBB, CD3(^, and / or CD28. In some embodiments, the endodomain comprises 4-1BB. In some embodiments, the endodomain comprises CD3(^. In some embodiments, the endodomain comprises CD28.

[0343] In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises a102MF-365319601260132004340nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4-1BB endodomain comprises the nucleotide sequence of SEQ ID NOS: 82 or 83. In some embodiments, the 4- IBB endodomain consists of the nucleotide sequence of SEQ ID NOS: 82 or 83.

[0344] In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises 103MF-365319601260132004340an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4-1BB endodomain comprises the amino acid sequence of SEQ ID NOS: 84 or 113. In some embodiments, the 4- IBB endodomain consists of the amino acid sequence of SEQ ID NOS: 84 or 113.

[0345] In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain comprises the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88. In some embodiments, the CD3(^ endodomain consists of the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88.

[0346] In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 85% identical to 104MF-365319601260132004340the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD3(^ endodomain consists of the amino acid sequence of SEQ ID NO: 89.e. Exemplary CAR Polynucleotides

[0347] In some embodiments the CAR is an anti-CD19 CAR, and in these embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a CD 19 CAR. In some embodiments, the CD 19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD 19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth heterologous POI encodes an anti-CD19 CAR with features set forth in Table 6.105MF-365319601260132004340

[0348] In some embodiments the CAR is an anti-CD20 CAR, and in these embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a CD20 CAR. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth heterologous POI encodes an anti-CD20 CAR with features set forth in Table 7 (anti-CD20 CAR with flag) or Table 8 (anti-CD20 CAR without flag).106MF-365319601260132004340

[0349] In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth 107MF-365319601260132004340heterologous POI comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI comprises the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140. In some embodiments, the fourth heterologous POI consists of the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140.

[0350] In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 80% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 85% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 90% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 95% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 96% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 97% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 98% identical to the nucleotide 108MF-365319601260132004340sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 99% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence at least 100% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence comprising the sequence of SEQ ID NOS: 94, 141, 95, 96 or 142. In some embodiments, the fourth heterologous POI encodes an amino acid sequence consisting of the sequence of SEQ ID NOS: 94, 141, 95, 96 or 142.

[0351] In further embodiments, an illustrative nucleotide sequence encoding a CAR may comprise SEQ ID NO: 92, and an illustrative CAR amino acid sequence may comprise SEQ ID NO: 95.

[0352] An illustrative nucleotide insert may comprise SEQ ID NO: 130.

[0353] In some embodiments, the CAR may be encoded by a nucleic acid sequence that encodes a signal peptide to signal transport of the CAR in the cell. It is understood that typically the signal peptide is removed from the protein.

[0354] An illustrative CAR amino acid sequence without a signal peptide may comprise SEQ ID NO: 131.

[0355] An illustrative CAR amino acid sequence signal peptide may comprise SEQ ID NO:75. In various embodiments, CAR-expressing cells comprising the nucleic acid of SEQ ID NO:92 or 130 are provided. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO:95 is contemplated. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 131 is contemplated. In some embodiments, a vector is contemplated comprising SEQ ID NO: 92 or 130. In some embodiments, a lentiviral vector is contemplated comprising SEQ ID NO: 92 or 130. In some embodiments, SEQ ID NO: 95 can comprise or consist of human or humanized amino acid sequences. In some embodiments, SEQ ID NO: 131 can comprise or consist of human or humanized amino acid sequences.

[0356] In some embodiments, variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 130 or SEQ ID NO: 131 are contemplated.

[0357] While the affinity at which the CARs, expressed by the lymphocytes, bind to the targeted moiety can vary, and in some cases low affinity binding may be preferable (such as 109MF-365319601260132004340about 50 nM), the binding affinity of the CARs to the targeted ligand will generally be at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM or 10 fM, even more preferably at least about 100 fM.D. Cleavable Linkers

[0358] As provided herein, expression cassettes of the polynucleotide constructs can be separated by linkers. In some embodiments, the linkers comprise sites for cleavage, making them cleavable linkers.

[0359] Cleavage sites can be used in the design of polynucleotide constructs to achieve coexpression of multiple genes. In some embodiments, the cleavage sites comprise self-cleaving sites. In some embodiments, the self-cleaving site comprises a 2A site. 2A peptides are a class of 18-22 amino acid-long peptides that can induce ribosomal skipping during translation resulting in loss of a peptide bond between glycine and proline residues, which allows a proteolytic enzyme to recognize the 2A site. The most commonly used 2A peptides in molecular biology include T2A, P2A, E2A, and F2A.

[0360] In some embodiments, the polynucleotide constructs provided herein comprise one or more cleavable linkers. In some embodiments, the one or more cleavable linkers separating the expression cassettes are the same. In some embodiments, the cleavable linkers separating the expression cassettes are different. In some embodiments, the one or more cleavable linkers separating the expression cassettes comprise one or more cleavage sites. In some embodiments, the one or more cleavage sites are the same. In some embodiments, the one or more cleavage sites are different.

