Polynucleotide constructs and related viral vectors and methods

Abstract

Provided herein are polycistronic constructs for the co-expression of synthetic cytokine receptor complexes and chimeric antigen receptor systems, as well as vectors, e.g., viral vectors, containing the constructs, cells containing the constructs, and methods of using the constructs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 422,920, filed November 4, 2022, U.S. Provisional Application No. 63 / 449,289, filed March 1, 2023, and U.S. Provisional Application No. 63 / 466,714, filed May 15, 2023, all entitled "POLYNUCLEOTIDE CONSTRUCT AND RELATED VIRAL VECTORS AND METHODS," the contents of which are incorporated by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (260132000940SEQLIST.xml; size: 246,342 bytes; created on October 31, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present disclosure provides polycistronic constructs for the co-expression of synthetic cytokine receptor complexes and chimeric antigen receptor systems, as well as vectors, e.g., viral vectors, containing same, cells containing same, and methods of using same. [Background technology]

[0004] background Chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited efficacy against solid tumors, in part due to the difficulty of overcoming solid tumor heterogeneity and CAR T-cell exhaustion associated with the immunosuppressive tumor microenvironment (TME). In addition to this challenge, there are also challenges in how to deliver CAR-expressing cells to a subject in a manner that can provide a controllable and sustained therapeutic effect of the CAR in the treatment of various diseases, including cancer. Provided herein are embodiments that address this need. Summary of the Invention

[0005] overview Provided herein is a polycistronic construct containing, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding an FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a cleavage site sequence.

[0006] In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 3, 13, or 50. In some of any of the embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID No: 3, 13, or 50. In some of any of the embodiments, FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51. In some of any of the embodiments, FRB comprises the amino acid sequence of SEQ ID NO: 4, 14, or 51.

[0007] In some of the embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 15. In some of the embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID No: 15.

[0008] In some of the embodiments, the synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). In some embodiments, IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 16. In some of the embodiments, IL2RG comprises the amino acid sequence of SEQ ID No: 16.

[0009] In some of the embodiments, the second expression cassette further comprises a nucleotide sequence encoding FRB. In some of the embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:13. In some of the embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO:13. In some of the embodiments, FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14. In some of the embodiments, FRB comprises the amino acid sequence of SEQ ID NO:14.

[0010] In some of the optional embodiments, the second expression cassette is codon optimized.

[0011] In some of any embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:11. In some of any embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO:11. In some of any embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:12. In some of any embodiments, the second expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO:12.

[0012] In some of any of the embodiments, the second expression cassette further comprises a nucleotide sequence encoding FKBP12. In some of the embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 21 or 55. In some of any of the embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID No: 21 or 55. In some of any of the embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some of any of the embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO: 22.

[0013] In some of any embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some of any embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO:53 or 56. In some of any embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some of any embodiments, the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:54, 57, or 128.

[0014] In some of any of the embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB). In some of any of the embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 23 or 61. In some of any of the embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide comprise the nucleotide sequence of SEQ ID No: 23 or 61. In some of the embodiments, IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 24 or 62. In some of any of the embodiments, IL2RB comprises the amino acid sequence of SEQ ID No: 24 or 62.

[0015] In some of any of the embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12. In some of the embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:21. In some of any of the embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO:21. In some of any of the embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22. In some of any of the embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO:22.

[0016] In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 55. In some of the embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 55.

[0017] In some of the optional embodiments, the third expression cassette is codon optimized.

[0018] In some of the embodiments, the third expression cassette further contains a nucleotide sequence encoding FRB. In some of the embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:13. In some of the embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO:13. In some of the embodiments, FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14. In some of the embodiments, FRB comprises the amino acid sequence of SEQ ID NO:14.

[0019] In some of any embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 19. In some of any embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 19. In some of any embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some of any embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 20.

[0020] In some of any embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 59. In some of any embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 59. In some of any embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60 or 129. In some of any embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO: 60 or 129.

[0021] In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an endodomain containing a costimulatory signaling domain and a primary activation signaling domain, such as a CD3 zeta signaling domain. In some embodiments, the extracellular antigen-binding domain and the transmembrane domain are separated by a spacer sequence, such as a hinge domain. In some embodiments, the extracellular antigen-binding domain comprises an scFv.

[0022] In some of the optional embodiments, the CAR comprises an scFv domain. In some embodiments, the scFv domain comprises anti-fluorescein isothiocyanate (FITC) E2.

[0023] In some of the embodiments, the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH). In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 30 or 65. In some of the embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID No: 30 or 65. In some of the embodiments, the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some of the embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO: 31.

