Cytokine armored engineered innate lymphoid cells and related methods

Abstract

Provided herein are fusion proteins of an interleukin- 15 (IL- 15) peptide and an interleukin-2 receptor (IL-2R) subunit, polynucleotide constructs for expression of same on cells, and engineered cells comprising the fusion proteins or polynucleotides. In some aspects, the fusion proteins are expressed with a synthetic cytokine receptor complex and / or a chimeric antigen receptor system. Also provided are vectors, such as viral vectors comprising same, cells comprising same, and methods of using same.

Description

260132004240CYTOKINE ARMORED ENGINEERED INNATE LYMPHOID CELLS AND RELATED METHODSCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 729,310 filed on December 6, 2024, entitled “CYTOKINE ARMORED ENGINEERED INNATE LYMPHOID CELLS AND RELATED METHODS,” the contents of which are incorporated by reference in their entirety.Reference to an Electronic Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 260132004240SeqList.xml created on December 5, 2025 which is 235,155 bytes in size bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entiretyField

[0003] The present disclosure provides fusion proteins of an interleukin- 15 (IL- 15) peptide and an interleukin-2 receptor (IL-2R) subunit, polynucleotide constructs for expression of same on cells, and engineered cells comprising the fusion proteins or polynucleotides. In some aspects, the fusion proteins are expressed with a synthetic cytokine receptor complex and / or a chimeric antigen receptor system. Also provided by the disclosure are vectors, such as viral vectors comprising same, cells comprising same, and methods of using same.Background

[0004] The process of deriving differentiated cells from induced pluripotent stem cells (iPSCs) requires addition of many different exogenous growth factors at different times during the differentiation process. Common gamma chain cytokines are some of the most important cytokines used in deriving innate lymphocytes (ILCs, NKs) or T cells and require multi-step addition of these cytokines during cell production. The use of constitutive rapamycin-activated cytokine receptor (RACR) signaling to derive cell differentiation and growth is known, including using RACR to induce iPSCs to induced cytotoxic innate lymphocytes (iCILs).1MF-364712232260132004240However, there is a need for alternative or additional differentiation methods that can efficiently derive synthetic cells. Provided herein are embodiments that address such needs.Summary

[0005] Provided herein is a fusion protein comprising an IL- 15 peptide and a subunit of an interleukin-2 (IL-2) receptor, wherein the fusion protein comprises from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL-2 receptor.

[0006] In some of any embodiments, the IL- 15 peptide 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: 14. In some embodiments, the IL- 15 peptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, a nucleotide sequence encoding the IL-15 peptide 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 sequence encoding the IL-15 peptide comprises the nucleotide sequence of SEQ ID NO: 2.

[0007] In some of any embodiments, the subunit of the IL-2 receptor is an IL-2 receptor beta chain. In some embodiments, the IL-2 receptor beta chain 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: 21. In some embodiments, the IL-2 receptor beta chain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding the IL-2 receptor beta chain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the IL-2 receptor beta chain comprises the nucleotide sequence of SEQ ID NO: 10.

[0008] In some of any embodiments, the subunit of the IL-2 receptor is an IL-2 receptor gamma chain. In some embodiments, the IL-2 receptor gamma chain 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: 16. In some embodiments, the IL-2 receptor gamma chain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, a nucleotide sequence encoding the IL-2 receptor gamma chain 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 IL-2 receptor gamma chain comprises the nucleotide sequence of SEQ ID NO: 4.2MF-364712232260132004240

[0009] In some of any embodiments, the linker is a peptide linker. In some embodiments, the linker is between four to 30 residues. In some embodiments, the linker comprises an amino acid sequence from the group consisting of (GGGGS)n (SEQ ID NO: 65), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3, (GGGGGS)n (SEQ ID NO: 66), wherein n is 1 to 4, such as 1 to 3, GGS, GGGGS (SEQ ID NO: 8), and GGGGGS (SEQ ID NO: 19). In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 15).

[0010] In some aspects, provided herein is a polynucleotide comprising a nucleic acid or nucleotide sequence encoding the fusion protein of any one of preceding embodiments. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleotide sequence encoding the IL- 15 peptide and the subunit of the interleukin-2 (IL-2) receptor.

[0011] In some embodiments, the promoter is a CMV, EFl alpha, CAG, PGK, or U6 promoter. In some embodiments, the polynucleotide is encoded by a multicistronic construct further encoding components of a chimeric antigen receptor (CAR). In some embodiments, the promoter comprises the sequence set forth in SEQ ID NO: 25.

[0012] In some aspects, provided herein is a viral vector comprising a polynucleotide of any one of the preceding embodiments. In some embodiments, the viral vector is a lentiviral vector.

[0013] In some aspects, provided herein is a cell comprising the fusion protein of any one of the preceding embodiments or the polynucleotide of the preceding embodiments, or the viral vector of the preceding embodiments.

[0014] In some embodiments, the cell comprises an endogenous IL-2R subunit. In some embodiments, the fusion protein is capable of heterodimerizing with the endogenous subunit of IL-2R. In some embodiments, the cell is a stem cell or a progenitor cell.

[0015] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the progenitor cell is a peripheral blood mononuclear cell (PBMC). In some embodiments, the peripheral blood mononuclear cell (PBMC) comprises lymphocytes. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the cell is a differentiated hematopoietic progenitor cell. In some embodiments, the cell is an induced cytotoxic innate lymphocyte (iCIL). 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.3MF-364712232260132004240

[0016] In some embodiments, the cell comprises a rapamycin-activated cytokine receptor (RACR) system.

[0017] In some embodiments, the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

[0018] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin- 21 receptor subunit P (IL-21RB) intracellular domain.

[0019] In some of any embodiments, the first dimerization domain is a FKBP12-rapamycin- binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain. In some of any embodiments, the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

[0020] In some of any embodiments, the cell further comprises a cytosolic FRB protein.

[0021] In some embodiments, the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0022] In some of any embodiments, a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108. In some of any embodiments, the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0023] In some of any embodiments, the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111. In some of any embodiments, a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%,4MF-36471223226013200424096%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108. In some of any embodiments, the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

[0024] In some of any embodiments, the FKBP12 dimerization 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: 28. In some of any embodiments, the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28. In some of any embodiments, a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116. In some of any embodiments, the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

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

[0026] 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 NO: 30. In some of any embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NO: 30. In some of any embodiments, the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29. In some of any embodiments, the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

[0027] In some of any embodiments, the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

[0028] 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 NO: 32. In some of any embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32. In some of any embodiments, the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31. In some of any embodiments, the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.5MF-364712232260132004240

[0029] In some of any embodiments, the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151. In some of any embodiments, the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

[0030] In some of any embodiments, the cell further comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some of any embodiments, the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0031] In some aspects, provided herein is a method of transducing a cell comprising contacting a target cell with any of the fusion proteins of any one of preceding embodiments, the polynucleotide constructs of any one of preceding embodiments, or viral vectors of any one of preceding embodiments.

[0032] In some embodiments, the target cell is a stem cell. In some embodiments, the target cell is a progenitor cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0033] In some embodiments, the target cell further comprises a rapamycin-activated cytokine receptor (RACR) system.

[0034] In some embodiments, the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

[0035] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin- 21 receptor subunit P (IL-21RB) intracellular domain.

[0036] In some of any embodiments, the first dimerization domain is a FKBP12-rapamycin- binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size6MF-36471223226013200424012 kD (FKBP12) domain. In some of any embodiments, the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

[0037] In some of any embodiments, the cell further comprises a cytosolic FRB protein.

[0038] In some embodiments, the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0039] In some of any embodiments, a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108. In some of any embodiments, the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0040] In some of any embodiments, the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111. In some of any embodiments, a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108. In some of any embodiments, the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

[0041] In some of any embodiments, the FKBP12 dimerization 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: 28. In some of any embodiments, the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28. In some of any embodiments, a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116. In some of any embodiments, the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

[0042] In some of any embodiments, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).7MF-364712232260132004240

[0043] 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 NO: 30. In some of any embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NO: 30. In some of any embodiments, the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29. In some of any embodiments, the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

[0044] In some of any embodiments, the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

[0045] 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 NO: 32. In some of any embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32. In some of any embodiments, the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31. In some of any embodiments, the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.

[0046] In some of any embodiments, the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151. In some of any embodiments, the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

[0047] In some embodiments, the target cell further comprises a CAR. In some embodiments, the CAR is a CD19-targeted CAR. In some embodiments, the CAR is a CD20- targeted CAR. In some embodiments, the target cell comprises a dual-targeting CAR. In some embodiments, the dual-targeting CAR targets CD19 and CD20.

[0048] In some embodiments, the fusion proteins of any one of preceding embodiments, the fusion proteins of the polynucleotide constructs of any one of preceding embodiments, or the fusion proteins of any one of the viral vectors of any one of preceding embodiments are capable of heterodimerizing with the endogenous subunit of IL-2R.

[0049] In some aspects, provided herein is a method of increasing differentiation efficiency of a cell expressing a rapamycin activated cytokine receptor (RACR) system, the method comprising culturing a cell comprising a RACR system in which also comprises the fusion protein of any one of preceding embodiments, the polynucleotide construct of any one of8MF-364712232260132004240 preceding embodiments, or the viral vectors of any one of preceding embodiments. In some embodiments, the cell is an induced pluripotent stem cell (iPSC).

[0050] In some embodiments, the iPSC is differentiated to an induced cytotoxic innate lymphocyte (iCIL). In some embodiments, the method comprises adding rapamycin to the culture of cells.

[0051] In some embodiments, the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

[0052] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin- 21 receptor subunit P (IL-21RB) intracellular domain.

[0053] In some of any embodiments, the first dimerization domain is a FKBP12-rapamycin- binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain. In some of any embodiments, the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

[0054] In some of any embodiments, the method further comprises a cytosolic FRB protein.

[0055] In some embodiments, the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0056] In some of any embodiments, a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108. In some of any embodiments, the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.9MF-364712232260132004240

[0057] In some of any embodiments, the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111. In some of any embodiments, a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108. In some of any embodiments, the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

[0058] In some of any embodiments, the FKBP12 dimerization 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: 28. In some of any embodiments, the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28. In some of any embodiments, a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116. In some of any embodiments, the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

[0059] In some of any embodiments, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0060] 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 NO: 30. In some of any embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NO: 30. In some of any embodiments, the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29. In some of any embodiments, the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

[0061] In some of any embodiments, the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

[0062] 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 NO: 32. In some of any embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32. In some of any embodiments, the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence10MF-364712232260132004240 set forth in SEQ ID NO: 31. In some of any embodiments, the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.

[0063] In some of any embodiments, the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151. In some of any embodiments, the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

[0064] In some embodiments, the method further comprises a chimeric antigen receptor (CAR). In some of any embodiments, the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

[0065] In some embodiments, the cell comprises an endogenous IL-2R subunit.

[0066] In some aspects, provided herein is a method of differentiating an induced cytotoxic lymphocyte (iCIL) from a cell, the method comprising culturing a cell comprising a RACR system in which also comprises the fusion protein of any one of preceding embodiments, the polynucleotide construct of any one of preceding embodiments, or the viral vectors of any one of preceding embodiments.

[0067] In some embodiments, the cell is an iPSC. In some embodiments, the cell is a hematopoietic progenitor.

[0068] In some embodiments, the cell expresses a rapamycin activated cytokine receptor (RACR) system.

[0069] In some embodiments, the method differentiates the cell iCIL without added IL- 15 or rapamycin. In some of any embodiments, the method differentiates the cell iCIL only with added rapamycin. In some of any embodiments, the method differentiates the cell iCIL without added IL- 15.

[0070] In some of any embodiments, the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

[0071] In some embodiments, wherein the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 211MF-364712232260132004240 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin- 21 receptor subunit P (IL-21RB) intracellular domain.

[0072] In some of any embodiments, the first dimerization domain is a FKBP12-rapamycin- binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain. In some of any embodiments, the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

[0073] In some of any embodiments, the cell further comprises a cytosolic FRB protein.

[0074] In some embodiments, the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0075] In some of any embodiments, a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108. In some of any embodiments, the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

[0076] In some of any embodiments, the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111. In some of any embodiments, a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108. In some of any embodiments, the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

[0077] In some of any embodiments, the FKBP12 dimerization 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: 28. In some of any embodiments, the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28. In some of any embodiments, a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%,12MF-36471223226013200424085%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116. In some of any embodiments, the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

[0078] In some of any embodiments, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).

[0079] 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 NO: 30. In some of any embodiments, the IL2RG comprises the amino acid sequence of SEQ ID NO: 30. In some of any embodiments, the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29. In some of any embodiments, the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

[0080] In some of any embodiments, the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

[0081] 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 NO: 32. In some of any embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32. In some of any embodiments, the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31. In some of any embodiments, the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.

[0082] In some of any embodiments, the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151. In some of any embodiments, the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

[0083] In some of any embodiments, the method further comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some of any embodiments, the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH).

[0084] In some embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).13MF-364712232260132004240

[0085] In some embodiments, the cell comprises an endogenous IL-2R subunit.

[0086] In some aspects, provided herein is a method of targeting cancer cells, wherein the cancer cells are exposed to methods comprising the fusion protein of any one of preceding embodiments, the polynucleotide construct of any one of preceding embodiments, or the viral vector of any one of preceding embodiments.Brief Description of the Drawings

[0087] FIGS. 1A-1C show exemplary schematics of IL- 15 signaling and rapamycin activated cytokine receptor (RACR) signaling. FIG. 1A depicts IL- 15 bound to an IL- 15 receptor, which presents in trans to cells that express IL-2 receptor. FIG. IB depicts an interleukin-15-interleukin-2-receptor (IL-15-IL-2R) synthetic fusion receptor composed of (1) an IL- 15 peptide that is fused to one of the two subunits of an IL-2 receptor, IL-2RG common gamma chain or IL-2RB common beta chain units. In this schematic, as an exemplar, IL- 15 is linked to IL-2RB. FIG. 1C depicts RACR signaling in the presence of rapamycin.

[0088] FIG. 2 shows RACR-engineered hematopoietic progenitor cells (HPs) transduced at varying levels of MOI with the polynucleotide construct encoding IL-15-IL-2-R-beta or IL-15- IL-2-R-gamma. Cells were analyzed by flow cytometry and transduced cells were detected using anti-GS4 linker.

[0089] FIG. 3 shows the total number of cells (HPs) at different points during differentiation (e.g., day of differentiation). HPs transduced with IL-15-IL-2R synthetic fusion receptor were cultured under the following conditions: 1) rapamycin and added IL-15, 2) rapamycin only, or 3) neither rapamycin nor added IL- 15.

[0090] FIGS. 4A-4D show a serial killing assay using iCILs differentiated and expanded under different culturing conditions. iCILs expressing 1) RACR only (e.g., RACR1 only) or 2) RACR and IL-15-IL-2R fusion synthetic receptor (e.g., IL-15-IL-2R-gamma or IL-15-IL-2R- beta) were tested. The tested conditions included: 1) unstimulated, 2) rapamycin only, 3) added IL- 15 only, or 4) rapamycin and added IL- 15 conditions.

[0091] FIG. 5 shows the total number of cells (HPs) at different points during differentiation (e.g., day of differentiation). HPs were engineered with RACR-only and culture supplemented with nothing (no IL- 15 and no rapamycin) or supplemented with IL- 15 and rapamycin, or the RACR- HPs were further engineered with IL-15-IL-2R-beta but not cultured with rapamycin or added IL- 15.14MF-364712232260132004240

[0092] FIG. 6 shows the fold change in expansion based number of cells on day 29 versus day 15 (Day 29 / 15). iCILs containing RACRl-only, RACR2-only, or RACRl-IL-15-IL-2R-beta were expanded with or without added IL- 15.

[0093] FIG. 7 shows the ratio of the number of iCILs at day 29 divided by the number of iPSCs at the start of the differentiation. Data for iCILs containing RACRl-only or RACR1-IL- 15-IL-2R-beta expanded with or without added IL- 15 is shown.

[0094] FIG. 8 shows an in vitro assay that combines Raji tumor cells and iCILs expressing CD20-CAR or CD20-CAR and IL-15-IL-2R-beta. iCILs and tumor cells were combined at a 4:1 ratio, and several stimulatory conditions were tested by adding: 1) unstimulated, 2) rapamycin, or 3) rapamycin and IL- 15 during the serial killing assay.

[0095] FIG. 9 depicts experimental schematic for in vivo functional validation of iCILs expressing an IL-15-IL-2R synthetic fusion receptor. Mice were injected by tail vein (i.v.) with 100,000 Raji tumor cells on day -1, and received iCILs expressing CD20-CAR or iCILs expressing CD20-CAR and IL-15-IL-2R-beta. Rapamycin was administered three times per week, and tumor imaging using luciferase was done on day 5 and day 12.

[0096] FIG. 10 shows measured firefly luciferase (ffLuc) of tumor cells in conditions where mice received rapamycin and 1) CD20-CAR iCILs, 2) dual-CAR iCILs (e.g., CD20 and CD 19, and 3) iCILs expressing CD20-CAR and IL-15-IL-2R-beta. Fluorescence was measured at study day 5 and 12.

[0097] FIGS. 11A-11D show flow cytometry analysis of blood from mice injected with iCILs. iCILs harvested at day 30 or day 40 were tested and the number of iCILs detected at day 15 was quantified for iCILs expressing 1) CD20-CAR or 2) CD20-CAR and IL-15-IL-2R-beta.Detailed Description

[0098] This disclosure relates generally to cells encoding at least a fusion protein of a synthetic receptor and methods for uses thereof. Provided herein are fusion proteins comprising an interleukin- 15 (IL- 15) peptide and an interleukin-2 receptor (IL-2R) subunit. In some aspects, the fusion protein is an IL-15-IL-2R synthetic fusion receptor. The fusion protein may comprise from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL-2 receptor. In some aspects, the fusion proteins are expressed in a cell with a synthetic cytokine receptor complex system. In other aspects, the fusion proteins are expressed in a cell with a chimeric antigen receptor (CAR) system.15MF-364712232260132004240

[0099] Provided herein are also exemplary synthetic fusions, exemplary polynucleotide constructs and vectors, such as viral vectors comprising same. Further, provided herein are engineered cells for use with an IL-15-IL-2R synthetic fusion receptor and additional receptors, including a synthetic cytokine receptor complex and / or CAR system. Lastly, methods of use, are provided herein.