[0361] In some embodiments, in addition to a 2A site, the cleavable linker may also comprise another cleavage site. In some embodiments, the additional cleavage site comprises a furin site. There are three known furin sites, including FC1, FC2, and FC3.

[0362] In some embodiments, the polynucleotide construct provided herein comprises a T2A, P2A, E2A, or F2A cleavage site in the cleavable linker. In some embodiments, the polynucleotide construct comprises a T2A cleavage site in a cleavable linker. In some embodiments, the polynucleotide construct comprises a P2A cleavage site in a cleavable linker. In some embodiments, the polynucleotide construct comprises a furin cleavage site in a cleavable linker. In some embodiments, the polynucleotide construct comprises a T2A cleavage site and a furin cleavage site in a cleavable linker.110MF-365319601260132004340

[0363] In some embodiments, the polynucleotide construct provided herein comprises at least one, at least two or at least three 2A cleavable linker sequences. In some embodiments, the polynucleotide construct herein comprises a T2A cleavage site and a P2A, E2A or F2A cleavage site. In some embodiments, the polynucleotide construct herein comprises a P2A cleavage site and a T2A, E2A or F2A cleavage site. In some embodiments, the polynucleotide construct herein comprises a E2A cleavage site and a P2A, T2A or F2A cleavage site. In some embodiments, the polynucleotide construct herein comprises a F2A cleavage site and a P2A, E2A or T2A cleavage site.

[0364] In some embodiments, the polynucleotide construct provided herein comprises a 2 A cleavable linker sequence. In some embodiments, each nucleotide sequence encoding the 2A cleavable linker sequences is different. In some embodiments, the 2 A cleavable linker is independently a T2A, P2A, E2A or F2A cleavage site. In some embodiments, the 2A cleavable linker is independently a P2A or a T2A.

[0365] In some embodiments, the 2 A cleavable linker is a P2A and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 50, 51, 52, or 53. In some embodiments, the nucleotide sequence encoding the P2A cleavable linker is set forth in SEQ ID NOS: 50, 51, 52, or 53. In some embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54. In some embodiments, the P2A cleavable linker comprises the sequence set forth in SEQ ID NO: 54.

[0366] In some embodiments, at least one 2 A cleavable linker is a T2A and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO: 55. In some embodiments, the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56. In some embodiments, the T2A cleavable linker comprising the sequence set forth in SEQ ID NO: 56.

[0367] In some embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence. In some embodiments, the furin cleavage site sequence is located between the first expression cassette and the second expression cassette. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is 111MF-365319601260132004340at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 58.

[0368] In some embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). In some embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 59.

[0369] In some embodiments, the cleavage site sequence comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO:60.

[0370] In some embodiments, the first fusion, the second fusion, and the third heterologous POI are separated by encoded cleavable signal peptides. In some embodiments, the first fusion, the second fusion, the third heterologous POI, and the fourth heterologous POI are separated by encoded cleavable signal peptides. In some embodiments, the first fusion and the second fusion are separated by a P2A, and the first fusion and the third heterologous POI are separated by a furin T2A. In some embodiments, the second fusion and the fourth heterologous POI are separated by a P2A.E. Exemplary Polynucleotide Constructs

[0371] In some aspects, the polynucleotide constructs of the present disclosure encode synthetic fusion proteins comprising RACR. In some embodiments, the polynucleotide constructs further comprise a CAR.

[0372] In some aspects, the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARA112MF-365319601260132004340(SEQ ID NO: 7), and wherein the heterologous POI comprises a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain.

[0373] In some aspects, the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD8 signal peptide comprising the amino acid sequence MAEPVTAEEEPEAEEEHAARA (SEQ ID NO: 7), and wherein the heterologous POI comprises a chimeric protein comprising a FKBP12 domain.

[0374] In some aspects, the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHEEEVEQEAEEPAATQA (SEQ ID NO: 24), and wherein the heterologous POI comprises a chimeric protein comprising a FKBP12 domain.

[0375] In some aspects, the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24), and wherein the heterologous POI comprises a chimeric protein comprising a FRB domain. In some embodiments, the polynucleotide construct encodes a fusion protein comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some embodiments, the polynucleotide construct encodes a fusion protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some embodiments, the polynucleotide construct encodes a fusion protein comprising an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22. In some embodiments, the polynucleotide construct encodes a fusion protein of an amino acid sequence of SEQ ID NO: 22. In some aspects, the polynucleotide construct encodes a heterologous POI comprising a chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor, wherein a linker links 113MF-365319601260132004340the FRB domain and the signaling domain from the cytokine receptor. In some embodiments, the amino acid sequence of the linker comprises a sequence selected from the SEQ ID NOS: 10-21. In some embodiments, the linker comprises an amino acid sequence of EAAAK (SEQ ID NO: 20). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 21).