[0024] In some of any of the embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 34 or 67. In some of any of the embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID No: 34 or 67. In some of any of the embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In some of any of the embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO: 35.

[0025] In some of any of the embodiments, the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 32 or 66. In some of any of the embodiments, the scFv linker comprises the nucleotide sequence of SEQ ID No: 32 or 66. In some of any of the embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some of any of the embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO: 33.

[0026] In some of any of the embodiments, the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 28 or 64. In some of any of the embodiments, the scFv comprises the nucleotide sequence of SEQ ID No: 28 or 64. In some of the embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some of any of the embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 29.

[0027] In some of the optional embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises a short hinge or a medium hinge domain.

[0028] In some of the optional embodiments, the hinge domain comprises CD8 or IgG. In some embodiments, the CD8 hinge comprises a CD8α hinge.

[0029] In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some of any of the embodiments, the CD8 alpha hinge comprises the nucleotide sequence of SEQ ID NO:38. In some of any of the embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some of any of the embodiments, the CD8 alpha hinge comprises the amino acid sequence of SEQ ID NO:39 or 115.

[0030] In some of the optional embodiments, the CAR contains a transmembrane domain. In some embodiments, the transmembrane domain comprises CD8 or CD28. In some embodiments, the CD8 transmembrane domain comprises a CD8α transmembrane domain.

[0031] In some of any of the embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 40. In some of any of the embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 40. In some of any of the embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some of any of the embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 41.

[0032] In some of the optional embodiments, the CAR comprises an endodomain. In some embodiments, the endodomain comprises a costimulatory molecule signaling domain.

[0033] In some of any of the embodiments, the endodomain comprises the signaling domain of 4-1BB, CD3ζ, and / or CD28.

[0034] In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 42 or 69. In some of any of the embodiments, the 4-1BB endodomain comprises the nucleotide sequence of SEQ ID No: 42 or 69. In some of any of the embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some of any of the embodiments, the 4-1BB endodomain comprises the amino acid sequence of SEQ ID NO: 43.

[0035] In some of any of the embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 46, 70, 100 or 118. In some of any of the embodiments, the CD3 zeta endodomain comprises the nucleotide sequence of SEQ ID No: 46, 70, 100 or 118. In some of any of the embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some of any of the embodiments, the CD3 zeta endodomain comprises the amino acid sequence of SEQ ID NO: 47.

[0036] In some of any embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID No: 26, 63, 71, or 82. In some of any embodiments, the fourth expression cassette comprises the nucleotide sequence of SEQ ID No: 26, 63, 71, or 82. In some of any embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127. In some of any embodiments, the fourth expression cassette encodes the amino acid sequence of SEQ ID NO: 27, 72 or 127.

[0037] In some of the optional embodiments, each of the cleavage site sequences comprises a 2A cleavable linker sequence.

[0038] In some of the optional embodiments, each nucleotide encoding the 2A cleavable linker sequence is different.

[0039] In some of the optional embodiments, the 2A cleavable linker is independently a T2A, P2A, E2A, or F2A cleavage site.

[0040] In some of the optional embodiments, the 2A cleavable linker is independently P2A or T2A.

[0041] In some of the optional embodiments, at least one 2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52, or 58.

[0042] In some optional embodiments, the nucleotide sequence encoding the P2A cleavable linker is set forth in SEQ ID NO:17, 25, 52, or 58.

[0043] In some of the optional embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18.

[0044] In some of the optional embodiments, the P2A cleavable linker comprises the sequence shown in SEQ ID NO:18.

[0045] In some of the optional embodiments, at least one T2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9.

[0046] In some optional embodiments, the nucleotide sequence encoding the T2A cleavable linker is shown in SEQ ID NO:9.

[0047] In some of the optional embodiments, the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

[0048] In some of the optional embodiments, the T2A cleavable linker comprises the sequence shown in SEQ ID NO:10.

[0049] In some optional embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence.

[0050] In some of the optional embodiments, a furin cleavage site sequence is located between the first expression cassette and the second expression cassette.

[0051] In some optional embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7.

[0052] In some of the embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence shown in SEQ ID NO: 7. In some of the embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0053] In some of the optional embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO:8.

[0054] In some optional embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furin T2A).

[0055] In some optional embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:5.

[0056] In some of the optional embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO:5.

[0057] In some of the optional embodiments, the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6.

[0058] In some of the optional embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO:6.