[0100] Cytokines regulate immune responses and several share a common-gamma-chain among their receptor subunits including interleukin-2 (IL-2) and IL- 15, which have pivotal roles in the control of the life and death of lymphocytes. In addition to the common gamma chain, the heterotrimeric receptors for IL-2 and IL15 share another subunit referred to as IL-2 / IL15RP (also known as IL-2RP, CD122). Furthermore, the interacting forms of IL-2R and IL15R contain a third cytokine-specific receptor a subunit IL15Ra (CD215) (FIG. 1A). IL-2 and IL15 signaling pathways also share JAK1 (Janus Kinase 1), JAK3 and STAT3 / 5 (signal transducer and activator of transcription 3 and 5) molecules and both cytokines stimulate the proliferation of T cells, induce the generation of cytotoxic T lymphocytes (CTL), and facilitate the maintenance of natural killer (NK) cells.

[0101] Interleukin-2 receptor (IL-2R) is a composed of up to three different subunit complexes, the a- (CD25), P- (CD122) and y-chains (CD132), where the latter is expressed constitutively and the former two are inducible. The IL-2P and y subunits compose the intermediate-affinity IL-2 receptor, while the IL-2Ra alone represents the low-affinity receptor, and expression of all three chains has the highest binding affinity to IL-2, which is predominantly a secreted cytokine that binds to preformed high-affinity heterotrimeric receptors. Endogenously, IL-2R also binds IL-15, which is a membrane- associated molecule that signals at an immunological synapse between antigen-presenting cells and CD8 T cells or NK cells. IL- 15Ra on the surface of activated monocytes or dendritic cells presents IL 15 in trans to cells that express IL-2 / IL-15RP and yc, thereby allowing signaling through these complexes (FIG. 1A).

[0102] The binding of IL-2 or IL- 15 to the IL-2 / IL-15RP and yc heterodimer induces JAK1 activation via the P chain and JAK3 via the y chain that together phosphorylate tyrosine on the cytokine receptors and induce the tyrosine phosphorylation of STAT3, STAT5A and STAT5B that via SH2 domain interactions homodimerize, translocate to the nucleus and bind to regulatory regions of target genes. Additional IL-2 and IL- 15 signaling mechanisms include the adaptor protein She that binds to a pho sphotyro sine residue on IL-2 / IL15RP resulting in the activation of Grb2 and Akt via the She, Grb2, Gab2, PI3K, PIP3, Akt, mTOR, p70, S6 signaling16MF-364712232260132004240 pathway. In a third signaling pathway IL-2 / IL15 signaling is associated with activation of SOS and Grb2 to form a Grb2 / SOS complex that in turn activates the Ras, Raf, MEK, MAPK ERK pathway involved in cellular proliferation. Collectively these signaling pathways induce the expression and activation of c-myc, c-fos, c-jun, Bcl-2, Bcl-xL and NF-kB as well as decrease expression of proapoptotic Bim and PUMA.

[0103] Cytokine receptors also drive cellular differentiation and the process of deriving differentiated cells from stem cells (e.g., induced pluripotent stem cells (iPSCs)) requires addition of many different exogenous growth factors at different times during differentiation. Common gamma chain cytokines are some of the most important cytokines used in deriving innate lymphocytes (ILCs, NKs) or T cells and require multi-step addition of these cytokines during cell production. These additions increase manufacturing complexity, cost, and variability when producing cell therapies, especially for use in hematologic and solid tumors.

[0104] This disclosure provides compositions and methods of use of fusion proteins comprising an interleukin- 15 (IL- 15) peptide and an interleukin-2 receptor (IL-2R) subunit. In some embodiments, the fusion protein is an IL-15-IL-2R synthetic fusion receptor that presents IL- 15 to IL-2R in cis. In some embodiments, the fusion protein is an IL-15-IL-2R synthetic fusion receptor that presents IL- 15 to IL-2R in trans. The fusion protein may comprise from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL-2 receptor. Introducing IL- 15 in this manner improves IL- 15 stimulation and lowers or removes the need for use of recombinant IL- 15. As further described below, improved IL- 15 signaling improves proliferation and cytotoxicity of manufactured cells and may be used in conjunction with other differentiation methods.

[0105] Further, IL-15-IL-2R synthetic fusion receptor may be expressed in cells with a synthetic cytokine receptor complex system, such as rapamycin activated cytokine receptor (RACR) complex. RACR also provides a cytokine-free manufacturing system for cell differentiation. IL-15-IL-2R synthetic fusion receptor may also be expressed in cells with a chimeric antigen receptor (CAR) system, enhancing anti-tumor activity.

[0106] 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 herein17MF-364712232260132004240 incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0107] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. IL-15-IL-2R SYNTHETIC FUSION RECEPTOR

[0108] Provided herein are fusion proteins comprising an interleukin- 15 (IL- 15) peptide and an interleukin-2 receptor (IL-2R) subunit. In some embodiments, the fusion protein is an IL- 15- IL-2R synthetic fusion receptor. In some embodiments, the fusion protein comprises from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL-2 receptor. In some embodiments, the subunit of the IL-2 receptor is an IL-2 receptor beta chain. In some embodiments, the linker is a peptide linker. In some embodiments, the subunit of the IL-2 receptor is an IL-2 receptor gamma chain. In some embodiments, the fusion protein is an IL-15- IL-2R synthetic fusion receptor comprising an IL- 15 peptide, a peptide linker, and an IL-2 receptor beta chain. In some embodiments, the fusion protein is an IL-15-IL-2R synthetic fusion receptor comprising an IL- 15 peptide, a peptide linker, and an IL-2 receptor gamma chain. The IL-15-IL-2R synthetic fusion can heterodimerize with the other IL-2R subunit not fused to an IL- 15, which may be endogenous.

[0109] The IL-15-IL-2R synthetic fusion receptors provided herein present IL-15 by linking a membrane-bound IL- 15 to IL-2R (FIG. IB). In endogenous conditions, IL-2R binds IL- 15, which is a membrane-associated molecule that signals at an immunological synapse between antigen-presenting cells and CD8 T cells or NK cells. IL-15Ra on the surface of activated monocytes or dendritic cells presents IL- 15 in trans to cells that express IL-2 / IL-15RP and yc, thereby allowing signaling through these complexes. By linking IL- 15 to IL-2R using IL-15-IL- 2R synthetic fusion receptors, there is improved IL- 15 stimulation and a lowered need for recombinant IL- 15.A. Receptor Components

[0110] IL-2 receptor (IL-2R) is a composed of up to three different subunit complexes, the a- (CD25), P- (CD122) and y-chains (CD132), where the latter is expressed constitutively and the former two are inducible producing three different IL-2R complex combinations. The isolated IL-2R-alpha(a) subunit that is transiently expressed following T-cell receptor (TCR) activation or by contact of IL-2 with the other subunits binds IL-2 with low affinity without18MF-364712232260132004240 transducing a signal. The heterodimeric IL-2R-beta(P)-gamma (y) complex bind IL-2 with intermediate affinity, while the heterotrimeric IL-2R-aPy bind IL-2 with high affinity. Both the heterodimeric and heterotrimeric receptors signal. IL-2 is predominantly a secreted cytokine that binds to preformed high-affinity heterotrimeric receptors. Furthermore, IL-2 and IL- 15 share the common yc and IL-2 / IL-15RP chains and bind IL- 15. In contrast to IL-2, IL- 15 is a membrane- associated molecule that signals at an immunological synapse between antigen-presenting cells and CD8 T cells or NK cells. IL-15Ra on the surface of activated monocytes or dendritic cells presents IL- 15 in trans to cells that express IL-2 / IL-15RP and gamma chain, thereby allowing signaling through these complexes.

[0111] The IL-15-IL-2R synthetic fusion receptors provided herein present IL-15 by linking a membrane-bound IL- 15 to IL-2R (FIG. IB). The IL-15-IL-2R synthetic fusion receptor is composed of (1) an IL- 15 peptide that is fused to one of the two subunits of an IL-2 receptor, IL- 2RG common gamma chain or IL-2RB common beta chain units. In some embodiments, the IL- 15-IL-2R synthetic fusion receptor is composed of (1) an IL-15 peptide that is fused to IL-2RG common gamma chain. In some embodiments, the IL-15-IL-2R synthetic fusion receptor is composed of (1) an IL- 15 peptide that is fused to IL-2RB common beta chain. The IL-15-IL-2R synthetic fusion can heterodimerize with the other IL-2R subunit not fused to an IL- 15, which may be endogenous.

[0112] Exemplary IL-2 receptor subunits that may be linked with an IL- 15 peptide are described below. Exemplary IL- 15 peptide and linkers are also described.1. IL-2 Receptor Beta Subunit

[0113] Interleukin-2 receptor beta (IL-2R-P), also known as CD122, IMD63, IL15RB, and P70-75, is one of the three receptors that can form the IL-2R complex. IL-2R- is an 11-exon gene encoding a beta subunit and a type I membrane protein primarily expressed in the hematopoietic system. The intermediate affinity form of IL-2R consists of an alpha / beta subunit heterodimer, while the high affinity form of IL-2R consists of an alpha / beta / gamma subunit hetero trimer. Both the intermediate and high affinity forms of the receptor are involved in receptor-mediated endocytosis and transduction of mitogenic signals from interleukin 2. Further, IL-2R-P is involved in signaling of IL- 15 via IL-2R.19MF-364712232260132004240

[0114] In some embodiments, the fusion protein comprises an IL- 15 linked to IL-2R-P. In some embodiments, the fusion protein comprises an IL-15 linked to IL-2R-gamma (y) that fuses with an endogenous IL-2R-0 subunit.

[0115] In some embodiments, IL-2R-P is encoded by an amino acid sequence with at least 75% sequence identity to the amino acid sequence of accession number: AAM54040. In some embodiments, IL-2R-P is encoded by an amino acid sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of accession number: AAM54040.

[0116] In some embodiments, IL-2R-P is encoded by an amino acid sequence of about 280 to 320 amino acids. In some embodiments, IL-2R-P is encoded by an amino acid sequence of 318 amino acids. In some embodiments, the IL-2 receptor beta chain 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: 21. In some embodiments, the IL-2 receptor beta chain comprises an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-2 receptor beta chain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-2 receptor beta chain is the amino acid sequence of SEQ ID NO: 21.

[0117] In some embodiments, IL-2R-P is encoded by a nucleotide sequence with at least 75% sequence identity to a nucleotide sequence with a genomic location NC_000022.l l (37125838..37175118, complement) based on the human GRCh38.pl4 assembly. In some embodiments, IL-2R-P is encoded by a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence with a genomic location NC_000022.l l (37125838..37175118, complement) based on the human GRCh38.pl4 assembly. In some embodiments, IL-2R-P is encoded by a nucleotide sequence with at least 75% sequence identity to a nucleotide sequence found in gene ID: 3560 of NCBI database. In some embodiments, IL-2R-P is encoded by a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,20MF-36471223226013200424096%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence found in gene ID: 3560 of NCBI database.

[0118] In some embodiments, IL-2R-P is encoded by a nucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, IL-2R-p is encoded by a nucleotide sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the IL-2 receptor beta chain comprises the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the IL-2 receptor beta chain is encoded by the nucleotide sequence of SEQ ID NO: 10.2. IL-2 Receptor Gamma Subunit

[0119] Interleukin-2 receptor gamma (IL-2R-y), also known as P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, and SCIDX1, is one of the three receptors that can form the IL-2R complex. IL-2R-y is an 8-exon gene encoding a gamma subunit and integral membrane protein common to the receptor complexes for at least six different IL receptors: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptor. Further, IL-2R- y is involved in signaling of IL- 15 via IL-2R.

[0120] In some embodiments, the fusion protein comprises an IL- 15 linked to IL-2R-y. In some embodiments, the fusion protein comprises an IL-15 linked to IL-2R- p that fuses with an endogenous IL-2R- y subunit.

[0121] In some embodiments, IL-2R-gamma is encoded by an amino acid sequence with at least 75% sequence identity to the amino acid sequence of accession number: AAA59145.1. In some embodiments, IL-2R-gamma is encoded by an amino acid sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of accession number: AAA59145.1.

[0122] In some embodiments, IL-2R-gamma is encoded by an amino acid sequence of about 100 to 130 amino acids. In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence of 123 amino acids. In some embodiments, the IL-2R-gamma chain 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: 16. In some embodiments, the IL-2R-gamma chain comprises an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,21MF-36471223226013200424079%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-2R-gamma chain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-2R-gamma chain is the amino acid sequence of SEQ ID NO: 16.

[0123] In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence with at least 75% sequence identity to a nucleotide sequence with a genomic location NC_000023.l l (71107404..71111577, complement) based on the human GRCh38.pl4 assembly. In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence with a genomic location NC_000023.l l (71107404..71111577, complement) based on the human GRCh38.pl4 assembly. In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence with at least 75% sequence identity to a nucleotide sequence found in gene ID: 3561 of NCBI database. In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence found in gene ID: 3561 of NCBI database.

[0124] In some embodiments, IL-2R-gamma is encoded by a nucleotide sequence 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, IL-2R-gamma is encoded by a nucleotide sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the IL-2R-gamma chain comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the IL-2R- gamma chain is encoded by the nucleotide sequence of SEQ ID NO: 4.3. IL-15 peptide

[0125] The IL-15-IL-2R synthetic fusion receptors provided herein present IL-15 by linking an IL-15 peptide to IL-2R (FIG. IB). Mature human IL-15 is a 14-15 kDa glycoprotein and a member of the four a-helix bundle family of cytokines and alternative splicing results in the22MF-364712232260132004240 production of two mature isoforms of the IL- 15 protein that differ only in the length of their signal peptides. Despite these differences both isoforms (e.g., human and mouse) produce a 114- amino acids containing IL- 15 protein. IL- 15 with the long signal peptide follows the secretory pathway leading to the secretion of IL- 15, whereas IL- 15 with the short signal peptide is not secreted, but is stored intracellularly in the cytoplasm. In contrast to other cytokines, IL- 15 also has an unusual mechanism of transport to the cell surface. While the high affinity IL- 15 binding protein, IL-15 receptor a (IL-15Ra) is not necessary for IL-15 to translocate into the endoplasmic reticulum, IL- 15 is transported through the Golgi apparatus to the cell surface as a complex bound to IL-15Ra. The IL- 15 complexed to IL-15Ra is then presented by membrane bound IL-15Ra in trans by a mechanism known as trans-presentation, to cells that express the intermediate affinity P (CD122) and y (CD132) signaling chains, such as IL-2R-beta-gamma complex.

[0126] IL- 15 is an inflammatory cytokine with structural similarity to IL-2 and induces the proliferation of natural killer cells. IL- 15 is also critical for the maintenance of long-lasting, high-avidity T-cell responses to invading pathogens, a function that it achieves by supporting the survival of CD8 memory T cells.

[0127] Various IL- 15 peptide for use in the provided fusion proteins are known in the art (see, e.g., US Patent No. US20190263877; Hu, Q., Ye, X., Qu, X. et al. Discovery of a novel IL- 15 based protein with improved developability and efficacy for cancer immunotherapy. Sci Rep 8, 7675 (2018). doi.org / 10.1038 / s41598-018-25987-4; Zhao, L„ Hu, B„ Zhang, Y. et al. An activation-induced IL- 15 isoform is a natural antagonist for IL- 15 function. Sci Rep 6, 25822 (2016). doi.org / 10.1038 / srep25822; and US Patent No. US11673932, the entire contents of each of which are incorporated by reference herein).

[0128] In some embodiments, the IL- 15 peptide provided herein is encoded by an amino acid sequence of about 100 to 120 amino acids. In some embodiments, IL-15 peptide provided herein is encoded by an amino acid sequence of about 100, 101, 102, 103, 104, 105, 106, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 amino acids. In some embodiments, fusion protein provided herein is encoded by an amino acid sequence of 114 amino acids.

[0129] In some aspects, the disclosure herein provides for an IL-15 peptide to be linked with the IL-2R subunit for in cis presentation. In some aspects, the disclosure herein provides for an IL- 15 peptide to be linked with the IL-2R subunit for in trans presentation. In some23MF-364712232260132004240 embodiments, IL- 15 is a native or wild-type IL- 15 protein. In some embodiments, IL- 15 is an IL- 15 variant or mutant. In some embodiments, the IL- 15 variant has a different amino acid sequence than the native or wild type IL- 15 protein. In some embodiments, the IL- 15 variant or IL- 15 mutant binds the IL-2R and functions as an IL- 15 agonist. In some embodiments, the IL- 15 variant or IL- 15 mutant binds the IL-2R and functions as an IL- 15 antagonist. In some embodiments, IL- 15 peptide of the disclosure suitably corresponds to the amino acid sequence naturally occurring in IL- 15 molecules, e.g. IL- 15 molecules of a human, mouse or other rodent, or other mammal.

[0130] In some embodiments, the IL-15 peptide forming part of the IL-15-IL-2R synthetic fusion receptor comprises an interleukin- 15 (IL-15) domain. In some embodiments, the IL-15 peptide is a variant with increased agonist activity. In some embodiments, the IL- 15 peptide is a variant with antagonist activity. IL- 15 agonists are exemplified by comparable or increased biological activity compared to wild type IL- 15. IL- 15 antagonists are exemplified by decreased biological activity compared to wild type IL- 15 or by the ability to inhibit IL-15-mediated responses. In some embodiments, the IL- 15 variant binds with increased or decreased activity to the IL-2RPyC receptors.

[0131] In some embodiments, the sequence of the IL- 15 peptide has at least one amino acid change, e.g. substitution or deletion, compared to the native IL-2 sequence, such changes resulting in IL- 15 agonist or antagonist activity. In some embodiments, the amino acid substitutions / deletions are in the domains of IL- 15 that interact with IL-15RP and / or yC. In some embodiments, the amino acid substitutions / deletions do not affect binding to the IL-2R or the ability to produce the IL- 15 variant. Suitable amino acid substitutions / deletions to generate IL- 15 variants can be identified based on putative or known IL- 15 structures, comparisons of IL- 15 with homologous molecules such as IL-2 with known structure, through rational or random mutagenesis and functional assays, or other empirical methods known in the art. Additionally suitable amino acid substitutions can be conservative or non-conservative changes and insertions of additional amino acids. In some embodiments, IL- 15 variants of the disclosure contain one or more than one amino acid substitutions / deletions at position 8, 61, 65, 72, 92, 101, 108, or 111 of the mature human IL- 15 sequence. In some embodiments, the IL- 15 variants comprise an amino acid mutation at D8N (“D8” refers to the amino acid and residue position in the native mature human IL- 15 sequence and “N” refers to the substituted amino acid residue at that position in the IL-15 variant), D8A, D61A, N65A, N72R or Q108A substitutions result in IL-1524MF-364712232260132004240 variants with antagonist activity and N72D substitutions result in IL- 15 variants with agonist activity.