[0376] In some aspects, the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24), and wherein the heterologous POI comprises a chimeric protein comprising a FKBP12 domain; and the polynucleotide construct encodes a fusion protein of a signal peptide and a heterologous protein of interest (POI), wherein the signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24), and wherein the heterologous POI comprises a chimeric protein comprising a FRB domain.IL VECTORS AND ENGINEERED RETROVIRAL PARTICLES

[0377] Also provided herein are vectors and engineered viral vectors including engineered retroviral vectors containing any of the polynucleotide constructs described herein, such as any described in Section I. These include viral vector particles, such as engineered retroviral vector particles, comprising and / or packaging any of the polynucleotide constructs and that may also be engineered with additional particle-surface proteins.

[0378] In some embodiments, the polynucleotide constructs can be inserted into a nucleic acid vector. As used herein, the term “nucleic acid vector” is intended to mean any nucleic acid that functions to carry, harbor, or express a nucleic acid of interest. Nucleic acid vectors can have specialized functions such as expression, packaging, pseudotyping, transduction or sequencing, for example. Nucleic acid vectors also can have, for example, manipulatory functions such as a cloning or shuttle vector. The structure of the vector can include any desired form that is feasible to make and desirable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms. A nucleic acid vector can be composed of, for example, DNA or RNA, as well as contain partially or fully, nucleotide derivatives, analogs, and mimetics. Such nucleic acid vectors can be obtained from natural sources, produced recombinantly or chemically synthesized.114MF-365319601260132004340

[0379] Non-limiting examples of vector systems of the present disclosure include a retrovirus, a lentivirus, a foamy virus, and a Sleeping Beauty transposon.

[0380] In some embodiments, the polynucleotide is incorporated into a viral vector. As it is well known in the art, a viral particle is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the disclosure, and by way of example, some viral particles used in recombinant DNA techniques allow entities, such as a segment of DNA, to be transferred into a host cell. Examples of vectors used in recombinant DNA techniques include but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" means a construct capable of in vivo or in vitro / ex vivo expression.

[0381] In some aspects, provided herein is a virus particle encapsulating the polynucleotide constructs disclosed herein. In some embodiments, any of the polynucleotide constructs can be provided as a payload in the generation of a viral particle. Also provided herein are viral particles, such as lentiviral vectors, incorporating any of the provided polynucleotide constructs for delivery of components of the rapamycin activated cytokine receptor (RACR) system, including FRB, chimeric protein comprising the synthetic cytokine receptor, and CAR, to a target cell. In further embodiments, the virus particles can be engineered to express one or more surface T cell activating agents. In some embodiments, the one or more surface T cell activating agents comprise a T cell surface receptor. In some embodiments, the T cell surface receptor comprises CD58, anti-CD3, or CD80.

[0382] Further, in some aspects, the virus particles encapsulating nucleotide vectors provided herein can comprise engineered viral envelopes. In some embodiments, the viral envelope comprises a transduction enhancer. In some embodiments, the viral envelope comprises at an immune cell- activating protein. In some embodiments, the viral envelope comprises a co- stimulation molecule. In some embodiments, the viral envelope comprises an immune cell-activating protein, and a co- stimulation molecule.A. Retroviral Vectors

[0383] Also provided herein are retroviral particles, such as lentiviral vectors, incorporating any of the provided polynucleotide constructs for delivery of components of the rapamycin activated cytokine receptor (RACR) system, including a chimeric protein comprising the synthetic cytokine receptor, to a target cell. In some embodiments, the polynucleotide construct115MF-365319601260132004340also encodes FRB and / or a CAR for delivery to the target cell. In further embodiments, the virus particles can be engineered to express one or more surface T cell activating agents. In some embodiments, the one or more surface T cell activating agents comprise a T cell surface receptor. In some embodiments, the T cell surface receptor comprises CD58, anti-CD3, or CD80.

[0384] Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which may be used to deliver polynucleotides to cells using methods known in the art.Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV-1 and HIV-2) and the Simian Immunodeficiency Virus (SIV). Retroviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.

[0385] In some embodiments, the virus particle comprises a retroviral particle. In some embodiments, the virus is a retrovirus. A large number of different retroviruses have been identified. Examples of retrovirus include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al., 1997, "Retroviruses", Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0386] A lentiviral vector (LVV) of the disclosure may be derived from or may be derivable from any suitable lentivirus. A recombinant retroviral vector particle is capable of transducing a recipient cell with a nucleotide of interest (NOI). Once within the cell, the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the DNA of the recipient cell. In some embodiments of the disclosure, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication defective. Portions of the viral genome may also be replaced by an NOI in order to generate a116MF-365319601260132004340vector comprising an NOI which is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome.