[0059] In some optional embodiments, the first expression cassette and the second expression cassette are separated by Furin T2A, the second expression cassette and the third expression cassette are separated by P2A, and the third expression cassette and the fourth expression cassette are separated by P2A.

[0060] In some of the embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some of the embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 1.

[0061] In some of the embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In some of the embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0062] In some of the embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 48. In some of the embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 48.

[0063] In some of the embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49. In some of the embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49.

[0064] In some aspects, provided herein is a viral vector containing any one of the polycistronic constructs disclosed herein. In some embodiments, the viral vector is a lentiviral vector.

[0065] In some of the optional embodiments, the viral vector further contains one or more surface T cell activators, hi some embodiments, the one or more surface T cell activators comprise CD58, anti-CD3, or CD80.

[0066] In some aspects, provided herein is a cell that contains any one of the viral vectors disclosed herein.In some embodiments, the cell comprises stem cell or progenitor cell.In some embodiments, the stem cell comprises induced pluripotent stem cell (iPSC).

[0067] In some of the embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs). In some of the embodiments, the cells comprise T cells. In some of the embodiments, the cells comprise cytotoxic innate lymphoid (CIL) cells. In some of the embodiments, the cells comprise natural killer (NK) cells.

[0068] In some aspects, provided herein is a method for transducing cells, comprising contacting target cells with any of the polycistronic constructs disclosed herein.In some aspects, provided herein is a method for transducing cells, comprising contacting target cells with any of the viral vectors disclosed herein.In some of any embodiments, the target cells comprise stem cells.In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs).

[0069] In some of the optional embodiments, the target cells comprise progenitor cells, hi some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

[0070] In some of the optional embodiments, the target cells comprise T cells. In some embodiments, the T cells comprise CD4+ or CD8+ T cells.

[0071] In some of any of the embodiments, the method further comprises contacting the target cell with (i) a guide RNA (gRNA) that targets a target site within the endogenous gene and (ii) an RNA-guided endonuclease, thereby inserting the nucleotide sequence into the endogenous gene.

[0072] In some aspects, provided herein are methods for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell. In some aspects, provided herein are methods for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell, comprising contacting the target cell with any one of the viral vectors disclosed herein. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC).

[0073] In some embodiments, the target cells comprise progenitor cells, hi some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

[0074] In some embodiments, the target cells comprise T cells. In some embodiments, the T cells comprise CD4+ or CD8+ T cells.

[0075] In some of the optional embodiments, the method is performed ex vivo or in vitro.

[0076] In some of the optional embodiments, the method is performed in vivo.

[0077] In some aspects, provided herein are methods of transducing T cells, comprising contacting the T cells with a viral vector comprising one or more T cell activators and any one of the polycistronic constructs disclosed herein, wherein the one or more T cell activators bind to a receptor on the T cells. In some aspects, provided herein are methods of expressing chimeric antigen receptors and / or synthetic cytokine receptors in T cells, comprising contacting the T cells with a viral vector comprising one or more T cell activators and any one of the polycistronic constructs disclosed herein, wherein the one or more T cell activators bind to a receptor on the T cells. In some aspects, provided herein are methods of delivering a payload to T cells, comprising contacting the T cells with a viral vector comprising one or more T cell activators and any one of the polycistronic constructs disclosed herein, wherein the one or more T cell activators bind to a receptor on the T cells. In some of any of the embodiments, the T cells comprise CD4+ or CD8+ T cells. In some of the embodiments, the methods are performed ex vivo or in vitro. In some of the embodiments, the method is performed in vivo. In some of the embodiments, the one or more T cell activators comprise CD58, anti-CD3, or CD80. In some of the embodiments, the viral vector comprises a lentiviral vector.

[0078] In some aspects, provided herein are cells produced by any one of the methods disclosed herein.

[0079] In some aspects, provided herein is a method for administering any one of the cells disclosed herein to a subject. In some embodiments, the chimeric antigen receptor can be targeted to an antigen associated with a disease or condition of the subject, and the subject has a disease or condition that can be treated by the chimeric antigen receptor. In some embodiments, the disease or condition is cancer.

[0080] In some aspects, provided herein is a method of administering any one of the viral vectors disclosed herein to a subject. In some of any embodiments, the method treats a disease or condition of the subject. In some of any embodiments, the disease or condition can be treated by a chimeric antigen receptor (CAR) encoded by a polycistronic construct. In some of any embodiments, the CAR is an anti-FITC CAR, and the CAR is targeted to cells of the disease or condition by administering a bifunctional ligand comprising FITC and a ligand that specifically binds to a molecule expressed on cells of the disease or condition. In some of any embodiments, the disease or condition is cancer. In some of any embodiments, the cancer is a solid tumor. In some of any embodiments, the cell is a cancer cell.