[0132] In some embodiments, the IL- 15 peptide 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: 14. In some embodiments, the IL-15 peptide comprises an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the IL- 15 peptide comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the IL- 15 peptide is encoded by the amino acid sequence of SEQ ID NO: 14.

[0133] In some embodiments, the IL- 15 peptide comprises a nucleotide sequence encoding the IL- 15 peptide 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 IL- 15 peptide comprises a nucleotide sequence encoding the IL-15 peptide is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the IL-15 peptide comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the IL- 15 peptide is the nucleotide sequence of SEQ ID NO: 2.4. Linkers

[0134] The IL-15-IL-2R synthetic fusion receptors provided herein link IL-15 to IL-2R subunit. The fusion proteins, polypeptide constructs, and polynucleotide constructs contain a linker that joins or couples the IL-15 peptide to the IL-2R subunit (e.g., beta or gamma). In some embodiments, the linker is positioned at the end of the C-terminal region of the IL- 15 peptide, such that the IL-15 is N-terminal to the IL-2R-subunit. In some embodiments, the fusion protein comprises from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL- 2 receptor.

[0135] Various polypeptide linkers for use in fusion proteins are known in the art (see e.g., Chen et al. (2013) Adv. Drug. Deliv. 65:1357-1369; and International PCT publication No. WO 2014 / 099997, W02000 / 24884; U.S. Pat. No. 5,258,498; U.S. Pat. No. 5,525,491; U.S. Pat. No.25MF-3647122322601320042405,525,491, U.S. Pat. No. 6,132,992, which are each incorporated herein by reference in their entireties).

[0136] Typically, the linker also is one that ensures correct folding of the polypeptide construct, does not exhibit a charge that would be inconsistent with the activity or function of the linked polypeptides or form bonds or other interactions with amino acid residues in one or more of the domains that would impede or alter activity of the linked polypeptides. In some embodiments, the linker is a peptide linker. The linker can be a flexible linker or a rigid linker or a combination of both. In some aspects, the linker is a short, medium, or long linker. In some embodiments, the linker is up to 40 amino acids in length. In some embodiments, the linker is between four to 30 residues. In some embodiments, the linker is up to 25 amino acids in length. In some embodiments, the linker is at least or is at least about 2 amino acids in length. In some aspects, a suitable length is, e.g., a length of at least one and typically fewer than about 40 amino acid residues, such as 2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, 8-12 amino acid. In some embodiments, the linker is from or from about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acids or 20 to 24 amino acids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

[0137] In certain aspects, the longer the linker length, the greater the IL- 15 binding to IL-2R subunit. Thus, in some aspects, the linker is greater than 12 amino acids in length, such as greater than 13, 14, 15, 16, 17 or 18 amino acids in length. In some embodiments, the linker is 12 to 40 amino acids in length, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30 amino acids or 30 to 40 amino acids.26MF-364712232260132004240

[0138] The linkers can be naturally occurring, synthetic or a combination of both.Particularly suitable linker polypeptides predominantly include amino acid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala), and Threonine (Thr). For example, the linker may contain at least 75% (calculated on the basis of the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala and / or Thr residues only. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, a peptide linker comprises glycine residues only. In some embodiments, a peptide linker comprises glycine and serine residues only.

[0139] In some embodiments, these linkers are composed predominately of the amino acids Glycine and Serine, denoted as GS-linkers herein. In some embodiments, the linker contains (GGS)n, wherein n is 1 to 10, such as 1 to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO: 112), wherein n is 0 to 10. In particular embodiments, the linker contains the sequence (GGGGS)n (SEQ ID NO: 65), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3. In further embodiments, the linker contains (GGGGGS)n (SEQ ID NO: 66), wherein n is 1 to 4, such as 1 to 3. The linker can include combinations of any of the above, such as repeats of 2, 3, 4, or 5 GS, GGS, GGGGS (SEQ ID NO: 8), and / or GGGGGS (SEQ ID NO: 19) linkers may be combined. In some embodiments, such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids in length.

[0140] In some embodiments, the linker is (in one-letter amino acid code): GGS, GGGGS (SEQ ID NO: 8), or GGGGGS (SEQ ID NO: 19). In some embodiments, the GS-linker comprises an amino acid sequence of GGSGGS, i.e., (GGS)2 (SEQ ID NO: 67); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 68); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 69); GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 70); GGGGGSGGGGGSGGGGGS, i.e., (G5S)3(SEQ ID NO: 71), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 72) and GGGGSGGGGSGGGGS (SEQ ID NO: 73). In some embodiments, the linker is GGGGG (SEQ ID NO: 74). In some embodiments, the linker is PGGGG (SEQ ID NO: 75). In some embodiments, the linker is GGGG (SEQ ID NO: 76). In some of any of the above examples, serine can be replaced with alanine (e.g., (Gly 4 Ala) or (Gly3Ala)). In some embodiments, the27MF-364712232260132004240 linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 15).

[0141] In some embodiments, the linker includes a peptide linker having the amino acid sequence GlyxXaa-Glyy-Xaa-Glyz(SEQ ID NO: 77), wherein each Xaa is independently selected from Alanine (Ala), Valine (Vai), Leucine (Leu), Isoleucine (He), Methionine (Met), Phenylalanine (Phe), Tryptophan (Trp), Proline (Pro), Glycine (Gly), Serine (Ser), Threonine (Thr), Cysteine (Cys), Tyrosine (Tyr), Asparagine (Asn), Glutamine (Gin), Lysine (Lys), Arginine (Arg), Histidine (His), Aspartate (Asp), and Glutamate (Glu), and wherein x, y, and z are each integers in the range from 1-5. In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala, and Thr. In a specific variation, each of x, y, and z is equal to 3 (thereby yielding a peptide linker having the amino acid sequence Gly-Gly-Gly- Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO: 78), wherein each Xaa is selected as above.

[0142] In some embodiments, the linker is serine-rich linkers based on the repetition of a (SSSSG)n (SEQ ID NO: 79) motif where n is at least 1, though n can be 2, 3, 4, 5, 6, 7, 8 and 9.

[0143] In some cases, it may be desirable to provide some rigidity into the peptide linker. This may be accomplished by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, a linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, a peptide linker can have an amino acid sequence wherein at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In one particular embodiment, the peptide linker comprises proline residues only.

[0144] In some aspects, a peptide linker comprises at least one cysteine residue, such as one cysteine residue. For example, in some embodiments, a linker comprises at least one cysteine residue and amino acid residues selected from the group consisting of Gly, Ser, Ala, and Thr. In some such embodiments, a linker comprises glycine residues and cysteine residues, such as glycine residues and cysteine residues only. Typically, only one cysteine residue will be included per peptide linker. One example of a specific linker comprising a cysteine residue includes a peptide linker having the amino acid sequence Glym-Cys-Glyn, wherein n and m are each integers from 1-12, e.g., from 3-9, from 4-8, or from 4-7. In a specific variation, such a peptide linker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO: 80).

[0145] In some embodiments, the linker of the fusion protein is a structured or constrained linker. In particular embodiments, the structured linker contains the sequence (AP)n or28MF-364712232260132004240(EAAAK)n (SEQ ID NO: 81), wherein n is 2 to 20, preferably 4 to 10, including but not limited to, AS-(AP)n-GT (SEQ ID NO: 82) or AS-(EAAAK)n-GT (SEQ ID NO: 83), wherein n is 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments, the linker comprises the sequences (GGGGA)n (SEQ ID NO: 84), (PGGGS)n (SEQ ID NO: 85), (AGGGS)n (SEQ ID NO: 86) or GGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 87, wherein n is 2 to 20), (ADAAP)n (SEQ ID NO: 88, wherein n is 2 to 20), (ADAAP)n-G (SEQ ID NO: 89, wherein n is 2 to 20), (GEPQG)n (SEQ ID NO: 90, wherein n is 2 to 20), (GEPQG)n-G (SEQ ID NO: 91, wherein n is 2 to 20), (AGGEP)n (SEQ ID NO: 92, wherein n is 2 to 20), (AGGEP)n-G (SEQ ID NO: 93, wherein n is 2 to 20), (AGSEP)n (SEQ ID NO: 94, wherein n is 2 to 20), (AGSEP)n-G (SEQ ID NO: 95, wherein n is 2 to 20), (GGGEQ)n (SEQ ID NO: 96, wherein n is 2 to 20), (GGGEQ)n-G (SEQ ID NO: 97, wherein n is 2 to 20). In some embodiments, the linker is SSSASASSA (SEQ ID NO: 98), GSPGSPG (SEQ ID NO: 99), ATTTGSSPGPT (SEQ ID NO: 100), ADAAPADAAPG (SEQ ID NO: 101), GEPQGGEPQGG (SEQ ID NO: 102), AGGEPAGGEPG (SEQ ID NO: 103), AGSEPAGSEPG (SEQ ID NO: 104), or GGGEQGGGEQG (SEQ ID NO: 105).B. Exemplary Synthetic Fusion Protein

[0146] The synthetic fusion proteins of the present disclosure comprise a synthetic IL- 15 peptide and a synthetic IL-2-receptor subunit, linked by a linker. In some aspects, the synthetic fusion proteins encoding an interleukin-15-interleukin-2-receptor (IL-15-IL-2R) synthetic fusion receptor are composed of (1) an IL- 15 peptide that is fused to one of the two subunits of an IL-2 receptor, IL-2RG common gamma chain or IL-2RB common beta chain units. In some embodiments, the IL-15-IL-2R synthetic fusion receptor comprises IL-15 linked to IL-2R beta subunit, also referred to as IL-15-IL-2R-beta. In some embodiments, the IL-15-IL-2R synthetic fusion receptor comprises IL- 15 linked to IL-2R gamma subunit, also referred to as IL-15-IL- 2R-gamma. In any of the embodiments, the IL-15-IL-2R synthetic fusion can heterodimerize with the other IL-2R subunit not fused to an IL- 15, which may be endogenous.

[0147] In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-beta is encoded by the amino acid set forth in SEQ ID NO: 22. In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-beta comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, an amino acid sequence encoding the synthetic fusion protein encoding IL-15-IL-2-R-beta comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,29MF-36471223226013200424078%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 22.

[0148] In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-beta is encoded by the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-beta comprises the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, a nucleotide sequence encoding the synthetic fusion protein encoding IL-15-IL-2-R-beta comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 12.

[0149] In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-gamma is encoded by the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-gamma comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, an amino acid sequence encoding the synthetic fusion protein encoding IL-15-IL-2-R-gamma comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 17.

[0150] In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-gamma is encoded by the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, synthetic fusion protein encoding IL-15-IL-2-R-gamma comprises the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, a nucleotide sequence encoding the synthetic fusion protein encoding IL-15-IL-2-R-gamma comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6.

[0151] In any of the embodiments herein, the synthetic fusion protein may include additional elements, such as a signal peptide. Signal peptide may be included to ensure proper export of the fusion protein to the cells surface. In some embodiments, the signal peptide may be heterologous to the components in the IL-15-IL-2R synthetic fusion. In some embodiments, the signal peptide may be endogenous to the components in the IL-15-IL-2R synthetic fusion. In some aspects, the30MF-364712232260132004240 disclosure herein relates to fusion proteins that comprise signal peptides for improving cell surface expression of the encoded polypeptides. In some embodiments, improving cell surface expression improves engineered cell functionality.

[0152] In some embodiments, the synthetic fusion proteins provided herein comprise the features set forth in Table 1 or Table 2.C. Polynucleotide and Vectors

[0153] Nucleic acid molecules comprising polynucleotides that encode any of the provided IL-15-IL-2R synthetic fusion receptors are provided herein. In some embodiments, the provided nucleic acid sequences and (e.g., DNA sequences) encode fusion proteins as provided herein. In31MF-364712232260132004240 some embodiments, the nucleic acid sequences encoded by a polynucleotide construct are codon optimized.

[0154] 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, cDNA, 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. Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art.

[0155] The polynucleotide constructs also included elements necessary for transcription and translation of the encoded proteins (e.g., protein-coding sequence), such as a promoter to signal transcription and a polyA element for proper mRNA processing. In some embodiments, the nucleic acid sequence comprises a promoter and / or translation initiation signal operably linked to the sequence encoding the fusion protein. In another embodiment, any of the nucleic acid sequences as described above are contained in a DNA vector.

[0156] In one embodiment of the above described aspects, the disclosure features a nucleic acid sequence encoding the IL- 15 peptide (e.g., wild type or variant) of any of the aspects or embodiments as described herein. In one embodiment of the above described aspects, the disclosure features a nucleic acid sequence encoding the linker (e.g., linker peptide) of any of the aspects or embodiments as described herein. In one embodiment of the above described aspects, the disclosure features a nucleic acid sequence encoding the IL-2R-subunit (e.g., beta or gamma) of any of the aspects or embodiments as described herein. In one embodiment of the above described aspects, the disclosure features a nucleic acid sequence encoding the IL-15-IL- 2R-beta fusion protein of any of the aspects or embodiments as described herein. In one embodiment of the above described aspects, the disclosure features a nucleic acid sequence encoding the IL-15-IL-2R-gamma fusion protein of any of the aspects or embodiments as described herein.32MF-364712232260132004240

[0157] In some embodiments, the polynucleotide constructs encode one of the polypeptide chains of the IL-2 receptor linked to IL- 15 via a peptide linker. In some embodiments, the linker nucleotide sequence encoded is set forth in SEQ ID NO: 3. In some embodiments, the exemplary IL- 15 nucleotide sequence encoded is set forth in SEQ ID NO: 2. In some embodiments, the polynucleotide constructs encode a nucleotide sequence as set forth in Section I.A.l and I.A.2.

[0158] In some embodiments, the polynucleotide construct encodes a fusion protein containing in N- to C- terminal order: the IL- 15 peptide, a peptide linker, and one of the two IL- 2-R subunits. In some embodiments, the polynucleotide construct encodes a fusion protein containing in N- to C- terminal order: the IL- 15 peptide, a peptide linker, and IL-2-R beta subunit. In some embodiments, the polynucleotide construct encodes a fusion protein containing in N- to C- terminal order: the IL-15 peptide, a peptide linker, and IL-2-R gamma subunit. In some embodiments, a polynucleotide construct encoding nucleotide sequences and amino acid sequences of a synthetic fusion receptor IL-15-IL-2R-gamma are set forth in Table 1. In some embodiments, a polynucleotide construct encoding nucleotide sequences and amino acid sequences of a synthetic fusion receptor IL-15-IL-2R-beta are set forth in Table 2.

[0159] Non-limiting examples of vector systems of the present disclosure include a retrovirus, a lentivirus, a foamy virus, and a Sleeping Beauty transposon. Vectors comprising nucleic acids that encode the IL-15-IL-2R fusion proteins described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell. In some embodiments, a vector is selected that is optimized for expression of polypeptides in a desired cell type, such as iPSC or hematopoietic (HP) cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0160] In some embodiments, vectors encode a gene of interest (GOI). In some embodiments, the gene of interest (GOI) is the nucleotide sequence encoding the IL-15-IL-2R subunit (e.g., IL-2R subunit linked with IL- 15 peptide).

[0161] In some embodiments, the GOI is the nucleotide sequence encoding the IL-15-IL- 2R-beta sequence as set forth in SEQ ID NO: 12. In some embodiments, the GOI is the nucleotide sequence encoding the IL-15-IL-2R-beta sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 12. In some33MF-364712232260132004240 embodiments, the gene of interest is the nucleotide sequence encoding the amino acid sequence of IL-15-IL-2R-beta as set forth in SEQ ID NO: 22. In some embodiments, the gene of interest is the nucleotide sequence encoding the amino acid sequence of IL-15-IL-2R-beta at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 22.

[0162] In some embodiments, the GOI is the nucleotide sequence encoding the IL-15-IL- 2R-gamma sequence as set forth in SEQ ID NO: 6. In some embodiments, the GOI is the nucleotide sequence encoding the IL-15-IL-2R-gamma sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the gene of interest is the nucleotide sequence encoding the amino acid sequence of IL-15-IL-2R-gamma as set forth in SEQ ID NO: 17. In some embodiments, the gene of interest is the nucleotide sequence encoding the amino acid sequence of IL-15-IL-2R-gamma at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 17.I. Viral Vectors

[0163] In some embodiments, the provided nucleic acid sequences (e.g., DNA sequences) encoding IL-15-IL-2R synthetic fusion proteins can be inserted into a viral vector. In some embodiments, the viral vectors are retroviruses. Retroviruses include lentiviruses, gammaretroviruses, 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.

[0164] 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 essential34MF-364712232260132004240 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.

[0165] 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 one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. For safety reasons, transfer plasmids are generally designed to make the resulting vector replication incompetent. For example, the transfer plasmid lacks gene elements necessary for generation of infective particles in the host cell. In addition, the transfer plasmid may be designed with a deletion of the 3' LTR, rendering the virus “selfinactivating” (SIN). See Dull et al. (1998) J. Virol. 72:8463-71; Miyoshi et al. (1998) J. Virol. 72:8150-57. The viral particle may also comprise a 3' untranslated region (UTR) and a 5' UTR. The UTRs comprise retroviral regulatory elements that support packaging, reverse transcription, and integration of a proviral genome into a cell following contact of the cell by the retroviral particle.

[0166] Third-generation systems also generally include two “packaging plasmids” and an “envelope plasmid.” The “envelope plasmid” generally encodes an Env gene operatively linked to a promoter. In an illustrative third-generation system, the Env gene is VSV-G and the promoter is the CMV promoter. The third- generation system uses two packaging plasmids, one encoding gag and pol and the other encoding rev as a further safety feature; an improvement over the single packaging plasmid of so-called second-generation systems. Although safer, the third-generation system can be more cumbersome to use and result in lower viral titers due to the addition of an additional plasmid. Illustrative packing plasmids include, without limitation, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI (e.g., exemplary GOIs are described above).

[0167] Many retroviral vector systems rely on the use of a “packaging cell line.” In general, the packaging cell line is a cell line whose cells are capable of producing infectious retroviral particles when the transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cells. Various methods of introducing the plasmids into the cells may be used, including transfection or electroporation. In some cases, a packaging cell line is adapted for high- efficiency packaging of a retroviral vector system into retroviral particles.35MF-364712232260132004240

[0168] As used herein, the terms “retroviral vector” or “lentiviral vector” is intended to mean a nucleic acid that encodes a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequence to be delivered into the target cell. Retroviral particles and lentiviral particles generally include an RNA genome (derived from the transfer plasmid), a lipid-bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix protein. As used herein, the terms “retroviral particle” and “lentiviral particle” refers a viral particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell. Such characteristics include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing a retroviral or lentiviral virion including one or more of the gag structural polypeptides, containing a retroviral or lentiviral envelope including one or more of the env encoded glycoproteins, containing a genome including one or more retrovirus or lentivirus cis-acting sequences functioning in replication, proviral integration or transcription, containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase or integrase, or containing a genome encoding regulatory activities such as Tat or Rev. The transfer plasmids may comprise a cPPT sequence, as described in U.S. Patent No. 8,093,042.