[0387] Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, which are each incorporated herein by reference in their entireties. In general, these vectors are configured to carry the essential sequences for selection of cells containing the vector, for incorporating foreign nucleic acid into a lentiviral particle, and for transfer of the nucleic acid into a target cell.

[0388] In some embodiments, the LVV is replication incompetent. In some embodiments, the LVV is self-inactivating and replication incompetent. Such LVV particles are desirable for safety reasons for embodiments that include introducing cells transduced with such LVV particles into a subject. When replication incompetent retroviral particles are used to transduce a cell, LVV particles are not produced from the transduced cell. Modifications to the retroviral genome are known in the art to assure that retroviral particles that include the genome are replication incompetent. However, it will be understood that in some embodiments for any of the aspects provided herein, replication competent recombinant retroviral particles can be used.

[0389] In some embodiments, the replication incompetent LVV particle can be derived from HIV, SIV, or FIV. In some embodiments, the replication incompetent LVV particle can be derived from the human immunodeficiency virus (HIV).

[0390] A commonly used lentiviral vector system is the so-called third- generation system. Third- generation lentiviral vector systems include four plasmids. The “transfer plasmid” encodes the polynucleotide sequence that is delivered by the lentiviral vector system to the target cell. The transfer plasmid generally has...

Claims

260132004340CLAIMS1. A polynucleotide construct comprising:(1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein comprising a FK506-binding protein (FKBP12)-rapamycin-binding (FRB) domain and a signaling domain from a first cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and(2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a second cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor,wherein the encoded first fusion protein and the encoded second fusion protein are separated by an encoded cleavable peptide linker; andthe first linker and / or the second linker each independently comprise an amino acid sequence set forth in SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158.

2. The polynucleotide construct of claim 1, wherein the first linker and / or the second linker independently comprise an amino acid sequence selected from the group consisting of EAAAK (SEQ ID NO: 20), EAAAKEAAAK (SEQ ID NO: 145), EAAAKEAAAKEAAAK (SEQ ID NO: 12), GGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGS (SEQ ID NOS: 16 or 17), and EAAAKEAAAKEAAAKGGGGS (SEQ ID NOS: 13 or 19).

3. The polynucleotide construct of claim 1 or claim 2, wherein the first linker is the amino acid sequence of EAAAKEAAAKEAAAK (SEQ ID NO: 12) and the second linker is the amino acid sequence of EAAAKEAAAKEAAAK (SEQ ID NO: 12).

4. A polynucleotide construct comprising:(1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein comprising a FK506-binding protein (FKBP12)-rapamycin- 229MF-365319601260132004340binding (FRB) domain and a signaling domain from a first cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and(2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a second cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor,wherein the encoded first fusion protein and the encoded second fusion protein are separated by a encoded cleavable peptide linker; andthe first linker and the second linker independently comprise an amino acid sequence selected from the sequence set forth in any of SEQ ID NOS: 10-21 and 145.

5. The polynucleotide construct of any one of claims 1 to 4, wherein the first linker and the second linker are the same amino acid sequence.

6. The polynucleotide construct of any one of claims 1 to 4, wherein the first linker and the second linker are different amino acid sequences.

7. The polynucleotide construct of any one of claims 1 to 6, wherein the first signal peptide is a modified CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

8. The polynucleotide construct of any one of claims 1 to 7, wherein the second signal peptide is a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24) or a CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQG (SEQ ID NO: 23).

9. The polynucleotide construct of any one of claims 1 to 8, wherein the first cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain.

10. The polynucleotide construct of claim 9, wherein the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).230MF-36531960126013200434011. The polynucleotide construct of any one of claims 1 to 10, wherein the FRB domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

12. The polynucleotide construct of any one of claims 1 to 11, wherein the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

13. The polynucleotide construct of any one of claims 1 to 12, wherein the FRB domain encoded by a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

14. The polynucleotide construct of any one of claims 1 to 13, wherein the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

15. The polynucleotide construct of any one of claims 10 to 14, wherein the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149.

16. The polynucleotide construct of any one of claims 10 to 15, wherein the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

17. The polynucleotide construct of any one of claims 1 to 16, wherein the second cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain.

18. The polynucleotide construct of claim 17, wherein the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB).

19. The polynucleotide construct of claim 18, wherein the IL-2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

20. The polynucleotide construct of claim 18 or claim 19, wherein the IL-2RB protein comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.231MF-36531960126013200434021. The polynucleotide construct of any one of claims 1 to 20, wherein the FKBP12 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37.

22. The polynucleotide construct of any one of claims 1 to 21, wherein the FKBP12 protein comprises the amino acid sequence of SEQ ID NO: 37.

23. A polynucleotide construct comprising:(1) a nucleotide sequence encoding a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first signal peptide encodes a modified CD8 signal peptide, and wherein the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain; and(2) a nucleotide sequence encoding a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain,wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide.