[0081] In some of the embodiments, the chimeric antigen receptor can be targeted to an antigen associated with a disease or condition of a subject, and the subject has a disease or condition that can be treated by the chimeric antigen receptor. In some of the embodiments, the disease or condition is cancer.

[0082] In some embodiments, the CAR is a CAR that targets a ligand capable of binding to a cell surface antigen associated with a disease or pathology. In some embodiments, the CAR of any of the provided embodiments is an anti-FITC CAR directed against FITC, and the ligand is a bifunctional ligand composed of FITC and a binding molecule capable of binding to a surface molecule or receptor on a target cell. In some embodiments, the method further comprises administering a bifunctional ligand to tag cancer cells in a subject, wherein the bifunctional ligand specifically binds to a molecule expressed on tumors. In some embodiments, the bifunctional ligand is FITC-folate. In some embodiments, the cancer is osteosarcoma. In some embodiments, the bifunctional ligand comprises a fluorescein isothiocyanate (FITC) moiety, and the chimeric antigen receptor (CAR) encoded by the polycistronic construct is an anti-FITC CAR.

[0083] In some of any of the embodiments, the method further comprises administering a non-physiological ligand to the subject. In some of any of the embodiments, the non-physiological ligand is capable of binding to the synthetic cytokine receptor and inducing gamma cytokine signaling in the cell. In some of any of the embodiments, the non-physiological ligand. In some of any of the embodiments, the non-physiological ligand binds to a synthetic cytokine receptor composed of a synthetic gamma chain polypeptide and a synthetic cytokine beta chain polypeptide encoded by a polycistronic construct. In some of any of the embodiments, the non-physiological ligand comprises rapamycin or a rapamycin analog. In some of any of the embodiments, the binding of the non-physiological ligand to the synthetic cytokine receptor stimulates an intracellular cytokine signal in cells transduced to express the synthetic cytokine receptor. In some of any of the embodiments, the binding of the non-physiological ligand to the synthetic cytokine receptor promotes the proliferation of cells transduced to express the synthetic cytokine receptor. [Brief explanation of the drawings]