[0169] The efficiency of the system is an important concern in vector engineering. The efficiency of a retroviral or lentiviral vector system may be assessed in various ways known in the art, including measurement of vector copy number (VCN) or vector genomes (vg) such as by quantitative polymerase chain reaction (qPCR), or titer of the virus in infectious units per milliliter (lU / mL). For example, the titer may be assessed using a functional assay performed on the cultured tumor cell line HT1080 as described in Humbert et al. Development of third- generation Cocal Envelope Producer Cell Lines for Robust Retroviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy 24:1237-1246 (2016). When titer is assessed on a cultured cell line that is continually dividing, no stimulation is required and hence the measured titer is not influenced by surface engineering of the retroviral particle. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. Comparison of retroviral vector titration methods. BMC Biotechnol. 6:34 (2006).

[0170] In some embodiments, provided herein is a viral vector comprising any of the polynucleotide constructs provided herein. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector further comprises one or more surface T cell36MF-364712232260132004240 activating agents. In some embodiments, the one or more surface T cell activating agents comprise CD58, anti-CD3, or CD80.2. Viral Particles

[0171] In some embodiments, the polynucleotide vectors described above are encapsulated in a viral particle. In some embodiments, any of the polynucleotide constructs described herein 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 IL-15-IL-2R synthetic fusion receptor.

[0172] 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.

[0173] In some embodiments, viral particles are used to deliver additional receptors (e.g., in addition to IL-15-IL-2R synthetic fusion receptor). In some embodiments, viral particles encapsulate rapamycin activated cytokine receptor (RACR) system, which is described below in Section ILA. In some embodiments, viral particles encapsulating the RACR system comprise a synthetic cytokine receptor (see, e.g., Section II.A.2). In some embodiments, viral particles encapsulating the RACR system further comprise a cytosolic FRB. In some embodiments, viral particles encapsulating the RACR system are delivered to cells prior to the delivery of viral particles encapsulating the IL-15-IL-2R synthetic fusion receptor. In some embodiments, viral particles encapsulating the RACR system are delivered to cells at the same time as delivering the viral particles encapsulating the IL-15-IL-2R synthetic fusion receptor.

[0174] In some embodiments, viral particles are used to deliver additional receptors such as a CAR to a target cell (see, e.g., Section II.B). In some embodiments, viral particles encapsulate components of a CAR. 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. In some embodiments, viral particles37MF-364712232260132004240 encapsulating the CAR system are delivered to cells prior to the delivery of viral particles encapsulating the IL-15-IL-2R synthetic fusion receptor. In some embodiments, viral particles encapsulating the CAR system are delivered to cells at the same time as delivering the viral particles encapsulating the IL-15-IL-2R synthetic fusion receptor.

[0175] In some embodiments, the virus particle comprises a retroviral particle. In some embodiments, the disclosure provides a method for preparing a viral formulation. 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-MEV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MEV), 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 Eaboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0176] 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.

[0177] A lentiviral particle 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 encoding, for example, a GOI), e.g., polynucleotide constructs provided herein. 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 a38MF-364712232260132004240 vector comprising an NOI which is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome.

[0178] 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.

[0179] In some embodiments, the retroviral particles and / or lentiviral particles of the disclosure comprise a polynucleotide comprising a sequence encoding an IL-15-IL-2R fusion protein. In some embodiments, the retroviral particles and / or lentiviral particles of the disclosure comprise a polynucleotide comprising a sequence encoding an IL-15-IL-2R fusion protein and a sequence encoding a heterologous protein CAR that specifically binds to the target antigens. In some embodiments, a sequence encoding a heterologous protein CAR that specifically binds to the target antigens is operatively linked to a promoter. In some embodiments, a sequence encoding an IL-15-IL-2R fusion protein is operatively linked to a promoter. Illustrative promoters include, without limitation, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and a MND promoter.

[0180] In some embodiments, the retroviral particles comprise transduction enhancers. In some embodiments, the retroviral particles comprise tagging proteins.

[0181] In some embodiments, each of the retroviral particles comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding an IL-15-IL-2R fusion protein, and (iv) a 3' LTR or UTR. In some embodiments, each of the retroviral particles comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding an IL-15-IL-2R fusion protein, (iv) a promoter, (v) a sequence encoding a CAR, and (vi) a 3' LTR or UTR.

[0182] In some embodiments, the retroviral particles comprise a cell surface receptor that binds to a surface marker on a target host cell, allowing host cell transduction. In some embodiments, the cell surface receptor is a T cell surface receptor. The viral vector may comprise a heterologous viral envelope glycoprotein giving a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its variants,39MF-364712232260132004240VSV-G, Gibbon-ape leukaemia virus (GALV), or is the Amphotropic envelope, Measles envelope or baboon retroviral envelope glycoprotein. In some embodiments, the cell-surface receptor is a VSV G protein from the Cocal strain or a functional variant thereof.

[0183] In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, a viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a nipha virus envelope protein, or a cocal virus G protein. In some embodiments, the viral particle comprises a modified VSV G protein that lacks LDLR binding affinity. In some embodiments, these mutations comprise mutations at positions 47 (for example, K47Q) and / or 354 (for example, R354A).

[0184] In some embodiments, the viral envelope protein is a protein from the Cocal strain (Cocal glycoprotein). In some embodiments, the protein is a Cocal envelope protein containing a mutation at position 354 (R354). In some embodiments, the protein is a Cocal envelope protein containing a mutation at position 47 (K47). In some embodiments, the protein is a Cocal envelope variant containing a R354Q mutation. In some embodiments, the protein is a Cocal envelope variant containing a K47Q mutation. In some embodiments, this variant may be referred to as “blinded” Cocal envelope. Illustrative Cocal envelope variants are provided in, e.g., US 2020 / 0216502 Al, which is incorporated herein by reference in its entirety.

[0185] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. While the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0186] In some embodiments, pseudotyping a fusion glycoprotein or functional variant thereof facilitates targeted transduction of specific cell types, including, but not limited to, innate lymphoid cells, cytotoxic innate lymphoid cells, or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is / are full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of Human immunodeficiency virus (HIV) gpl60, Murine leukemia virus (MLV) gp70, Gibbon ape leukemia virus (GALV) gp70, Feline leukemia virus (RD114) gp70, Amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, Ecotropic retrovirus (Eco) gp70, Baboon ape leukemia virus (BaEV) gp70, Measles virus (MV) H and F, Nipah virus (NiV) H and F, Rabies virus (RabV) G, Mokola virus (MOKV) G,40MF-364712232260132004240Ebola Zaire virus (EboZ) G, Lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, Baculovirus GP64, Chikungunya virus (CHIKV) El and E2, Ross River virus (RRV) El and E2, Semliki Forest virus (SFV) El and E2, Sindbis virus (SV) El and E2, Venezualan equine encephalitis virus (VEEV) El and E2, Western equine encephalitis virus (WEEV) El and E2, Influenza A, B, C, or D HA, Fowl Plague Virus (FPV) HA, Vesicular stomatitis virus VSV-G, or Chandipura virus and Piry virus CNV-G and PRV-G.

[0187] In some embodiments, the fusion glycoprotein or functional variant thereof is a full- length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is the Cocal virus G protein.

[0188] The disclosure further provides various retroviral particles, including but not limited to gamma-retroviral particles, alpha-retroviral particles, and lentiviral particles. In some embodiments, the particle may be a viral particle, a retroviral particle, a lentiviral particle, a gamma-retroviral particle. In some embodiments, the viral particle comprises a VSV G-protein or functional variant thereof. In some embodiments, the viral particle comprises a Cocal G- protein or functional variant thereof.II. ADDITIONAL RECEPTORS

[0189] Provided herein are fusion proteins compatible with expression of additional synthetic receptors when expressed on cells. In some embodiments, IL-15-IL-2R synthetic fusion receptor is expressed in cells expressing a synthetic cytokine receptor complex system, such as rapamycin activated cytokine receptor (RACR) complex (see, e.g., Section ILA). In some embodiments, IL-15-IL-2R synthetic fusion receptor is expressed in cells expressing a chimeric antigen receptor (CAR) system, enhancing anti-tumor activity (see, e.g., Section II.B).A. Rapamycin Activated Cytokine Receptor (RACR) System

[0190] Disclosed herein are fusion proteins that may be used in combination with rapamycin activated cytokine receptor (RACR) complex system. RACR, as previously disclosed, provides a cytokine-free manufacturing system for cell differentiation (see e.g., WO 2023 / 240282, the entire contents of each of which are incorporated by reference herein). In some embodiments,41MF-364712232260132004240 the IL-15-IL-2R synthetic fusion receptor is expressed in cells comprising polynucleotide constructs encoding one or more of the polypeptide chains of a rapamycin activated cytokine receptor (RACR) complex. In some embodiments, the IL-15-IL-2R synthetic fusion receptor is expressed in cells comprising one or more of the polypeptides of a rapamycin activated cytokine receptor (RACR) complex. In some embodiments, the IL-15-IL-2R synthetic fusion receptor is expressed in cells further comprising a cytosolic FRB.

[0191] RACR is a synthetic cytokine receptor composed of two polypeptides that mimics the JAK / STAT signal downstream to induce STAT5 activation (e.g., which is associated with cytokine signaling in T cells) in the presence of rapamycin or an analog and induce cell survival and growth (FIG. 1C). 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 RACR system further comprises a free intracellular FRB (e.g., cytosolic FRB). In some embodiments, cytosolic FRB binds intracellular rapamycin-FKBP12 complexes and prevents their inhibition of mTOR.1. Cytosolic FRB

[0192] In some embodiments, the IE-15-IE-2-R synthetic fusion protein is expressed in cells comprising a cytosolic FRB. In some embodiments, the cells comprise an expression cassette of the polynucleotide construct 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.

[0193] In some embodiments, polynucleotide constructs related to RACR comprise a nucleotide sequence encoding an FRB. In some embodiments, when the FRB domain is expressed, it is a freely diffusible soluble protein.

[0194] 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: 106, 107 (or SEQ ID NO:33), or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In42MF-364712232260132004240 some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NOs: 106, 107, or 108. In some embodiments, the nucleotide sequence encoding the FRB consists of the nucleotide sequence of SEQ ID NOs: 106, 107, or 108.

[0195] 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: 109, 110 (or SEQ ID NO: 34), or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises an amino acid sequence at least43MF-364712232260132004240100% identical to the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NOs: 109, 110, or 111. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NOs: 109, 110, or 111.

[0196] Advantageously, the cytosolic FRB confers resistance to the immunosuppressive effect of the non-physiological ligand (e.g., rapamycin or rapalog).2. RACR Cytokine Receptors

[0197] IL-15-IL-2R synthetic fusions can be co-expressed with a synthetic cytokine receptor, such as a rapamycin activated cytokine receptor(RACR). The 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. RACR comprises two polypeptide chains each with a signaling domain from a cytokine receptor, wherein the cytokine receptor 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), each 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. For instance, each polypeptide chain is a chimeric protein composed of an extracellular head domain of FKBP 12 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 FKBP 12 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. The dimerization domains controllably dimerize in the present of a non-physiological ligand, thereby activating and signaling the synthetic cytokine receptor.

[0198] The synthetic cytokine receptor can include transmembrane receptor proteins that include the cytokine gamma chain polypeptide and the cytokine beta chain polypeptide, such as provided as a first transmembrane receptor and a second transmembrane receptor. In some44MF-364712232260132004240 embodiments, 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. In some embodiments, the dimerization domain is 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). In some embodiments, 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. In some embodiments, the dimerization domain is 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 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).

[0199] 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.

[0200] 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.

[0201] 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.

[0202] In some embodiments, the RACR polynucleotide constructs provided herein comprise one or more nucleotide sequences encoding a synthetic cytokine receptor. In some embodiments, the one or more nucleotide sequences correspond to one or more fusions. In some45MF-364712232260132004240 embodiments, the polynucleotide construct provided herein comprises a first fusion encoding IL-2RG chain of the synthetic cytokine receptor and a second fusion encoding IL-2RB chain of the synthetic cytokine receptor.

[0203] In some embodiments, the polynucleotide encoding the synthetic cytokine receptor encodes the two distinct polypeptide chains, each encoded by a single polycistronic 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.

[0204] 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.

[0205] Exemplary features of the synthetic cytokine receptor (e.g. RACR) system are described in the following subsections. a. Dimerization Domain

[0206] 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, which are known in the art to dimerize in the presence of rapamycin or a rapalog. In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are FRB and FKBP 12. In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are FKBP 12 and FRB.

[0207] In some embodiments, the FKBP 12 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: 28. In some embodiments, the FKBP12 domain is a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NOS: 28. In some embodiments, the FKBP12 domain comprises a variant, derivative, or fragment of SEQ ID NO: 28 (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity46MF-364712232260132004240 thereto) that retains the ability to heterodimerize with an FRB domain in the presence of rapamycin or an analog thereof.

[0208] 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: 27 or 116. In some embodiments, the FKBP12 domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 27. In some embodiments, the FKBP12 domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 116. 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: 27 or 116 (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: 28. 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: 28.

[0209] 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: 34 (or SEQ ID NO: 110), 109, or 111. In some embodiments, the FKBP12 domain is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34, 109 or 111. In some embodiments, the FRB domain is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34, 109 or 111. In some embodiments, the FRB domain comprises or consists of a variant, derivative, or fragment of SEQ ID NOS: 34, 109 or 111 (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. In some embodiments, the FKBP12 domain is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the FRB domain is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 34.

[0210] 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: 33 (or SEQ ID NO: 107), 106, or 108. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NOS: 33, 106, 108. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NOS: 33, 106, 108. In some embodiments, the47MF-364712232260132004240 nucleotide sequence encoding the FRB domain comprises or consists of a variant, homolog, derivative, or codon-optimized sequence of SEQ ID NOS: 33 106, or 108 (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: 34, 109 or 111. 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 NOS: 34, 109, or 111. In some embodiments, the FRB domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 33.

[0211] 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 NO: 34. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises a variant, derivative, or fragment of SEQ ID NO: 34 (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.

[0212] 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: 33. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NO: 33. 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: 33 (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: 34. 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: 34.

[0213] 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.48MF-364712232260132004240

[0214] 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 (FKBP) and a calcineurin domain, which are known in the art to dimerize in the presence of FK506 or an analogue thereof.

[0215] In some embodiments, the dimerization domains involve alternative heterodimerization systems that allow ligand-dependent association of two polypeptide chains with different dimerization domains.

[0216] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a cyclophilin domain.

[0217] 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.

[0218] In some embodiments, the first and second dimerization domains of the transmembrane receptor proteins are a calcineurin domain and a cyclophilin domain.

[0219] 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).

[0220] In some embodiments, a first synthetic cytokine polypeptide composed of an FRB domain is encoded with an IL2Ry signaling domain. In some embodiments, the IL2Ry signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 29. In some embodiments, the IL2Ry signaling domain comprises the polypeptide set forth in SEQ ID NO: 30. In some embodiments, a second synthetic cytokine polypeptide composed of an FKBP12 domain is encoded with an IL2R-beta signaling domain. In some embodiments, the IL2R-beta signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the IL2R-beta signaling domain comprises the polypeptide set forth in SEQ ID NO: 32.

[0221] In some embodiments, a first synthetic cytokine polypeptide composed of an FKBP 12 domain is encoded with an IL2Ry signaling domain. In some embodiments, the IL2Ry signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 29. In some embodiments, the IL2Ry signaling domain comprises the polypeptide set forth in SEQ ID NO: 30. In some embodiments, a second synthetic cytokine polypeptide composed of an FRB domain is encoded with an IL2R-beta signaling domain. In some embodiments, the IL2R-beta signaling49MF-364712232260132004240 domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the IL2R-beta signaling domain comprises the polypeptide set forth in SEQ ID NO: 32. b. Transmembrane Domain

[0222] The polypeptide chains of the synthetic cytokine receptor include a transmembrane domain sequence that is a sequence that facilitates delivery and incorporation of the synthetic cytokine receptor (e.g., RACR) into the membrane when expressed by a cell transduced with a viral vector particle. In some embodiments, the transmembrane domain (TM) is the sequence of the synthetic cytokine receptor (e.g., RACR complex) that spans the membrane.

[0223] 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.

[0224] In some embodiments, the transmembrane domain comprises a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein. 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.

[0225] 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 encoded within 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. In some embodiments, the TM domain and the intracellular50MF-364712232260132004240 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.

[0226] 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. c. Intracellular Domain

[0227] In some embodiments, the intracellular domain of RACR 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 the dimerization 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.51MF-364712232260132004240

[0228] 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.

[0229] In some embodiments, the intracellular signaling domain from a cytokine receptor comprises an interleukin-2 receptor subunit gamma (IL-2Rg) domain. The IL-2RG domain provides the y-chain signaling function that participates in, for example, JAK3 recruitment and activation following receptor dimerization.

[0230] In some embodiments, the intracellular signaling domain from a cytokine receptor comprises interleukin-2 receptor subunit beta (IL-2RB) 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 (IL-2RB) intracellular domain. IL-2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL-2RB 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 in terleukin- 21 receptor subunit beta (IL-21RB).

[0231] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL-2Rg) domain. In some embodiments, the intracellular signaling domain of the second transmembrane receptor protein comprises an interleukin-2 receptor subunit beta (IL-2RB) 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.

[0232] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization52MF-364712232260132004240 domain (e.g., FRB), a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain (e.g., FKBP12). 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). 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.

[0233] 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: 113, 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: 113. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 113. In some embodiments, the IL-2RG transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 113. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 113 (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).

[0234] 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 aminoacid sequence of SEQ ID NO: 30, 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: 30. In some embodiments, the53MF-364712232260132004240IL-2RG transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 30 (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).

[0235] 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: 30 or SEQ ID NO: 113, 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: 30 or SEQ ID NO: 113, 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: 30 or SEQ ID NO: 113. In some embodiments, the nucleotide sequence comprises or consists of SEQ ID NO: 29, 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: 29, and encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 30 or a variant, derivative, or fragment thereof retaining IL-2RG signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization.