24. The polynucleotide construct of claim 23, wherein the first signal peptide comprises a modified CD8 signal peptide comprising an amino acid sequence MALPVTALLLPLALLLHAARA (SEQ ID NO: 7).

25. The polynucleotide construct of claim 23 or claim 24, wherein the second signal peptide comprises a modified CD4 signal peptide comprising the amino acid sequence MNRGVPFRHLLLVLQLALLPAATQA (SEQ ID NO: 24).

26. The polynucleotide construct of any one of claims 1 to 25, wherein the FRB domain is the amino terminal protein of the first chimeric protein.

27. The polynucleotide construct of any one of claims 1 to 26, wherein the FKBP12 domain is the amino terminal protein of the second chimeric protein.

28. The polynucleotide construct of any one of claims 1 to 27, wherein the first chimeric protein is a synthetic cytokine receptor comprising in N to C terminal order the FRB domain, a transmembrane domain, and a signaling domain from a first cytokine receptor.232MF-36531960126013200434029. The polynucleotide construct of claim 28, wherein the first cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain.

30. The polynucleotide construct of claim 29, wherein the gamma chain signaling domain is an interleukin 2 receptor subunit y (IL2RG).

31. The polynucleotide construct of claim 30, wherein the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 34 or 149.

32. The polynucleotide construct of claim 30 or claim 31, wherein the IL2RG comprises the amino acid sequence of SEQ ID NOS: 34 or 149.

33. The polynucleotide construct of any one of claims 23 to 32, wherein the FRB domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

34. The polynucleotide construct of any one of claims 23 to 33, wherein the FRB domain comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

35. The polynucleotide construct of any one of claims 1 to 34, wherein the second chimeric protein comprises in N to C terminal order the FKBP12 domain, a transmembrane domain, and a signaling domain from a second cytokine receptor.

36. The polynucleotide construct of claim 35, wherein the second cytokine receptor is a cytokine beta chain polypeptide comprising a beta chain signaling domain.

37. The polynucleotide construct of claim 36, wherein the beta chain signaling domain is an interleukin-2 receptor subunit beta (IL-2RB).

38. The polynucleotide construct of claim 37, wherein the IL-2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 38, 49, or 148.

39. The polynucleotide construct of claim 37 or claim 38, wherein the IL-2RB comprises the amino acid sequence of SEQ ID NOS: 38, 49, or 148.233MF-36531960126013200434040. The polynucleotide construct of any one of claims 23 to 39, wherein the FKBP12 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 37.

41. The polynucleotide construct of any one of claims 23 to 40, wherein the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 37.

42. The polynucleotide construct of any one of claims 1 to 41, wherein the polynucleotide construct further comprises a nucleotide sequence encoding a third heterologous POI comprising an FRB protein.

43. The polynucleotide construct of claim 42, wherein the FRB protein is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

44. The polynucleotide construct of claim 42 or claim 43, wherein the FRB protein is encoded by the nucleotide sequence of SEQ ID NOS: 4, 32, or 39.

45. The polynucleotide construct of any one of claims 42 to 44, wherein the FRB protein comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

46. The polynucleotide construct of any one of claims 42 to 45, wherein the FRB protein comprises the amino acid sequence of SEQ ID NOS: 29, 33, or 40.

47. The polynucleotide construct of any one of claims 1 to 46, wherein the polynucleotide construct further comprises a nucleotide sequence encoding a fourth heterologous POI comprising a chimeric antigen receptor (CAR).

48. The polynucleotide construct of claim 47, wherein the CAR comprises an scFv domain.

49. The polynucleotide construct of claim 48, wherein the scFv domain comprises an anti-fluorescein isothiocyanate (FITC) E2.234MF-36531960126013200434050. The polynucleotide construct of claim 48 or claim 49, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

51. The polynucleotide construct of claim 50, wherein the scFv VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 64 or 65.

52. The polynucleotide construct of claim 50 or claim 51, wherein the scFv VL is encoded by the nucleotide sequence of SEQ ID NOS: 64 or 65.

53. The polynucleotide construct of any one of claims 50 to 52, wherein the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66.

54. The polynucleotide construct of any one of claims 50 to 53, wherein the scFv VL comprises the amino acid sequence of SEQ ID NO: 66.

55. The polynucleotide construct of any one of claims 50 to 54, wherein the scFv VH is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 67 or 68.

56. The polynucleotide construct of any one of claims 50 to 55, wherein the scFv VH is encoded by the nucleotide sequence of SEQ ID NOS: 67 or 68.

57. The polynucleotide construct of any one of claims 50 to 56, wherein the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 69.

58. The polynucleotide construct of any one of claims 50 to 57, wherein the scFv VH comprises the amino acid sequence of SEQ ID NO: 69.

58. The polynucleotide construct of any one of claims 50 to 58, wherein the scFv linker is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 70 or 71.235MF-36531960126013200434060. The polynucleotide construct of any one of claims 50 to 59, wherein the scFv linker is encoded by the nucleotide sequence of SEQ ID NOS: 70 or 71.

61. The polynucleotide construct of any one of claims 50 to 60, wherein the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72.

62. The polynucleotide construct of any one of claims 50 to 61, wherein the scFv linker comprises the amino acid sequence of SEQ ID NO: 72.

63. The polynucleotide construct of any one of claims 50 to 62, wherein the scFv is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 73 or 74.

64. The polynucleotide construct of any one of claims 50 to 63, wherein the scFv is encoded by the nucleotide sequence of SEQ ID NOS: 73 or 74.

65. The polynucleotide construct of any one of claims 50 to 64, wherein the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 75.

66. The polynucleotide construct of any one of claims 48 to 65, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 75.

67. The polynucleotide construct of any one of claims 47 to 66, wherein the CAR comprises a hinge domain.

68. The polynucleotide construct of claim 67, wherein the hinge domain comprises a short hinge or a medium hinge domain.

69. The polynucleotide construct of claim 67 or claim 68, wherein the hinge domain comprises a CD8 or an IgG.

70. The polynucleotide construct of claim 69, wherein the CD8 hinge comprises CD8a hinge.236MF-36531960126013200434071. The polynucleotide construct of claim 70, wherein the CD8a hinge is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 76 or 77.

72. The polynucleotide construct of claim 70 or claim 71, wherein the CD8a hinge is encoded by the nucleotide sequence of SEQ ID NOS: 76 or 77.

73. The polynucleotide construct of any one of claims 70 to 72, wherein the CD8a hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 78 or 79.

74. The polynucleotide construct of any one of claims 70 to 73, wherein the CD8a hinge comprises the amino acid sequence of SEQ ID NOS: 78 or 79.

75. The polynucleotide construct of any one of claims 47 to 74, wherein the CAR comprises a transmembrane domain.

76. The polynucleotide construct of claim 75, wherein the transmembrane domain comprises a CD8 transmembrane domain or a CD28 transmembrane domain.

77. The polynucleotide construct of claim 76, wherein the CD8 transmembrane domain comprises a CD8a transmembrane domain.

78. The polynucleotide construct of any one of claims 75 to 77, wherein the transmembrane domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 80.

79. The polynucleotide construct of any one of claims 75 to 78, wherein the transmembrane domain is encoded by the nucleotide sequence of SEQ ID NO: 80.

80. The polynucleotide construct of any one of claims 75 to 79, wherein the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81.

81. The polynucleotide construct of any one of claims 75 to 80, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 81.237MF-36531960126013200434082. The polynucleotide construct of any one of claims 47 to 81, wherein the CAR comprises an endodomain.

83. The polynucleotide construct of claim 82, wherein the endodomain comprises a costimulatory molecule.

84. The polynucleotide construct of claim 82 or claim 83, wherein the endodomain comprises 4- IBB, CD3(^, and / or CD28.

85. The polynucleotide construct of claim 84, wherein the 4- IBB endodomain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 82 or 83.

86. The polynucleotide construct of claim 84 or claim 85, wherein the 4- IBB endodomain is encoded by the nucleotide sequence of SEQ ID NOS: 82 or 83.

87. The polynucleotide construct of any one of claims 84 to 86, wherein the 4- IBB endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 84 or 113.

88. The polynucleotide construct of any one of claims 84 to 87, wherein the 4- IBB endodomain comprises the amino acid sequence of SEQ ID NOS: 84 or 113.

89. The polynucleotide construct of any one of claims 84 to 88, wherein the CD3(^ endodomain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88.

90. The polynucleotide construct of any one of claims 84 to 89, wherein the CD3(^ endodomain is encoded by the nucleotide sequence of SEQ ID NOS: 85, 86, 87, or 88.

91. The polynucleotide construct of any one of claims 84 to 90, wherein the CD3(^ endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89.238MF-36531960126013200434092. The polynucleotide construct of any one of claims 84 to 91, wherein the CD3(^ endodomain comprises the amino acid sequence of SEQ ID NO: 89.

93. The polynucleotide construct of any one of claims 47 to 92, wherein the fourth heterologous POI is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140.

94. The polynucleotide construct of any one of claims 47 to 93, wherein the fourth heterologous POI is encoded by the nucleotide sequence of SEQ ID NOS: 90, 91, 92, 93 or 138-140.

95. The polynucleotide construct of any one of claims 47 to 94, wherein the fourth heterologous POI comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOS: 94, 141, 95, 96 or 142.

96. The polynucleotide construct of any one of claims 47 to 95, wherein the fourth heterologous POI comprises the amino acid sequence of SEQ ID NOS: 94, 141, 95, 96 or 142.