[0236] 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: 149, 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% sequence54MF-364712232260132004240 identity to SEQ ID NO: 149. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 149. In some embodiments, the IL-2RB transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 149. In some embodiments, the IL-2RB 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- 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).

[0237] 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 NO: 32 (or SEQ ID NO: 124), 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 NO: 32. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises the amino-acid sequence of SEQ ID NO: 32. In some embodiments, the IL-2RB transmembrane and intracellular domain consists of the amino-acid sequence of SEQ ID NO: 32. In some embodiments, the IL-2RB transmembrane and intracellular domain comprises a variant, derivative, or fragment of SEQ ID NO: 32 (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).

[0238] 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: 32 or SEQ ID NO: 149 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: 32 or SEQ ID NO: 149, and retains IL-2RB signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization. In some embodiments, the nucleotide55MF-364712232260132004240 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: 32 or SEQ ID NO: 149. In some embodiments, the nucleotide sequence comprises or consists of SEQ ID NO: 31, 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: 31, and encodes a polypeptide having the amino-acid sequence of SEQ ID NO: 32 or a variant, derivative, or fragment thereof retaining IL-2RB signaling activity, including JAK3 recruitment and STAT5 activation upon receptor dimerization.

[0239] 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. d. Exemplary Synthetic Cytokine Receptors

[0240] IL-15-IL-2R synthetic fusions can be co-expressed with exemplary synthetic cytokine receptor RACR provided herein. The synthetic cytokine receptors of the RACR system comprise a cytokine gamma chain and a cytokine beta chain, each comprising a dimerization domain (e.g., FRB and FKPB12). The dimerization domains controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0241] In some embodiments, 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. In some embodiments, the cytokine gamma chain polypeptide comprises an FRB:IL-2RG fusion protein. In some56MF-364712232260132004240 embodiments, the synthetic gamma chain polypeptide comprises an FKBP12:IL-2RG fusion protein. In some embodiments, 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 cytokine beta chain polypeptide comprises an FKBP12:IL-2RB fusion protein. In some embodiments, the cytokine beta chain polypeptide comprises an FRB:IL-2RB fusion protein.

[0242] In some embodiments, a first synthetic cytokine polypeptide composed of an FRB domain is encoded with an IL-2Ry signaling domain. In some embodiments, the IL-2Ry signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 29. In some embodiments, the IL-2Ry signaling domain comprises the polypeptide set forth in SEQ ID NO: 30. In some embodiments, a second synthetic cytokine polypeptide composed of an FKBP12 domain is encoded with an IL-2R-beta signaling domain. In some embodiments, the IL-2R-beta signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the IL-2R-beta signaling domain comprises the polypeptide set forth in SEQ ID NO: 32.

[0243] In some embodiments, a first synthetic cytokine polypeptide composed of an FKBP12 domain is encoded with an IL-2Ry signaling domain. In some embodiments, the IL- 2Ry signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 29. In some embodiments, the IL-2Ry signaling domain comprises the polypeptide set forth in SEQ ID NO: 30. In some embodiments, a second synthetic cytokine polypeptide composed of an FRB domain is encoded with an IL-2R-beta signaling domain. In some embodiments, the IL-2R-beta signaling domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 31. In some embodiments, the IL-2R-beta signaling domain comprises the polypeptide set forth in SEQ ID NO: 32.

[0244] 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, the57MF-364712232260132004240 cytokine receptor is a cytokine gamma chain polypeptide comprising a gamma chain signaling domain. In some embodiments, the synthetic cytokine receptor is an FRB:IL-2RG fusion protein. An exemplary FRB:IL2RG sequence and its components is set forth in Table 3 with respective SEQ ID Nos listed.

[0245] In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising 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: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID58MF-364712232260132004240NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain comprises the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain consists of the nucleotide sequence of SEQ ID NO: 29.

[0246] 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 (IL-2RG). In some embodiments, the IL-2RG 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: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG consists of the amino acid sequence of SEQ ID NO: 30.

[0247] 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 least59MF-36471223226013200424080%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence encoding the FRB domain consists of the nucleotide sequence of SEQ ID NO: 33.

[0248] 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: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 34. In60MF-364712232260132004240 some embodiments, the FRB domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the FRB domain consists of the amino acid sequence of SEQ ID NO: 34.

[0249] In some embodiments, the polynucleotide construct is codon optimized.

[0250] In some embodiments, the RACR polynucleotide construct (e.g., FRB:IL-2RG) 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: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 114.

[0251] In some embodiments, the RACR polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the RACR polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 114.

[0252] In some embodiments, the RACR polynucleotide construct (e.g., FRB:IL-2RG) 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: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino61MF-364712232260132004240 acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 115. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence consists of the sequence of SEQ ID NO: 115.

[0253] 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:IL-2RG fusion protein. Exemplary FKBP12:IL2RG sequences and their components are set forth in Table 4 with respective SEQ ID Nos listed.62MF-364712232260132004240

[0254] In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising 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:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the gamma chain signaling domain comprises the nucleotide sequence of SEQ ID NO:29. In some embodiments, the nucleotide encoding the synthetic cytokine receptor63MF-364712232260132004240 comprising the gamma chain signaling domain consists of the nucleotide sequence of SEQ ID NO:29.

[0255] 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 (IL-2RG). In some embodiments, the IL-2RG 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: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-2RG consists of the amino acid sequence of SEQ ID NO: 30.

[0256] 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: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the64MF-364712232260132004240 nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NOs: 27 or 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NOs: 27 or 116.

[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: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID65MF-364712232260132004240NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NO: 28.

[0258] In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NOs: 117 or 118. In some embodiments, the RACR polynucleotide construct consists of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NOs: 117 or 118.

[0259] In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 95% identical to66MF-364712232260132004240 the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 119, 120, or 121. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence consisting of the amino acid sequence of SEQ ID NOs: 119, 120, or 121.

[0260] 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:IL-2RB fusion protein. Exemplary FKBP12:IL2RB sequences and their components are set forth in Table 5 with respective SEQ ID Nos listed.

[0261] In some embodiments, the nucleotide encoding the synthetic cytokine receptor comprising the beta chain signaling domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,67MF-36471223226013200424099% or 100% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 80% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 85% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 96% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 97% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 98% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 99% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain comprises the nucleotide sequence of SEQ ID NOs: 31 or 123. In some embodiments, the nucleotide encoding the beta chain signaling domain consists of the nucleotide sequence of SEQ ID NOs: 31 or 123.

[0262] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit P (IL-2RB).

[0263] 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: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 97%68MF-364712232260132004240 identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL- 2RB comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB comprises the amino acid sequence of SEQ ID NOs: 32 or 124. In some embodiments, the IL-2RB consists of the amino acid sequence of SEQ ID NOs: 32 or 124.

[0264] In some embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding FKBP12.

[0265] 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: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NO: 27.

[0266] 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 sequence69MF-364712232260132004240 of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 80% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 85% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 90% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 95% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 96% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 97% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 98% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 99% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain is at least 100% identical to the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain comprises the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the FKBP12 domain consists of the nucleotide sequence of SEQ ID NO: 116.

[0267] 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: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain70MF-364712232260132004240 comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO :28. In some embodiments, the FKBP12 domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the FKBP12 domain consists of the amino acid sequence of SEQ ID NO: 28.

[0268] In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence encoding FKBP12:IL-2RB with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the RACR polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 125.

[0269] In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct71MF-364712232260132004240 encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 151. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 151.

[0270] In some embodiments, the RACR 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:IL-2RB fusion protein. Exemplary FRB:IL2RB sequences and their components are set forth in Table 6 with respective SEQ ID Nos listed.72MF-364712232260132004240

[0271] In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct comprises the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the RACR polynucleotide construct consists of the nucleotide sequence of SEQ ID NO: 126.

[0272] In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some73MF-364712232260132004240 embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 127 or 128. In some embodiments, the RACR polynucleotide construct encodes an amino acid sequence consisting of the sequence of SEQ ID NOs: 127 or 128.

[0273] In some embodiments, the RACR is a two chain polypeptide containing a FKBP12:IL-2Ry and a FRB:IL-2Rp. In some embodiments, RACR comprises a first synthetic cytokine polypeptide composed of a FKBP12 domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 27 encoding the polypeptide set forth in SEQ ID NO: 28) and an IL-2Ry signaling domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 29 encoding the polypeptide set forth in SEQ ID NO: 30), and a second synthetic cytokine polypeptide composed of an FRB domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 33 encoding the polypeptide set forth in SEQ ID NO: 34) and a beta chain signaling domain (e.g., signaling domain from IL-2RP; e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 31 encoding the polypeptide set forth in SEQ ID NO: 32). In some embodiments, the FKBP12:IL-2Ry comprises the sequence set forth in SEQ ID NO: 119, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 119, and the FRB:IL-2Rp. comprises the sequence set forth in SEQ ID NO: 128, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 128. In some embodiments, the FKBP12: IL-2Ry comprises the sequence set forth in SEQ ID NO: 119, and the FRB:IL-2Rp comprises the sequence set forth in SEQ ID NO: 128.

[0274] In some embodiments, the RACR is a two chain polypeptide containing a FKBP12:IL-2Rp and a FRB: IL-2Ry. In some embodiments, RACR comprises a first synthetic cytokine polypeptide composed of a FKBP12 domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 27 encoding the polypeptide set forth in SEQ ID NO: 28) and a beta chain signaling domain (e.g., signaling domain from IL-2RP; e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 31 encoding the polypeptide set forth in SEQ ID NO: 32), and a second synthetic cytokine polypeptide composed of an FRB domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 33 encoding the polypeptide set forth in SEQ ID NO: 34) and74MF-364712232260132004240 an IL-2Ry signaling domain (e.g., exemplary nucleotide sequence set forth in SEQ ID NO: 29 encoding the polypeptide set forth in SEQ ID NO: 30). In some embodiments, the FKBP12:IL- 2RP comprises the sequence set forth in SEQ ID NO: 151, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 151, and the FRB: IL-2Ry comprises the sequence set forth in SEQ ID NO:115, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 115. In some embodiments, the FKBP12:IL-2Rp comprises the sequence set forth in SEQ ID NO: 151, and the FRB: IL-2Ry comprises the sequence set forth in SEQ ID NO: 115.B. Chimeric Antigen Receptor (CAR)

[0275] Disclosed herein are IL-15-IL-2R synthetic fusion receptors that may be used in combination with one or more chimeric antigen receptor (CAR). In some embodiments, fusion proteins IL-15-IL-2R synthetic fusion receptor is expressed in cells comprising polynucleotide constructs encoding one or more of the polypeptide chains of a CAR. In some embodiments, the polynucleotide construct encoding the IL-15-IL-2R synthetic fusion receptor further comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR).1. CAR Constructs and Encoding Nucleotides

[0276] 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.

[0277] 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.75MF-364712232260132004240

[0278] 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.

[0279] In some embodiments of the disclosure, 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).

[0280] 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 disclosure, 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.

[0281] 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 hinge76MF-364712232260132004240 domain that imparts flexibility to the recognition region and allows strong binding to the targeted moiety.

[0282] 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.

[0283] 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 U SA. 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 Immuno ther. 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);(5) SCFV-2B4TM+IC-CD3^S (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866);77MF-364712232260132004240(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).2. Extracellular Domain

[0284] 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.

[0285] 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-l lRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, P-human chorionic gonadotropin, alphafetoprotein (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-78MF-364712232260132004240TES1, 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 and 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 comprises79MF-364712232260132004240 binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA.

[0286] 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.

[0287] 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.

[0288] 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 (VE) 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 7 with its different portions including the extracellular domain.

[0289] 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, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR80MF-364712232260132004240 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 8 including its extracellular domain. a. Universal CARs

[0290] 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.

[0291] 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.

[0292] 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).

[0293] 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.

[0294] In some embodiments, the CAR is an anti-FITC CAR. In some embodiments, anti- FITC CAR is encoded by the amino acid sequence of SEQ ID NO: 9. In some embodiments, anti-FITC CAR is encoded by an amino acid sequence of at least 70%, 71%, 72%, 73%, 74%,81MF-36471223226013200424015%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, anti-FITC CAR is encoded by the amino acid sequence of SEQ ID NO: 20. In some embodiments, anti-FITC CAR is encoded by an amino acid sequence of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0295] In one embodiment, the disclosure provides an illustration of this conjugate molecule / CAR system.

[0296] 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 FITC (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. Ato 2009. 1(3): 190-209).

[0297] 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.

[0298] 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).

[0299] 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 the82MF-364712232260132004240 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).

[0300] 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.

[0301] 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).

[0302] 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.

[0303] 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.

[0304] 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.

[0305] In some embodiments, the cell-targeting moiety is a PSMA ligand.

[0306] In some embodiments, the cell-targeting moiety is a FAP ligand.

[0307] 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 about83MF-3647122322601320042404000 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.

[0308] 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.

[0309] 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.

[0310] In some embodiments, the linker may be a divalent linker that may include one or more spacers.

[0311] An illustrative conjugate of the disclosure is FITC-Folate84MF-364712232260132004240An illustrative conjugate of the disclosure is FITC-CA9

[0312] 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.

[0313] 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.

[0314] 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.3. Spacer (e.g., hinge domain)

[0315] 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.

[0316] 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: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge85MF-364712232260132004240 comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID Nos: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge comprises the nucleotide sequence of SEQ ID NOs: 129 or 130. In some embodiments, the CD8a hinge consists of the nucleotide sequence of SEQ ID NOs: 129 or 130.

[0317] 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: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID Nos: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID Nos: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID Nos: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 131 or 132. In some embodiments, the CD8a hinge comprises the amino acid sequence of86MF-364712232260132004240SEQ ID Nos: 131 or 132. In some embodiments, the CD8a hinge consists of the amino acid sequence of SEQ ID NOs: 131 or 132.

[0318] 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 NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge comprises the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the CD8 hinge consists of the nucleotide sequence of SEQ ID NO: 133.

[0319] 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: 134. 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: 134. 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: 134. 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: 134. 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: 134. 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: 134. 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: 134. In87MF-364712232260132004240 some embodiments, the modified IgG4 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 134. 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: 134. 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: 134. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 134.

[0320] 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. 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: 135. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO: 135.

[0321] In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 136%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the88MF-364712232260132004240IgGl hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 136% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the IgGl hinge consists of the amino acid sequence of SEQ ID NO: 136.

[0322] 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: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the PD1 hinge consists of the amino acid sequence of SEQ ID NO: 137.89MF-364712232260132004240

[0323] In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 138% or 100% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 138% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO: 138.4. Transmembrane Domain

[0324] 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.

[0325] In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 139%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 139% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the90MF-364712232260132004240 transmembrane domain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 139. In some embodiments, the transmembrane domain consists of the nucleotide sequence of SEQ ID NO: 139.

[0326] 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: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain91MF-364712232260132004240 comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the transmembrane domain consists of the amino acid sequence of SEQ ID NO: 140.5. Intracellular domain (i.e., endodomain)

[0327] In some embodiments, the CAR comprises an endodomain. In some embodiments, the endodomain comprises a signaling domain of a costimulatory molecule. In some embodiments, the costimulatory molecule is 4- IBB or CD28. In some embodiments, the endodomain comprises 4- IBB. In some embodiments, the signaling domain comprises a CD3(^ signaling doman. In some embodiments, the endodomain comprises CD28.

[0328] 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: 141 or 142. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- IBB endodomain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- IBB endodomain comprises the nucleotide sequence of SEQ ID NOs: 141 or 142. In some embodiments, the 4- 1BB endodomain consists of the nucleotide sequence of SEQ ID NOs: 141 or 142.

[0329] 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 NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid92MF-364712232260132004240 sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 4- IBB endodomain consists of the amino acid sequence of SEQ ID NO: 143.

[0330] 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: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 98% identical to the nucleotide sequence of SEQ ID NOs: 144, 145,93MF-364712232260132004240146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain comprises the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147. In some embodiments, the CD3(^ endodomain consists of the nucleotide sequence of SEQ ID NOs: 144, 145, 146, or 147.

[0331] 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: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the CD3(^ endodomain consists of the amino acid sequence of SEQ ID NO: 148.6. Exemplary CAR Polynucleotides

[0332] In some embodiments the CAR is an anti-CD19 CAR, and in these embodiments, the polynucleotide construct further comprises a nucleotide sequence comprising a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a CD 19 CAR. In some94MF-364712232260132004240 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 CAR encodes an anti-CD19 CAR with features set forth in Table 7.

[0333] In some embodiments, the CAR is an anti-CD20 CAR, and in these embodiments, the polynucleotide construct further comprises a nucleotide sequence encoding a 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 CAR encodes an anti-CD20 CAR with features set forth in Table 8.95MF-364712232260132004240

[0334] In some embodiments, the CAR polynucleotide construct or sequence 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 in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 98% identical to 96MF-364712232260132004240 the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence comprises the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence consists of the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8.

[0335] In some embodiments, the CAR polynucleotide construct or sequence 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 in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 80% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 85% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 90% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 95% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 96% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 97% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 98% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 99% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence at least 100% identical to the nucleotide sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct or sequence encodes an amino acid sequence comprising the sequence of SEQ ID NOs in Table 7 or Table 8. In some embodiments, the CAR polynucleotide construct97MF-364712232260132004240 or sequence encodes an amino acid sequence consisting of the sequence of SEQ ID NOs in Table 7 or Table 8.

[0336] 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.

[0337] 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 about 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.C. Cleavable Linker

[0338] As provided herein, RACR or CAR polynucleotide constructs may comprise linkers. In some embodiments, the linkers comprise sites for cleavage, making them cleavable linkers.

[0339] 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.

[0340] 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.

[0341] 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 FC3.98MF-364712232260132004240

[0342] 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.

[0343] 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.

[0344] 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.

[0345] In some embodiments, the first RACR fusion, the second RACR fusion, and the third RACR component (e.g., FRB) are separated by encoded cleavable signal peptides. In some embodiments, the first fusion, the second fusion, the third RACR component, and a fourth heterologous protein (e.g., CAR) 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 RACR component (e.g., FRB) are separated by a furin T2A. In some embodiments, the second fusion and the fourth heterologous protein (e.g., CAR) are separated by a P2A.III. ENGINEERED CELLS

[0346] Provided herein are engineered cells comprising IL-15-IL-2R synthetic fusion protein (e.g., receptor). In some embodiments, the engineered cells comprise an IL-15-IL-2R-beta99MF-364712232260132004240 synthetic fusion receptor. In some embodiments, the engineered cells comprise an IL-15-IL-2R- gamma synthetic fusion receptor.