97. The polynucleotide construct of any one of claims 42 to 96, wherein the each of the first fusion, the second fusion, and the third heterologous POI is operably linked in series, and each adjacent pair of (i) the first fusion, (ii) the second fusion, and (iii) the third heterologous POI is separated by a cleavable linker.

98. The polynucleotide construct of any one of claims 47 to 97, wherein each of the first fusion, the second fusion, the third heterologous POI, and the fourth heterologous POI is operably linked in series, and each adjacent pair of (i) the first fusion, (ii) the second fusion, (iii) the third heterologous POI, and (iv) the fourth heterologous POI is separated by a cleavable linker.

99. The polynucleotide construct of any one of claims 1 to 98, wherein the cleavable linker comprises a 2A cleavable linker sequence.239MF-365319601260132004340100. The polynucleotide construct of claim 99, wherein the 2A cleavable linker sequences in the polynucleotide construct are different from one another when there are two or more cleavable linker sequences.

101. The polynucleotide construct of claim 99 or claim 100, wherein the 2A cleavable linker sequence is independently a T2A, P2A, E2A or F2A cleavable linker sequence.

102. The polynucleotide construct of any one of claims 99 to 101, wherein at least one of the 2A cleavable linkers is a P2A cleavable linker and the P2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 50, 51, 52, or 53.

103. The polynucleotide construct of any one of claims 99 to 102, wherein the P2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NOS: 50, 51, 52, or 53.

104. The polynucleotide construct of any one of claims 101 to 103, wherein the P2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54.

105. The polynucleotide construct of any one of claims 101 to 104, wherein the P2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 54.

106. The polynucleotide construct of any one of claims 99 to 101, wherein at least one of the 2A cleavable linkers is a T2A cleavable linker and the T2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55.

107. The polynucleotide construct of any one of claims 99 to 101 and 106, wherein the T2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 55.

108. The polynucleotide construct of any one of claims 99 to 101, 106 and 107, wherein the T2A cleavable linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56.240MF-365319601260132004340109. The polynucleotide construct of any one of claims any one of claims 99 to 101 and 106 to 108, wherein the T2A cleavable linker comprises the nucleotide sequence set forth in SEQ ID NO: 56.

110. The polynucleotide construct of any one of claims 99 to 109, wherein at least one of the 2A cleavable linkers comprises a furin cleavage site sequence.

111. The polynucleotide construct of claim 110, wherein the furin cleavage site sequence is located between the first fusion protein and the second fusion protein.

112. The polynucleotide construct of claim 110 or claim 111, wherein the furin cleavage site sequence comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57.

113. The polynucleotide construct of any one of claims 110 to 112, wherein the furin cleavage site sequence comprises the nucleotide sequence set forth in SEQ ID NO: 57.

114. The polynucleotide construct of any one of claims 110 to 113, wherein the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 58.

115. The polynucleotide construct of any one of claims 110 to 114, wherein the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 58.

116. The polynucleotide construct of any one of claims 101 to 115, wherein the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavable linker (furinT2A).

117. The polynucleotide construct of any one of claims 101 to 116, wherein the cleavage site sequence is comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59.

118. The polynucleotide construct of any one of claims 101 to 117, wherein the cleavage site sequence is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 59.241MF-365319601260132004340119. The polynucleotide construct of any one of claims 101 to 118, wherein the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60.

120. The polynucleotide construct of any one of claims 101 to 119, wherein the cleavage site sequence comprises the amino acid sequence of SEQ ID NO: 60.

121. The polynucleotide construct of any one of claims 97 to 120, wherein the first fusion and the second fusion are separated by a P2A, and the first fusion and the third heterologous POI are separated by a furin T2A.

122. The polynucleotide construct of any one of claims 1 to 121, wherein the polynucleotide construct comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 146.

123. The polynucleotide construct of any one of claims 1 to 122, wherein the polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 146.

124. The polynucleotide construct of any one of claims 1 to 123, wherein the chimeric protein or the first chimeric protein comprises the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151.

125. The polynucleotide construct of any one of claims 1 to 122, wherein the chimeric protein or the first chimeric protein comprises the amino acid sequence identical to the amino acid sequence of SEQ ID NOS: 31, 150, or 151.

126. A polynucleotide construct of SEQ ID NO: 146.

127. The polynucleotide construct of any one of claims 1 to 126, wherein one or more nucleotide sequences are codon optimized.

128. The polynucleotide construct of any one of claims 1 to 127, wherein the polynucleotide construct is encoded in a bicistronic construct.242MF-365319601260132004340129. The polynucleotide construct of any one of claims 1 to 128, wherein the polynucleotide construct is encoded in a polycistronic construct.

130. A viral vector particle comprising a polynucleotide construct of any one of claims 1 to 129.

131. The viral vector particle of claim 130, wherein the viral vector is a lentiviral vector.

132. The vector particle of claim 131, wherein the lentiviral vector is a selfinactivating and replication-incompetent lentiviral vector particle.

133. The vector particle of any of claims 130 to 132, wherein the viral vector comprises a pseudotyped viral envelope protein, optionally wherein the pseudotyped viral envelope protein is cocal virus glycoprotein (G) (Cocal G), optionally wherein the Cocal G is a Cocal envelope variant containing a R354Q mutation or a K47Q mutation.

134. The vector particle of any of claims 130 to 133, wherein the vector particle comprises a membrane-bound multidomain fusion (MDF) protein comprising a T cell activating domain and at least one costimulatory molecule on the surface of the lentiviral particle.

135. The vector particle of claim 134, wherein the MDF protein comprises a transmembrane domain that anchors the MDF protein to the lentiviral envelope membrane.

136. The vector particle of claim 134 or claim 135, wherein the MDF protein comprises:(a) a first costimulatory ligand comprising an extracellular domain portion of CD58 that binds to CD2;(b) a T-cell activating domain comprising a single-chain variable fragment (scFv) that specifically binds to CD3e; and(c) a second costimulatory ligand comprising CD80.

137. A cell comprising the polynucleotide construct of any one of claims 1 to 129.

138. A cell transduced by the viral vector particle of any one of claims 130 to 136.243MF-365319601260132004340139. A cell comprising a rapamycin activated cytokine receptor (RACR) system, wherein the RACR system comprises:(a) a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain and a signaling domain from a cytokine receptor, wherein a first linker links the FRB domain and the signaling domain from the cytokine receptor; and(b) a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain and a signaling domain from a cytokine receptor, wherein a second linker links the FKBP12 domain and the signaling domain from the cytokine receptor, wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide and the first linker and / or the second linker comprise an amino acid sequence represented by SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158.

140. A cell comprising a rapamycin activated cytokine receptor (RACR) system, wherein the RACR system comprises:(a) a first fusion protein comprising (a) a first signal peptide and (b) a first heterologous protein of interest (POI), wherein the first signal peptide encodes a modified CD8 signal peptide, and wherein the first heterologous POI comprises a first chimeric protein comprising a FKBP12-rapamycin-binding (FRB) domain; and(b) a second fusion protein comprising (a) a second signal peptide and (b) a second heterologous POI, w, and wherein the second heterologous POI comprises a second chimeric protein comprising an FKBP12 domain,wherein the first fusion protein and the second fusion protein are separated by an encoded cleavable signal peptide.

141. A cell comprising a polypeptide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set forth in SEQ ID NO: 144 or that is encoded by a polynucleotide that has at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to the sequence set forth in SEQ ID NO: 146.244MF-365319601260132004340142. The cell of any one of claims 137 to 141, wherein the cell comprises a stem cell or a progenitor cell.

143. The cell of claim 142, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).

144. The cell of claim 142, wherein the progenitor cell comprises a peripheral blood mononuclear cell (PBMC).

145. The cell of claim 144, wherein the peripheral blood mononuclear cell (PBMC) comprises lymphocytes.

146. The cell of any one of claims 137 to 141, wherein the cell comprises a T cell.

147. The cell of any one of claims 137 to 141, wherein the cell comprises a cytotoxic innate lymphocyte (CIL) cell.

148. The cell of any one of claims 137 to 141, wherein the cell comprises a natural killer (NK) cell.

149. The cell of any one of claims 137 to 141, wherein the cell comprises a white blood cell.

150. The cell of any one of claims 137 to 141, wherein the white blood cell comprises a monocyte and / or a macrophage.

151. A method of transducing a cell comprising contacting a target cell with the polynucleotide constructs of any one of claims 1 to 129.

152. A method of transducing a cell comprising contacting a target cell with the vector particle of any one of claims 130 to 136.

153. The method of claim 151 or claim 152, wherein the target cell comprises a stem cell.

154. The method of claim 153, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).245MF-365319601260132004340155. The method of claim 151 or claim 152, wherein the target cell comprises a progenitor cell.

156. The method of claim 155, wherein the progenitor cell comprises a peripheral blood mononuclear cell (PBMC).

157. The method of claim 151 or claim 152, wherein the target cell comprises a T cell.

158. The method of claim 157, wherein the T cell comprises a CD4+ or CD8+ T cell.

159. A method of increasing expression of a polynucleotide construct expressing a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of claims 1 to 129 or the vector particle of any one of claims 130 to 136.

160. A method of decreasing tonic signaling associated with a rapamycin activated cytokine receptor (RACR) system, the method comprising transducing a cell with a polynucleotide construct of any one of claims 1 to 129 or the vector particle of any one of claims 130 to 136.

161. A method of expressing a RACR system with decreased tonic signaling, the method comprising transducing a cell with a polynucleotide construct of any one of claims 1 to 129 or the vector particle of any one of claims 130 to 136.246MF-365319601

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