[0347] In some embodiments, the engineered cell is engineered to express an IL-15-IL-2R synthetic fusion comprising IL-15 peptide linked to an IL-2R-beta subunit. In embodiments, the engineered cell is engineered to express an IL-15-IL-2R synthetic fusion comprising IL- 15 peptide linked to an IL-2R-gamma subunit.

[0348] In some embodiments, the engineered cells comprising the IL-15-IL-2R synthetic fusion receptor further comprise the synthetic cytokine receptor RACR (e.g., see, Section II.A). In some embodiments, the engineered cells comprising the IL-15-IL-2R synthetic fusion receptor further comprise a CAR (e.g., see, Section II.B). In some embodiments, the engineered cells comprising the IL-15-IL-2R synthetic fusion receptor and the synthetic cytokine receptor RACR further comprise a CAR.

[0349] In some embodiments, the engineered cell further comprises a first CAR binding a first antigen and a second CAR binding a second CAR. In some embodiments, the one or more antigen is a solid tumor antigen. In embodiments, the engineered cell comprises a bispecific CAR binding two solid tumor antigens. In some embodiments, the one or more antigen is a white blood cell (WBC) antigen. In embodiments, the engineered cell comprises a bispecific CAR binding two WBC antigens. In embodiments, the WBC antigen is or comprises CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CDl lb, CD18, CD169, CDlc, CD33, CD38, CD138, or CD13. In embodiments, the WBC antigen is or comprises CD 19, CD20, CD22, or BCMA. In embodiments, the solid tumor antigen comprises tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, CLDN18.2, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-a, ErbB2, EpCAM, EGFRvIII, B7-H3, or EGFR. In embodiments, the solid tumor antigen comprises tumor-associated MUC1, ACPP, TSHR, GUCY2C, UPK2, CLDN18.2, PSMA, DPEP3, CXCR5, B7-H3, MUC16, SIGLEC-15, CLDN6, Mucl7, PRLR, or FZD10.100MF-364712232260132004240A. Types of Cells

[0350] Disclosed herein, but not limited to, are target cells for expression of IL-15-IL-2R synthetic fusion receptor.

[0351] In some aspects, the engineered cells are stem cells. In some embodiments, the stems cells are pluripotent stem cells. In some embodiments, the stems cells are induced pluripotent stem cells (iPSCs). In some embodiments, an iPSCs are obtained by introducing a specific factor (nuclear reprogramming factor) into a mammalian somatic cell or an undifferentiated stem cell to reprogram them. In some embodiments, the engineered cells are CIL cells. In some embodiments, the engineered cells are derived from peripheral blood cells.

[0352] In some embodiments, CIL cells may be generated from multiple sources, illustrative examples include: iPSCs, PBMCs, or UCBs. In some embodiments, the CIL source cells are autologous cells. In some embodiments, the CIL source cells are allogeneic cells. In some embodiments, the CIL source cells are heterologous cells. For example, when the subject being treated using the compositions of the present disclosure has received high-dose chemotherapy or radiation treatment to destroy the subject’s immune system, allogenic cells may be used.

[0353] As used herein, the term “peripheral blood cell” is used to refer to cells that originate from circulating blood and comprise hematopoietic stem cells that are capable of proliferation, selectable differentiation, and maturation. As such, peripheral blood NK cells may alternatively be referred to as differentiated blood-derived NK cells (bdNK).

[0354] In some embodiments, the lymphocytes used to generate engineered stem cells or CIL cells may be obtained from a donor or a subject (for autologous therapy) by various means well-known in the art. For example, lymphocytes can be obtained by collecting peripheral blood from the patient and subjecting the blood to Ficoll density gradient centrifugation and / or leukapheresis, and then using an isolation kit to isolate a population of lymphocytes from the peripheral blood. In one illustrative embodiment, the population of lymphocytes need not be pure of the selected cell type and may contain other cell types such as T cells, monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the cell population being collected can comprise at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0355] In some embodiments, the engineered cells are stem or progenitor cells containing a synthetic cytokine receptor (e.g., RACR). In some embodiments, the synthetic cytokine receptor is any as described in Section ILA.101MF-364712232260132004240

[0356] In some embodiments, the engineered cells are stem or progenitor cells containing a CAR. In some embodiments, the CAR is any as described in Section II.B.

[0357] Various types of iPSCs and methods for obtaining the same are known in the art. In some embodiments, iPSCs are established by introducing the four factors Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts, such as described by Yamanaka et al. (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676, herein incorporated by reference in its entirety). In some embodiments, iPSCs are derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872, herein incorporated by reference in its entirety). In some embodiments, the iPSCs are Nanog-iPS cells established by introducing the four factors, then selecting them by using the expression of Nanog as an index (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317, herein incorporated by reference in its entirety). In some embodiments, iPSCs are prepared by a method free of C-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106, herein incorporated by reference in its entirety). In some embodiments, iPSCs are established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66, herein incorporated by reference in its entirety). In some embodiments, iPSCs are established by introducing the four factors OCT3 / 4, SOX2, NANOG, and LIN28, such as described by Thomson et al. (Yu J., Thomson J A. et al., Science (2007) 318: 1917-1920, herein incorporated by reference in its entirety). In some embodiments, iPSCs are prepared by methods described in Daley et al. (Park I H, Daley G Q. et al., Nature (2007) 451: 141-146, herein incorporated by reference in its entirety). In some embodiments, iPSCs are prepared by methods described in Sakurada et al. (JP 2008-307007 A, herein incorporated by reference in its entirety). Other methods for inducing or obtaining iPSCs are well known, including any described in any published literature (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim J B., Scholer H R., et al., Nature, (2008) 454, 646-650; and Huangfu D., Melton, D A., et al., Nature Biotechnology, (2008) 26, No 7, 795-797), or patent (for example, JP 2008-307007 A, JP 2008-283972 A, US2008- 2336610, US2009-047263, W02007-069666, W02008-118220, WO2008-124133, W02008- 151058, W02009-006930, W02009-006997, and W02009-007852, all herein incorporated by reference in their entirety). In some embodiments, the cells are an established iPSC cell line. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-lA line, HiPS-RIKEN-2A line,102MF-364712232260132004240H1PS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's 253G1 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, and 648A1 line.

[0358] In some embodiments, to generate iPSCs, somatic cells may be reprogrammed through methods including, but not limited to, the transient expression of reprogramming factors, virus-free methods, adenoviruses, plasmids, minicircle vectors, episomal vectors, Sendai viruses, synthetic mRNAs, self-replicating RNAs, retroviruses, lentiviruses, PhiC31 integrases, excisable transposons, CRISPR-based gene editing, or recombinant proteins.

[0359] In some embodiments, iPSCs can be used to generate various types of blood cells. In some embodiments, iPSCs are differentiated to hematopoietic progenitor cells (HPs). In some embodiments, HP cell markers are CD34-positive and CD3-negative (CD34+CD3- cells). In some embodiments, positive HP cells markers include CD34, Cd90, CD49f, and EPCR. In some embodiments, positive HP cells markers further include RET, GPRC5C, CD117, CD133, CD59, CD45, GPI-80, CD43, CD44, CD9, CD48, CD84, and CD244. See, e.g., Markers for human haematopoietic stem cells: The disconnect between an identification marker and its function, Front. Physiol., 29 September 2022. Sec. Cell Physiology, Volume 13 - 2022, doi.org / 10.3389 / fphys.2022.1009160, which is herein incorporated by reference in its entirety.

[0360] Various methods of HP differentiation are known in the art, including RACR- mediated differentiation as described above. See, e.g., Zheng, H., Chen, Y., Luo, Q. et al. Generating hematopoietic cells from human pluripotent stem cells: approaches, progress and challenges. Cell Regen 12, 31 (2023). doi.org / 10.1186 / sl3619-023-00175-6, which is herein incorporated by reference in its entirety.

[0361] In some embodiments, RACR is used to induce iPSC to HP differentiation. In some embodiments, IL-15-IL-2R synthetic fusion receptor is introduced before initiating RACR- induced differentiation of iPSCs to HPs. In some embodiments, IL-15-IL-2R synthetic fusion receptor is introduced after RACR-induced differentiation of iPSCs to HPs. In some embodiments, after HPs are differentiated using RACR, HPs are transduced with a viral vector containing the polynucleotide construct encoding one of the two versions of IL-15-IL-2-R synthetic fusion receptors (e.g., beta or gamma).

[0362] In some embodiments, the synthetic cytokine receptor RACR contains a common gamma chain intracellular signaling domains (e.g. interleukin-2 receptor subunit gamma, IL- 2RG) and an intracellular domain from interleukin-2 receptor subunit beta (IL-2RB), interleukin-7 receptor subunit beta (IL-7RB) or in terleukin- 21 receptor subunit beta (IL-21RB).103MF-364712232260132004240In some embodiments, the synthetic cytokine receptor also contains an extracellular domain that is able to be bound by a non-physiological ligand (e.g. rapamycin or an analog). In this way, binding of the non-physiological ligand to the extracellular domain of the synthetic cytokine receptor activates cytokine receptor-mediated signaling to include JAK / STAT signaling, which is an important pathway for differentiation of stem cells, such as iPSCs or other pluripotent stem cells, to downstream cell lines, such as cytokine innate lymphoid (CILs). Thus, in the presence of a non-physiological ligand (e.g., rapamycin) the synthetic cytokine receptor can be engaged during cell differentiation removing the need for endogenous receptors or exogenous growth factors. In some embodiments, this increases the control and decreases the variability of JAK / STAT signaling during cell differentiation to thereby permit efficient generation of induced CILs (iCILs).

[0363] In some embodiments, iPSCs are cultured with polynucleotides or vectors for delivery of same (see, e.g., Section I.C) under conditions to introduce the polynucleotides or vectors for delivery of same into stem cells of the population to promote hematopoietic progenitor cell differentiation. In some embodiments, iPSCs are cultured with polynucleotides or vectors for delivery of same (see, e.g., Section II. A) under conditions to introduce the polynucleotides or vectors for delivery of same into stem cells of the population to promote hematopoietic progenitor (HP) cell differentiation using RACR.

[0364] In some of any embodiments, provided herein is a hematopoietic progenitor (HP) cell that has been differentiated from a pluripotent stem cell according to any of the methods provided herein. In some embodiments, the HPs express CD34. In some embodiments, the HPs express at least one marker from the group consisting of CD43, CD34, CD31, CD41, CD235 and CD45. In some embodiments, HPs express one or more of the cell surface markers selected from the group consisting of CD34, CD43, CD7, DLL4, CD 144, and CD235. In some embodiments, the HPs express CD 144, CD34, CD45, and CD7. In some embodiments, the HPs express CD 144, CD34, CD45, and CD7. In some embodiments, HPs express CD34 and CD43. In some embodiments, HPs express CD34 and CD43 and do not express CD235a.

[0365] In some of any embodiments, provided herein is a population of hematopoietic progenitor (HP) cells produced by any of the methods provided herein. In some of any embodiments, the population of HP cells comprise lower expression of HLF, HOXA9, and / or CD133 compared to a population of CD34+ cord blood cells. In some of any embodiments, the104MF-364712232260132004240 expression of HLF, H0XA9, and / or CD133 in HP cells is 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold lower compared to a population of CD34+ cord blood cells.

[0366] In some embodiments, IL-15-IL-2R synthetic fusion receptor is introduced in differentiated HPs. In some embodiments, iPSCs are differentiated to HPs using RACR prior to the introduction of the IL-15-IL-2R synthetic fusion receptor. In some embodiments, RACR- engineered iPSC clones are cultured in conditions that allow for differentiation into HPs. In some embodiments, IL-15-IL-2R synthetic fusion receptor is introduced in engineered cells after RACR-mediated HP differentiation. In some embodiments, the HP comprises a synthetic cytokine receptor and an IL-15-IL-2R synthetic fusion receptor.

[0367] As described above, provided herein are IL-15-IL-2R synthetic fusion receptors for use in stem or progenitor cells that may be differentiated into lymphoid cells comprising a synthetic cytokine receptor complex activated by a non-physiological ligand. In some embodiments, provided herein are IL-15-IL-2R synthetic fusion receptors for use in differentiated cells produced from those stem or progenitor cells for use in medical treatment. In some embodiments, differentiated cells may be, but are not limited to, iCIL cells. As a nonlimiting illustration of the compositions and methods described herein, cytotoxic innate lymphoid cells may be produced from pluripotent stem cells, such as induced pluripotent stem cells, engineered to express synthetic cytokine receptor able to be activated by a non- physiological ligand (e.g. rapamycin) as described to induce differentiation, in addition to or instead of an exogenous cytokine. In some embodiments, the synthetic cytokine receptor is a rapamycin activated cytokine receptor (RACR) using rapamycin or a rapalog to induce differentiation, in addition to or instead of an exogenous cytokine. Advantages of embodiments may include the ability to generate from a plentiful cell source (e.g., induced pluripotent stem cells) effector cells expressing synthetic cytokine receptor complex activated by a non- physiological ligand and IL-15-IL-2R synthetic fusion receptors, so that proliferation of the effector cells in patients may be controlled by administering or ceasing administration of the non-physiological ligand. Other advantages of embodiments include, but are not limited to, the ability to generate effector cells from source cells in media substantially free of cytokines conventionally used in the art for CIL cell differentiation, such as IL-2, IL-7, and / or IL- 15.

[0368] In any of the preceding embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for one or more of the markers of HPs. In105MF-364712232260132004240 some embodiments, the markers are selected from the group consisting of CD43, CD34, CD31, CD41, CD235 and CD45. In some embodiments, the method further includes isolating or enriching cells from the population of differentiated cells that are positive for one or more of the markers of HPs. In some embodiments, the markers are selected from the group consisting of CD43, CD34, CD31, CD41, CD235 and CD45.

[0369] In some embodiments, the differentiated HPs express CD34. In some embodiments, the methods differentiate the population of stem cells into a population of cells that are enriched for cells that express CD34. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more of the cells in the population of cells are positive for CD34. In some embodiments, the method further includes isolating or enriching cells from the population of differentiated cells that are positive for the marker CD34.

[0370] In any of the preceding embodiments, introducing IL-15-IL-2R synthetic fusion receptor increases differentiation efficiency. In any of the preceding embodiments, introducing IL-15-IL-2R synthetic fusion receptor increases expansion rates of differentiated cells.

[0371] In any of the preceding embodiments, IL-15-IL-2R synthetic fusion receptor is introduced into the cell and expression can be detected through various methods known in the art. In some embodiments, cells expressing IL-15-IL-2R synthetic fusion receptor can be analyzed using flow cytometry. In some embodiments, cells expressing IL-15-IL-2R synthetic fusion receptor can be analyzed for IL-15-IL-2R synthetic fusion receptor- specific markers. In some embodiments, the specific marker corresponds to components of the IL-15-IL-2R synthetic fusion receptor. In some embodiments, cells expressing IL-15-IL-2R synthetic fusion receptor can be analyzed to quantify linker expression. In some embodiments, cells expressing IL-15-IL-2R synthetic fusion receptor are analyzed for G4S linker expression.IV. METHODS OF EXPANDING CELLS

[0372] Provided herein are methods of differentiation and methods of expansion of cells expressing an IL-15-IL-2R synthetic fusion receptor. In some embodiments, the engineered cells described above (see, e.g., Section III) are expanded and comprise an IL-15-IL-2R-beta synthetic fusion receptor. In some embodiments, the engineered cells described above (see, e.g., Section III) are expanded and comprise an IL-15-IL-2R-gamma synthetic fusion receptor.106MF-364712232260132004240

[0373] In some aspects, the engineered cells are cultured in varying conditions to trigger differentiation and / or expansion. In some aspects, added culture agents include rapamycin and IL-15. In some aspects, added culture agents include rapamycin and IL-15. In some embodiments, a cytotoxic innate lymphoid (iCIL) that has been differentiated from any of the provided engineered stem cells further expanded. In some embodiments, the iCIL comprises an IL-15-IL-2R synthetic fusion receptor. In some embodiments, the iCIL comprises a synthetic cytokine receptor and an IL-15-IL-2R synthetic fusion receptor. In some embodiments, the iCIL comprises a synthetic cytokine receptor, a chimeric antigen receptor (CAR), and an IL-15-IL-2R synthetic fusion receptor.

[0374] In some aspects, methods provided herein are applied to, but not limited to, CIL cells, iCILs, iPSCs, differentiated HPs, and peripheral blood cells. In some aspects, methods provided herein are applied to cells comprising endogenous IL-2R subunits. In some aspects, methods provided herein are applied to cells described in Section III.

[0375] In some embodiments, culturing the engineered cells comprises adding rapamycin. In some embodiments, culturing the engineered cells comprises adding rapamycin and no added IL-15. In some embodiments, culturing the engineered cells comprises no added rapamycin and no added IL- 15. In some embodiments, culturing the engineered cells no added IL- 15.

[0376] In some embodiments, engineered cells comprise rapamycin activated cytokine receptor (RACR) system. In some embodiments, rapamycin or an analog is added to differentiate cells. In some embodiments, rapamycin induces RACR- mediated iPSC to HP differentiation. In some embodiments, engineered cells comprising IL-15-IL-2R fusion synthetic receptor are further expanded. In some embodiments, engineered cells are further expanded without IL- 15 added. In some embodiments, engineered cells are further expanded without rapamycin added. In some embodiments, engineered cells are further expanded without rapamycin or added IL-15. In some embodiments, engineered cells comprising an IL-15-IL-2R fusion receptor can differentiate and expand to iCILs without rapamycin or added IL-15. In some embodiments, engineered cells comprising an IL-15-IL-2R fusion receptor can differentiate and expand to iCILs without added IL- 15.

[0377] In some embodiments, the provided method includes culturing engineered stem cells, e.g. engineered with a synthetic cytokine receptor, with the non-physiological ligand for a first period of time sufficient to generate common lymphoid progenitor cells (CLPs), and contacting the CLPs with a differentiation media for a second period of time sufficient to generate iCILs.107MF-364712232260132004240

[0378] In some embodiments, conditions in addition to or other than activation with the synthetic cytokine receptor can be used in methods to differentiate the engineered stem cells to CILs.

[0379] In some embodiments, the provided stem cells, such as iPSCs, engineered with a synthetic cytokine receptor may instead or alternatively be differentiated via any other method known to differentiate CILs. In some embodiments, one or more growth factor or cytokine customarily used in connection with differentiation of CILs may be used in the provided methods in addition to the non-physiological ligand engagement of the synthetic cytokine receptor.

[0380] Various differentiation protocols for CIL cells are known in the art.

[0381] In some embodiments, the stem cells are adapted for feeder-free culture. As used herein, a “feeder-free” (FF) environment refers to an environment such as a culture condition, cell culture or culture media which is essentially free of feeder or stromal cells, and / or which has not been pre-conditioned by the cultivation of feeder cells. “Pre-conditioned” medium refers to a medium harvested after feeder cells have been cultivated within the medium for a period of time, such as for at least one day. Pre-conditioned medium contains many mediator substances, including growth factors and cytokines secreted by the feeder cells cultivated in the medium.

[0382] Broadly, techniques for differentiating a cell involve modulation of specific cellular pathways, either directly or indirectly, using polynucleotide-, polypeptide- and / or small molecule-based approaches. The developmental potency of a cell may be modulated, for example, by contacting a cell with one or more modulators. In some embodiments, cells are cultured in the presence of one or more agents to induce cell differentiation (such as, for example, small molecules, proteins, peptides, etc.). In some embodiments, the one or more differentiation agents are introduced to the cell during in vitro culture. The cell may be maintained in the culture medium comprising one or more agents for a period sufficient for the cell to achieve the differentiation phenotype that is desired.

[0383] In some embodiments, the culture platform comprises one or more of the following: nutrients, extracts, growth factors, hormones, cytokines and medium additives. Illustrative nutrients and extracts may include, for example, DMEM / F-12 (Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12), which is a widely used basal medium for supporting the growth of many different mammalian cells; KOSR (knockout serum replacement); L-glut;108MF-364712232260132004240NEAA (Non-Essential Amino Acids). Medium additives may include, but are not limited to, MTG, ITS, (ME, anti-oxidants (for example, ascorbic acid).

[0384] In some embodiments, the differentiation media contains supplements such as serums, extracts, growth factors, hormones, cytokines and the like.

[0385] In some embodiments, a culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet- derived growth factor (PDGF), transforming growth factor beta (TGF-P), bone morphogenetic protein (BMP4), vascular endothelial cell growth factor (VEGF) transferrin, various interleukins (such as IE-1 through IE- 18), various colony-stimulating factors (such as granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFNy) and other cytokines such as stem cell factor (SCF) and erythropoietin (EPO).

[0386] These cytokines may be obtained commercially and may be either natural or recombinant. In some other embodiments, the culture medium of the present disclosure comprises one or more of bone morphogenetic protein (BMP4), insulin-like growth factor- 1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factor (for example, SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms- Related Tyrosine Kinase 3 Ligand (FLT3L); and one or more cytokines from Leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors, mitogens, and cytokines are stage and / or cell type specific in concentrations that are determined empirically or as guided by the established cytokine art. Examples of exogenous cell culture media additives and supplements and cell selection kit components are provided in WO 2020 / 124256, the disclosure of which is incorporated by reference herein in its entirety.

[0387] In some other embodiments, the culture medium of the present disclosure comprises Roswell Park Memorial Institute (RPMI) media, cRPMI1640 media, fetal bovine serum (FBS), Glutamax, Penicillin, streptomycin, Rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination thereof. In some other embodiments, the culture medium of the present disclosure comprises AIM V media, fetal bovine serum (FBS), Glutamax, Penicillin, streptomycin, Rosuvastatin, BX795, protamine sulfate, brefeldin A, monensin, UM729, IL-2, IL-15, IL-21, IL-18, IL-7, or any combination109MF-364712232260132004240 thereof. In some other embodiments, the culture medium of the present disclosure comprises AIM V media STEMdiff APEL 2 Medium (STEMCELL Technologies).

[0388] Examples of methods for differentiating stem cells (e.g., iPSCs) into multipotent hematopoietic progenitor cells are provided in U.S. Patent No. 9,624,470, U.S. Patent Appl. No. 2020 / 0080059, as well as Mesquitta et al., Sci. Rep. 9:6622 (2019), the disclosures of which are incorporated by reference herein in their entireties.

[0389] In some embodiments, engineered cells (e.g., differentiated lymphoid cells) can be generated from induced pluripotent stem cells (iPSCs), such as in methods as previously described. Exemplary protocols are set forth in PCT publication nos. WO 2023 / 115049 and WO 2023 / 240282, including in Example 17 of WO 2023 / 240282, which are incorporated by reference in their entirety herein.

[0390] In some embodiments, the method of producing CIL cells of the disclosure comprises: forming embryoid bodies (EBs) comprising aggregates of stem cells; differentiating the cells into hematopoietic stem cells in a first differentiation medium; differentiating the cells into lymphoid progenitor cells in a second differentiation medium; and / or differentiating the cells into differentiated CIL cells in a third differentiation medium. In some embodiments, the methods provided herein results in EBs that completely dissociate into pure hematopoietic progenitors or hematopoietic stem cells without the need for an additional purification step.

[0391] In some embodiments, provided is a method for generating cytotoxic innate lymphoid (iCIL) cells, comprising culturing a cell population comprising engineered iPSCs as described under conditions to differentiate the iPSCs to cytotoxic innate lymphoid (iCILs), wherein a non-physiological ligand of the synthetic cytokine receptor is added during at least a portion of the culturing.

[0392] In some embodiments, one or more of the above steps of producing CIL cells can include addition of a non-physiological ligand of the synthetic cytokine receptor (e.g. rapamycin or analog) to the culture medium to induce differentiation.

[0393] In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is rapalog. In some embodiments, the non- physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of 2.5 nM and 200 nM, 2.5 nM and 150 nM, 2.5 nM and 100 nM, 2.5 nM and 50 nM, 2.5 nM and 20 nM, 2.5 nM and 10 nM, In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of between 5110MF-364712232260132004240 nM and 200 nM, 5 nM and 150 nM, 5 nM and 100 nM, 5 nM and 50 nM, 5 nM and 20 nM, 5 nM and 10 nM, 10 nM and 200 nM, 10 nM and 150 nM, 10 nM and 100 nM, 10 nM and 50 nM, 10 nM and 20 nM, 20 nM and 200 nM, 20 nM and 150 nM 20 nM and 100 nM, 20 nM and 50 nM, 50 nM and 200 nM, 50 nM and 150 nM, 50 nM and 100 nM, 100 nM and 200 nM, 100 nM and 150 nM and 150 nM and 200 nM.

[0394] In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of at or about 10 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog)is added to the media at a concentration of at or about 100 nM. In some embodiments, rapamycin is added to the media at a concentration of at or about 100 nM. In some embodiments, rapalog is added to the media at a concentration of at or about 100 nM.

[0395] In some embodiments, it is surprisingly found that low concentrations of the non- physiological ligand (e.g., rapamycin or a rapamycin analog) is able to support differentiation and / or expansion. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration at or less than 10 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration from 2.5 nM to 10 nM, such as 3 nM to 7 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of at or about 3 nM, at or about 4 nM, at or about 5 nM, at or about 6 nM, at or about 7 nM, at or about 8 nM, at or about 9 nM, or at or about 10 nM, or any value between any of the foregoing. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of at or about 3.1 nM. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the media at a concentration of at or about 6.2 nM. In some embodiments, rapamycin is added to the media at a concentration of at or about 3.1 nM. In some embodiments, rapamycin is added to the media at a concentration of at or about 6.2 nM. In some embodiments, rapalog is added to the media at a concentration of at or about 3.1 nM. In some embodiments, rapalog is added to the media at a concentration of at or about 6.2 nM.

[0396] In some embodiments, the method for generating cytotoxic innate lymphoid (iCIL) cells comprises: a) culturing a cell population comprising engineered iPSCs as described under conditions to form an aggregate; b) culturing the cells produced in a) under conditions to induce mesoderm formation in a plurality of the cells, wherein the initiation of the culturing in b) is day111MF-3647122322601320042400; c) culturing the cells produced in b) under conditions to differentiate cells into a population of hematopoietic progenitors (HP); and d) culturing the cells produced in c) under conditions to generate iCIL cells, wherein at least a portion of one or more of steps a)-d) are carried out in the presence of a non-physiological ligand of the synthetic cytokine receptor.

[0397] In some embodiments, the culturing in b) is for 2 to 4 days. In some embodiments, the cells are cultured in the differentiation media for 2 to 4 days. In some days, the culturing is for at or about 2 days, at or about 3 days or at or about 4 days. In some embodiments, the culturing is for at or about 3 days. In some embodiments, the concentration of the BMP4 in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, each inclusive. In some embodiments, the concentration of BMP4 in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, In some embodiments, the concentration of BMP4 in the media is about 10 ng / mL.

[0398] In some embodiments, the concentration of the FGF2 in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, each inclusive. In some embodiments, the concentration of FGF2 in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, In some embodiments, the concentration of FGF2 in the media is about 10 ng / mL.

[0399] In some embodiments, the concentration of the VEGF in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 5 ng / mL - 10 ng / mL, 5112MF-364712232260132004240 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL, each inclusive. In some embodiments, the concentration of VEGF in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, or 100 ng / mL, In some embodiments, the concentration of VEGF in the media is about 50 ng / mL.

[0400] In some embodiments, the concentration of the Y27632 in the media is from about 0.5 pM- 2.5 |iM, 0.5 pM - 5 pM, 0.5 pM - 10 pM, 0.5 pM - 15 pM, 0.5 pM - 20 pM, 0.5 pM - 30 pM, 0.5 pM - 50 pM, 2.5 pM - 5 pM, 2.5 pM - 10 pM, 2.5 pM - 15 pM, 2.5 pM - 20 pM, 2.5 pM - 30 pM, 2.5 pM - 50 pM, 5 pM - 10 pM, 5 pM - 15 pM, 5 pM - 20 pM, 5 pM - 30 pM, 5 pM - 50 pM, 10 pM - 15 pM, 10 pM - 20 pM, 10 pM - 30 pM, 10 pM - 50 pM, each inclusive. In some embodiments, the concentration of Y27632 in the media is at least about 0.5 pM, 2.5 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, or 50 pM, In some embodiments, the concentration of Y27632 in the media is about 10 |aM.

[0401] In some embodiments, mesoderm cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF, and SCF. In some embodiments, mesoderm cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, VEGF, SCF, TPO, and LDL. In some embodiments, mesoderm cells are differentiated into hematopoietic stem cells in a differentiation medium comprising BMP4, FGF2, and VEGF. In some embodiments, the differentiation media comprises a non-physiological ligand of the synthetic cytokine receptor (e.g. rapamycin or an analog).

[0402] In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF, and SCF. In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, and VEGF. In some embodiments, hematopoietic stem cells are differentiated into lymphoid progenitor cells in a differentiation medium comprising BMP4, FGF2, VEGF, SCF, TPO and LDL. In some embodiments, the differentiation media comprises a non-physiological ligand of the synthetic cytokine receptor (e.g. rapamycin or an analog).

[0403] In some embodiments, the culturing in c) is in a media comprising one or more of BMP4, FGF2, VEGF, TPO, SCF, and LDL. In some embodiments, the culturing in c) is in a113MF-364712232260132004240 media comprising one or more of BMP4, FGF2, VEGF and LDL. In some embodiments, the culturing in c) is in a media without SCF and TPO. In some of any embodiments, the culturing in c) is in a media comprising one or more of BMP4, FGF2, and a PI3K inhibitor. In some embodiments, the PI3K inhibitor is LY2940002. In some of any embodiments, the culturing in c) is in a media without LDL, VEGF, SCF and / or TPO. In some embodiments, the culturing in c) is in a media comprising the non-physiological ligand. In some embodiments, the culturing in c) is in a media comprising the non-physiological ligand without any additional growth factors, cytokines or both.

[0404] In some embodiments, the culturing in c) is on days 3 to 15 days. In some embodiments, during at least a portion of the culturing in c) the media comprises an aryl hydrocarbon receptor (AHR) antagonist (e.g. StemRegenin-1), a pyrimido-[4,5-b]-indole derivative (e.g. UM729) or both. In some embodiments, the portion of the culturing is on or about days 9-15. In some embodiments, the portion of the culturing is on or about days 6-15. In some embodiments, the culturing in c) is on day 6. In some embodiments, the culturing in c) is on day 9.

[0405] In some embodiments, the iCILs are prepared by culture for days 1-5 in STEMdiff APEL 2 medium containing BMP4, VEGF, FGF2, Y-27632, Rapamycin; then culture for days 5-15 in StemSpan SFEM II medium containing BMP4, VEGF-165, FGF2, LY294002, Rapamycin; with media change to StemSpan SFEM II medium containing BMP4, FGF2, SCF, LY294002 (PI3K inhibitor), Rapamycin, SRI (AhR antagonist / inhibitor), UM729 (pyrimido- [4,5-b]-indole derivative); and thereafter culturing for up to or about 40 days in AIMV + IX GlutaMAX + 10% KOSR + 1 g / L Glucose with IL15, SCF, rapamycin, SRI (AhR antagonist / inhibitor), and UM729 (pyrimido- [4, 5-b] -indole derivative), to form iCILs.

[0406] In some embodiments the following protocol or one substantially similar to the following is used: Culture for days 1-5 in STEMdiff APEL 2 medium containing 10 ng / mL BMP4, 50 ng / mL VEGF, 10 ng / mL FGF2, 10 uM Y-27632, 10 nM Rapamycin; then culture for days 5-15 in StemSpan SFEM II medium containing 10 ng / mL BMP4, 50 ng / mL VEGF- 165, 10 ng / mL FGF2, 4 uM LY294002, 10 nM Rapamycin; with media change to StemSpan SFEM II medium containing 10 ng / mL BMP4, 10 ng / mL FGF2, 50 ng / mL SCF, 4 uM LY294002 (PI3K inhibitor), 10 nM Rapamycin, 1 uM SRI (AhR antagonist / inhibitor), 1 uM UM729 (pyrimido- [4, 5-b] -indole derivative); and thereafter culturing for up to or about 40 days in AIMV + IX GlutaMAX + 10% KOSR + 1 g / L Glucose with 10 ng / mL IL15, 50 ng / mL SCF,114MF-36471223226013200424010 nM rapamycin, 1 uM SRI (AhR antagonist / inhibitor), and 1 uM UM729 (pyrimido-[4,5-b]- indole derivative), to form iCILs.

[0407] In some embodiments, the concentration of the BMP4 in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, each inclusive. In some embodiments, the concentration of BMP4 in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, In some embodiments, the concentration of BMP4 in the media is about 10 ng / mL.

[0408] In some embodiments, the concentration of the FGF2 in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, each inclusive. In some embodiments, the concentration of FGF2 in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL, In some embodiments, the concentration of FGF2 in the media is about 10 ng / mL.

[0409] In some embodiments, the concentration of the SCF in the media is from about 0.5 ng / mL- 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL, each inclusive. In some embodiments, the concentration of SCF in the media is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20115MF-364712232260132004240 ng / mL, 30 ng / mL, 50 ng / mL, or 100 ng / mL, In some embodiments, the concentration of SCF in the media is about 50 ng / mL.

[0410] In some embodiments, the concentration of the UM729 in the media is from about 0.5 pM- 1 |iM, 0.5 pM - 5 pM, 0.5 pM - 10 pM, 0.5 pM - 15 pM, 0.5 pM - 20 pM, 0.5 pM - 30 pM, 0.5 pM - 50 pM, 1 pM - 5 pM, 1 pM - 10 pM, 1 pM - 15 pM, 1 pM - 20 pM, 1 pM - 30 pM, 1 pM - 50 pM, 5 pM - 10 pM, 5 pM - 15 pM, 5 pM - 20 pM, 5 pM - 30 pM, 5 pM - 50 pM, 10 pM - 15 pM, 10 pM - 20 pM, 10 pM - 30 pM, 10 pM - 50 pM, each inclusive. In some embodiments, the concentration of UM729 in the media is at least about 0.5 pM, 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, or 50 pM, In some embodiments, the concentration of UM729 in the media is about 1 |aM.

[0411] In some embodiments, the concentration of the SRI in the media is about or less than about 0.5 pM- 1 pM, 0.5 pM - 5 pM, 0.5 pM - 10 pM, 0.5 pM - 15 pM, 0.5 pM - 20 pM, 0.5 pM - 30 pM, 0.5 pM - 50 pM, 1 pM - 5 pM, 1 pM - 10 pM, 1 pM - 15 pM, 1 pM - 20 pM, 1 pM - 30 pM, 1 pM - 50 pM, 5 pM - 10 pM, 5 pM - 15 pM, 5 pM - 20 pM, 5 pM - 30 pM, 5 pM - 50 pM, 10 pM - 15 pM, 10 pM - 20 pM, 10 pM - 30 pM, 10 pM - 50 pM, each inclusive. In some embodiments, the concentration of SRI in the media is at least about 0.5 pM, 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, or 50 pM, In some embodiments, the concentration of SRI in the media is about 1 |aM.

[0412] In some embodiments, the provided methods can result in increased yields greater than at or about 10-fold (10X), 20-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold, 150-fold, 200-fold, 250-fold, 300-fold of HP from iPSC. In some embodiments, the provided methods can result in increased yields greater than at or about 350-fold, 400-fold, 450- fold, 500-fold, 550-fold, 600-fold, 650-fold, 1,000-fold, 1,500-fold, 2,000-fold, 2,500-fold, 3,000-fold, 3,500-fold of HP from iPSC. In some embodiments, the provided methods can result in increased yields greater than at or about 630-fold of HP from iPSC. In some embodiments, the provided methods can result in increased yields greater than at or about 3,000-fold of HP from iPSC. This result represents a substantial improvement over other approaches to differentiation iPSC to HP. Furthermore, generation of iCIL from HP using the provided embodiments result in iCIL that are highly potent as demonstrated by killing of tumor target cells.

[0413] In some embodiments, the provided methods can result in increased yields greater than at or about 1 x 106-fold (l,000,000X), 1.5 x 106-fold, 2 x 106-fold, 2.5 x 106-fold,3 x 106-116MF-364712232260132004240 fold, 3.5 x 106-fold, 4 x 106-fold, 4.5 x 106-fold, 5 x 106-fold, 5.5 x 106-fold, 6 x 106-fold, 6.5 x 106-fold, 7 x 106-fold, 7.5 x 106-fold, 8 x 106-fold, 8.5 x 106-fold, 9 x 106...

Claims

260132004240CLAIMS1. A fusion protein comprising an IL- 15 peptide and a subunit of an interleukin-2 (IL-2) receptor, wherein the fusion protein comprises from amino to carboxy terminal order the IL- 15 peptide, a linker, and the subunit of the IL-2 receptor.

2. The fusion protein of claim 1, wherein the IL- 15 peptide 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: 14.

3. The fusion protein of claim 1 or claim 2, wherein the IL- 15 peptide comprises the amino acid sequence of SEQ ID NO: 14.

4. The fusion protein of any one of claims 1 to 3, wherein a nucleotide sequence encoding the IL-15 peptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 2.

5. The fusion protein of any one of claims 1 to 4, wherein the nucleotide sequence encoding the IL-15 peptide comprises the nucleotide sequence of SEQ ID NO: 2.

6. The fusion protein of any one of claims 1 to 5, wherein the subunit of the IL-2 receptor is an IL-2 receptor beta chain.

7. The fusion protein of claim 6, wherein the IL-2 receptor beta chain 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: 21.

8. The fusion protein of claim 6 or claim 7, wherein the IL-2 receptor beta chain comprises the amino acid sequence of SEQ ID NO: 21.

9. The fusion protein of any one of claims 6 to 8, wherein a nucleotide sequence encoding the IL-2 receptor beta chain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 10.

10. The fusion protein of any one of claims 6 to 9, wherein the nucleotide sequence encoding the IL-2 receptor beta chain comprises the nucleotide sequence of SEQ ID NO: 10.

11. The fusion protein of any one of claims 1 to 5, wherein the subunit of the IL-2 receptor is an IL-2 receptor gamma chain.174MF-36471223226013200424012. The fusion protein of claim 11, wherein the IL-2 receptor gamma chain 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: 16.

13. The fusion protein of claim 11 or claim 12, wherein the IL-2 receptor gamma chain comprises the amino acid sequence of SEQ ID NO: 16.

14. The fusion protein of any one of claims 11 to 13, wherein a nucleotide sequence encoding the IL-2 receptor gamma chain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 4.

15. The fusion protein of any one of claims 11 to 14, wherein the nucleotide sequence encoding the IL-2 receptor gamma chain comprises the nucleotide sequence of SEQ ID NO: 4.

16. The fusion protein of any one of claims 1 to 15, wherein the linker is a peptide linker.

17. The fusion protein of any one of claims 1 to 16, wherein the linker is between four to 30 residues.

18. The fusion protein of any one of claims 1 to 17, wherein the linker comprises an amino acid sequence from the group consisting of (GGGGS)n (SEQ ID NO: 65), wherein n is 1 to 10 or n is 1 to 5, such as 1 to 3, (GGGGGS)n (SEQ ID NO: 66), wherein n is 1 to 4, such as 1 to 3, GGS, GGGGS (SEQ ID NO: 8), and GGGGGS (SEQ ID NO: 19).

19. The fusion protein of any one of claims 1 to 18, wherein the linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 15).

20. A polynucleotide comprising a nucleic acid or nucleotide sequence encoding the fusion protein of any one of claims 1 to 19.

21. The polynucleotide of claim 20, wherein the polynucleotide comprises a promoter operably linked to the nucleotide sequence encoding the IL- 15 peptide and the subunit of the interleukin-2 (IL-2) receptor.

22. The polynucleotide of claim 20 or claim 21, wherein the promoter is a CMV, EFlalpha, CAG, PGK, or U6 promoter.

23. The polynucleotide of any one of claims 20 to 22, wherein the polynucleotide is encoded by a multicistronic construct further encoding components of a chimeric antigen receptor (CAR).175MF-36471223226013200424024. A viral vector comprising a polynucleotide of any one of claims 20 to 23.

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

26. A cell comprising the fusion protein of any one of claims 1 to 19 or the polynucleotide of claim 20 to 23 or the viral vector of claim 24 or claim 25.

27. The cell of claim 26, wherein the cell comprises an endogenous IL-2R subunit.

28. The cell of claim 26 or claim 27, wherein the fusion protein is capable of heterodimerizing with the endogenous subunit of IL-2R.

29. The cell of any one of claims 26 to 28, wherein the cell is a stem cell or a progenitor cell.

30. The cell of claim 29, wherein the stem cell is an induced pluripotent stem cell (iPSC).

31. The cell of claim 29, wherein the progenitor cell is a peripheral blood mononuclear cell (PBMC).

32. The cell of claim 31, wherein the peripheral blood mononuclear cell (PBMC) comprises lymphocytes.

33. The cell of any one of claims 26 to 32, wherein the cell is a hematopoietic progenitor cell.

34. The cell of any one of claims 26 to 33, wherein the cell is a differentiated hematopoietic progenitor cell.

35. The cell of any one of claims 26 to 34, wherein the cell is an induced cytotoxic innate lymphocyte (iCIL).

36. The cell of any one of claims 26 to 35, wherein the cell is a white blood cell.

37. The cell of any one of claims 26 to 36, wherein the cell is a natural killer (NK) cell.

38. The cell of claim 36, wherein the white blood cell is a monocyte and / or a macrophage.

39. The cell of any one of claims 26 to 38, wherein the cell comprises a rapamycin-activated cytokine receptor (RACR) system.

40. The cell of claim 39, wherein the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and176MF-364712232260132004240 a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

41. The cell of claim 40, wherein the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin-21 receptor subunit P (IL-21RB) intracellular domain.

42. The cell of claim 40 or claim 41, wherein the first dimerization domain is a FKBP12- rapamycin-binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain.

43. The cell of claim 40 or claim 41, wherein the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12- rapamycin binding (FRB) domain.

44. The cell of any one of claims 39 to 43, wherein the cell further comprises a cytosolic FRB protein.

45. The cell of claim 44, wherein the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

46. The cell of claim 44 or claim 45, wherein a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108.

47. The cell of any one of claims 42 to 46, wherein the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

48. The cell of any one of claims 42 to 47, wherein the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111.177MF-36471223226013200424049. The cell of any one of claims 42 to 48, wherein a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

50. The cell of any one of claims 42 to 49, wherein the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

51. The cell of any one of claims 42 to 50, wherein the FKBP12 dimerization 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: 28.

52. The cell of any one of claims 42 to 51, wherein the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28.

53. The cell of any one of claims 42 to 52, wherein a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

54. The cell of any one of claims 42 to 50, wherein the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

55. The cell of any one of claims 40 to 54, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).

56. The cell of claim 55, wherein 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 NO: 30.

57. The cell of claim 55 or claim 56, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 30.

58. The cell of any one of claims 52 to 57, wherein the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29.

59. The cell of any one of claims 52 to 58, wherein the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.178MF-36471223226013200424060. The cell of any one of claims 40 to 59, wherein the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

61. The cell of claim 60, wherein 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 NO: 32.

62. The cell of claim 60 or claim 61, wherein the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32.

63. The cell of any one of claims 60 to 62, wherein the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31.

64. The cell of any one of claims 60 to 63, wherein the nucleotide sequence encoding the IL- 2RB comprises the sequence set forth in SEQ ID NO: 31.

65. The cell of any one of claims 39-64, wherein the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151.

66. The cell of any one of claims 39-64, wherein the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

67. The cell of any one of claims 26 to 66, wherein the cell further comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR).

68. The cell of claim 67, wherein the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH).

69. The cell of claim 67, wherein the CAR comprises an scFv domain.

70. The cell of claim 69, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

71. A method of transducing a cell comprising contacting a target cell with any of the fusion proteins of any one of claims 1 to 19, the polynucleotide constructs of any one of claims 20 to 23, or the viral vectors of claim 24 or claim 25.

72. The method of claim 71, wherein the target cell is a stem cell.179MF-36471223226013200424073. The method of claims 71 or claim 72, wherein the target cell is a progenitor cell.

74. The method of claim 72 or claim 73, wherein the stem cell is an induced pluripotent stem cell (iPSC).

75. The method of any one of claims 71 to 74, wherein the target cell further comprises a rapamycin-activated cytokine receptor (RACR) system.

76. The method of claim 75, wherein the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

77. The method of claim 76, wherein the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin-21 receptor subunit P (IL-21RB) intracellular domain.

78. The method of claim 76 or claim 77, wherein the first dimerization domain is a FKBP12- rapamycin-binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain.

79. The method of claim 76 or claim 77, wherein the first dimerization domain is a FK506- Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

80. The method of any one of claims 71 to 79, wherein the cell further comprises a cytosolic FRB protein.

81. The method of claim 80, wherein the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.180MF-36471223226013200424082. The method of claim 80 or claim 81, wherein a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108.

83. The method of any one of claims 78 to 82, wherein the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

84. The method of any one of claims 78 to 83, wherein the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111.

85. The method of any one of claims 78 to 84, wherein a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

86. The method of any one of claims 78 to 85, wherein the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

87. The method of any one of claims 78 to 86, wherein the FKBP12 dimerization 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: 28.

88. The method of any one of claims 78 to 87, wherein the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28.

89. The method of any one of claims 78 to 88, wherein a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

90. The method of any one of claims 78 to 89, wherein the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

91. The method of any one of claims 76 to 90, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).181MF-36471223226013200424092. The method of claim 91, wherein 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 NO: 30.

93. The method of claim 91 or claim 92, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 30.

94. The method of any one of claims 91 to 93, wherein the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29.

95. The method of any one of claims 91 to 94, wherein the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

96. The method of any one of claims 76 to 95, wherein the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

97. The method of claim 96, wherein 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 NO: 32.

98. The method of claim 96 or claim 97, wherein the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32.

99. The method of any one of claims 96 to 98, wherein the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31.

100. The method of any one of claims 96 to 99, wherein the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.

101. The method of any one of claims 75 to 100, wherein the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151.

102. The method of any one of claims 75 to 101, wherein the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

103. The method of any one of claims 71 to 102, wherein the target cell further comprises a CAR.182MF-364712232260132004240104. The method of claim 103, wherein the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH).

105. The method of claim 103 or claim 104, wherein the CAR comprises an scFv domain.

106. The method of claim 105, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

107. The method of any one of claims 103 to 106, wherein the CAR is a CD19-targeted CAR.

108. The method of any one of claims 103 to 106, wherein the CAR is a CD20-targeted CAR.

109. The method of any one of claims 103 to 106, wherein the target cell comprises a dualtargeting CAR.

110. The method of claim 109, wherein the dual-targeting CAR targets CD 19 and CD20.

111. The method of any one of claims 71 to 110, wherein the fusion proteins of any one of claims 1 to 19, the fusion proteins of the polynucleotide constructs of any one of claims 20 to 23, or the fusion proteins of any one of the viral vectors of claim 24 or claim 25 are capable of heterodimerizing with the endogenous subunit of IL-2R.

112. A method of increasing differentiation efficiency of a cell expressing a rapamycin activated cytokine receptor (RACR) system, the method comprising culturing a cell comprising a RACR system in which also comprises the fusion protein of any one of claims 1 to 19, the polynucleotide construct of claims 20 to 23, or the viral vectors of claim 24 or claim 25.

113. The method of claim 112, wherein the method comprises adding rapamycin to the culture of cells.

114. The method of claim 112 or claim 113, wherein the cell is an induced pluripotent stem cell (iPSC).

115. The method of claim 114, wherein the method differentiates the iPSC to an induced cytotoxic innate lymphocyte (iCIL).

116. The method of any one of claims 112-115, wherein the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and183MF-364712232260132004240 a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

117. The method of any one of claims 112-116, wherein the RACR system comprises a synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL-2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin-21 receptor subunit P (IL-21RB) intracellular domain.

118. The method of claim 116 or claim 117, wherein the first dimerization domain is a FKBP12-rapamycin-binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain.

119. The method of claim 116 or claim 117, wherein the first dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

120. The method of any one of claims 112 to 119, wherein the cell further comprises a cytosolic FRB protein.

121. The method of claim 120, wherein the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

122. The method of claim 120 or 121, wherein a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108.

123. The method of any one of claims 118 to 122, wherein the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

124. The method of any one of claims 118 to 123, wherein the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111.184MF-364712232260132004240125. The method of any one of claims 118 to 124, wherein a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

126. The method of any one of claims 118 to 125, wherein the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

127. The method of any one of claims 118 to 126, wherein the FKBP12 dimerization 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: 28.

128. The method of any one of claims 118 to 127, wherein the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28.

129. The method of any one of claims 118 to 128, wherein a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

130. The method of any one of claims 118 to 129, wherein the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

131. The method of any one of claims 116 to 130, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).

132. The method of claim 131, wherein 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 NO: 30.

133. The method of claim 131 or claim 132, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 30.

134. The method of any one of claims 131 to 133, wherein the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29.

135. The method of any one of claims 131 to 134, wherein the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.185MF-364712232260132004240136. The method of any one of claims 116 to 135, wherein the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

137. The method of claim 136, wherein 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 NO: 32.

138. The method of claim 136 or claim 137, wherein the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32.

139. The method of any one of claims 136 to 138, wherein the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31.

140. The method of any one of claims 136 to 139, wherein the nucleotide sequence encoding the IL-2RB comprises the sequence set forth in SEQ ID NO: 31.

141. The method of any one of claims 112 to 140, wherein the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151.

142. The method of any one of claims 112 to 141, wherein the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

143. The method of any one of claims 112 to 142, wherein the method further comprises a chimeric antigen receptor (CAR).

144. The method of claim 143, wherein the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH).

145. The method of claim 143 or claim 144, wherein the CAR comprises an scFv domain.

146. The method of claim 145, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

147. The method of any one of claims 112 to 146, wherein the cell comprises an endogenous IL-2R subunit.

148. A method of differentiating an induced cytotoxic lymphocyte (iCIL) from a cell, the method comprising culturing a cell comprising a RACR system in which also comprises the186MF-364712232260132004240 fusion protein of any one of claims 1 to 19, the polynucleotide construct of any one of claims 20 to 23, or the viral vectors of claim 24 or claim 25.

149. The method of claim 148, wherein the cell is an iPSC.

150. The method of claim 148 or claim 149, wherein the cell is a hematopoietic progenitor.

151. The method of any one of claims 148 to 150, wherein the cell expresses a rapamycin activated cytokine receptor (RACR) system.

152. The method of any one of claims 112 to 151, wherein the method differentiates the iCIL from the cell without added IL- 15 or rapamycin.

153. The method of any one of claims 112 to 152, wherein the method differentiates the iCIL from the cell only with added rapamycin.

154. The method of any one of claims 112 to 153, wherein the method differentiates the iCIL from the cell without added IL- 15.

155. The method of any one of claims 148 to 154, wherein the RACR system comprises: a synthetic cytokine gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain from a gamma chain cytokine; and a synthetic cytokine beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain and an intracellular domain from a beta chain cytokine.

156. The method of claim 155, wherein the synthetic cytokine gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain and an interleukin 2 receptor subunit y (IL2RG) intracellular signaling domain and the synthetic cytokine beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain selected from an interleukin-2 receptor subunit P (IL- 2RB) intracellular signaling domain, an interleukin-7 receptor subunit P (IL-7RB) intracellular domain, and an interleukin- 21 receptor subunit P (IL-21RB) intracellular domain.

157. The method of claim 155 or 156, wherein the first dimerization domain is a FKBP12- rapamycin-binding (FRB) domain and the second dimerization domain is a FK506-Binding Protein of size 12 kD (FKBP12) domain.187MF-364712232260132004240158. The method of claim 155 or 156, wherein the first dimerization domain is a FK506- Binding Protein of size 12 kD (FKBP12) domain and the second dimerization domain is a FKBP12-rapamycin binding (FRB) domain.

159. The method of any one of claims 148 to 158, wherein the cell further comprises a cytosolic FRB protein.

160. The method of claim 159, wherein the cytosolic 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

161. The method of claim 159 or 160, wherein a nucleotide sequence encoding the cytosolic FRB domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 33, 106, 107, and 108.

162. The method of any one of claims 157 to 161, wherein the FRB dimerization 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 any one of SEQ ID NOs: 34, 109, 110, and 111.

163. The method of any one of claims 157 to 162, wherein the FRB dimerization domain comprises the amino acid sequence of any one of SEQ ID NOs: 34, 109, 110, and 111.

164. The method of any one of claims 157 to 163, wherein a nucleotide sequence encoding the FRB dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

165. The method of any one of claims 157 to 164, wherein the nucleotide sequence encoding the FRB dimerization domain comprises the nucleotide sequence of any one of SEQ ID NO: 33, 106, 107, and 108.

166. The method of any one of claims 157 to 165, wherein the FKBP12 dimerization 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: 28.

167. The method of any one of claims 157 to 166, wherein the FKBP12 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28.188MF-364712232260132004240168. The method of any one of claims 157 to 167, wherein a nucleotide sequence encoding the FKBP12 dimerization domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

169. The method of any one of claims 157 to 168, wherein the nucleotide sequence encoding the FKBP12 dimerization domain comprises the nucleotide sequence of SEQ ID NO: 27 or SEQ ID NO: 116.

170. The method of any one of claims 155 to 169, wherein the gamma chain intracellular signaling domain is an interleukin 2 receptor subunit y (IL2RG).

171. The method of claim 170, wherein 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 NO: 30.

172. The method of claim 170 or claim 171, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 30.

173. The method of any one of claims 170 to 172, wherein the IL2RG is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 29.

174. The method of any one of claims 170 to 173, wherein the nucleotide sequence encoding the IL2RG comprises the sequence set forth in SEQ ID NO: 29.

175. The method of any one of claims 155 to 174, wherein the beta chain intracellular signaling domain is an interleukin 2 receptor subunit beta (IL-2RB).

176. The method of claim 175, wherein 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 NO: 32.

177. The method of claim 175 or claim 176, wherein the IL-2RB comprises the amino acid sequence of SEQ ID NO: 32.

178. The method of any one of claims 175 to 177, wherein the IL-2RB is encoded by a nucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to nucleotide sequence set forth in SEQ ID NO: 31.189MF-364712232260132004240179. The method of any one of claims 175 to 178, wherein the nucleotide sequence encoding the IL2RB comprises the sequence set forth in SEQ ID NO: 31.

180. The method of any one of claims 151 to 179, wherein the RACR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 151.

181. The method of any one of claims 175 to 180, wherein the RACR comprises an amino acid sequence set forth in SEQ ID NO: 151.

182. The method of any one of claims 148 to 181, wherein the method further comprises a chimeric antigen receptor (CAR).

183. The method of claim 182, wherein the CAR comprises a light chain variable domain (VL) and a heavy chain variable domain (VH).

184. The method of claim 182 or claim 183, wherein the CAR comprises an scFv domain.

185. The method of claim 184, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

186. The method of any one of claims 150 to 185, wherein the cell comprises an endogenous IL-2R subunit.

187. A method of targeting cancer cells, wherein the cancer cells are exposed to methods comprising the fusion protein of claims 1 to 19, the polynucleotide construct of claims 20 to 23, or the viral vector of claim 24 or claim 25.190MF-364712232

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