[0084] [Figure 1A] Figure 1A shows lentiviral particles surface-engineered to engage and activate T cells to deliver a payload containing free FKBP12-rapamycin conjugate (FRB), rapamycin-activated cytokine receptor (RACR), and a chimeric antigen receptor (CAR) that binds to a tumor tag (TagCAR). Rapamycin, FRB, and RACR inhibit tumor growth and immune responses to the lentiviral particles while simultaneously driving the expansion of transduced TagCAR T cells. [Figure 1B] Figure 1B shows how the TagCAR T system targets tumor cells. The bispecific tumor tag contains a universal tag antigen on one end and an exchangeable ligand that associates with a tumor-associated antigen or tumor microenvironment-associated antigen on the other end. When T cells express the TagCAR, the TagCAR can bind to the universal tag (e.g., FITC-folate). [Figure 2] Figures 2A-2B show eight polycistronic constructs. The TagCAR polynucleotide encodes a CAR with the following components, from N- to C-terminus: an scFv (e.g., anti-FITC E2), a hinge (spacer), a transmembrane domain, and an endodomain with a costimulatory signaling domain and a CD3 zeta signaling domain (Z). The constructs differ in the hinge (spacer) domain, either an IgG4 hinge (IgG4H) or a CD8 alpha hinge (CD8H); the transmembrane domain, either a CD28 TM or a CD8 TM; and the costimulatory domain, either a 41BB costimulatory domain or a CD28 costimulatory domain. The constructs also differ in the placement of the TagCAR, which is present either at the beginning or end of the construct transgene sequence. The individual polynucleotide components of the constructs are separated by a 2A cleavage site sequence. [Figure 3-1] Figures 3A-3B show day 3 activation and day 7 transduction of CD8+ and CD4+ T cell populations cultured with the polynucleotide constructs of Figures 2A-2B at a multiplicity of infection (MOI) of 2 or 10. [Figure 3-2] FIG. 3C shows FRB expression in T cells when FRB is placed in the first or second position within the transgene as depicted in FIGS. 2A-2B. [Figure 4] Figure 4 shows the percentage of TagCAR+ T cells generated when transduced with construct D.2 or construct C.2 and cultured in IL-2 alone, rapamycin alone, or both. [Figure 5A] 5A-5B show tumor cell killing assays by PBMCs transduced with the constructs disclosed in FIGS. 2A-2B and treated with IL-2 alone or IL-2 and rapamycin (rapa). [Figure 5B] See legend to Figure 5A. [Figure 6A] Figure 6A shows CD19 chimeric antigen receptor (CAR) and TagCAR expression in PBMCs transduced with lentivirus encoding construct V or construct C.2 polynucleotides. [Figure 6B] Figure 6B shows the number of CD19-CAR+ (left panel) or FITC-folate+ (right panel) PBMCs over 11 days. PBMCs were transduced with lentivirus encoding either Construct V or Construct C.2 polynucleotides. [Figure 7-1] Figure 7A shows the N- to C-terminal orientation of the polynucleotide constructs. In construct C.2, FRB is encoded next to IL2Rβ. In construct C.2U, FRB is encoded next to IL2Rγ. Figure 7B shows the percentage of CD25 T cells in five donors 3 days after transfection with lentivirus encoding Construct V or Construct C.2 polynucleotides. [Figure 7-2] Figure 7C shows the percentage of TagCAR T cells on day 7. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides and stained on day 7. Figure 7D shows the TagCAR MFI in T cells on day 7. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. [Figure 7-3]Figure 7E shows an immunoblot depicting the expression of FKBP12:IL2Rβ and FKBP12:IL2Rγ in T cells. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. A rabbit pAb against FKBP12 was used to detect FKBP12. Cells were collected on day 8 for Western blot. Figure 7F shows an immunoblot depicting the expression of FKBP12:IL2Rβ and FKBP12:IL2Rγ in T cells. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. A mouse mAb against FKBP12 was used to detect FKBP12. Cells were collected on day 8 for Western blot. [Figure 8A] Figure 8A shows total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 in one donor over a 14-day period. Tag-CAR+ T cells were treated with either IL-2 (250 U / mL) or rapamycin (10 nM). [Figure 8B] Figure 8B shows total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 in one donor over a 12-day period. Tag-CAR+ T cells were treated with either IL-2 (250 U / mL), rapamycin (10 nM), rapamycin and IL-2, or AP21967 (50 nM). [Figure 9A] FIG. 9A shows day 3 activation and day 7 transduction of CD8+ and CD4+ T cell populations cultured with construct C.2 at a multiplicity of infection (MOI) of 2 or 10. [Figure 9B] Figure 9B shows a representative flow cytometry plot of CD8+ T cells expressing TagCAR transduced with construct C.2 at day 7 post-transduction. [Figure 10A]Figure 10A shows the timeline of the in vitro assay for measuring T cell activation and TagCAR T cell abundance. On day 0, PBMCs are transduced with lentiviral particles. On day 3, IL-2 or IL-2 and rapamycin are added to measure T cell activation. On days 7, 11, and 14, TagCAR T cell abundance was measured. [Figure 10B] Figure 10B shows the enrichment (left) and expansion (right) of TagCAR T cells over a 2-week period. Enrichment was measured by flow cytometry. Expansion was measured by flow cytometry using counting beads. [Figure 11A] Figure 11A shows a graph depicting tumor cell growth after incubation of breast cancer cells (MDA-MB-231 or MDA) with rapamycin, with TagCAR T cells and FITC-folate, or with TagCAR T cells, FITC-folate, and rapamycin. Breast cancer cells not incubated with TagCAR T cells, FITC-folate, or rapamycin are indicated by arrows. Tumor cells were reintroduced (i.e., tumor cell reimplantation) at 72, 144, and 216 hours. [Figure 11B] Figure 11B shows a graph depicting T cell proliferation after incubation of breast cancer cells (MDA-MB-231 or MDA) with rapamycin, TagCAR T cells and FITC-folate, or TagCAR T cells, FITC-folate, and rapamycin (rapa). Breast cancer cells not incubated with TagCAR T cells, FITC-folate, or rapamycin are indicated by arrows. Tumor cells were reintroduced (i.e., tumor cell reimplantation) at 72, 144, and 216 hours. [Figure 12A] Figure 12A shows the timeline of the in vivo mouse model of breast cancer. NSG MHCI / IIDKO mice were injected with FRα+ MDA-MB-231 cells 14 days before the start of the experiment. On the first day of the experiment (D0), mice were infused with ex vivo generated TagCAR T cells. Mice were injected subcutaneously with FITC-folate twice a week for 4 weeks. Blood was collected weekly for flow cytometry. [Figure 12B] Figure 12B shows a graph depicting the tumor volume of mice over a 4-week period. Mice were injected with (i) 10e6 T cells not transduced with lentiviral particles (mock T cells) with FITC-folate; (ii) 10e6 TagCAR T cells without FITC-folate; (iii) 5e6 TagCAR T cells with FITC-folate; (iv) 10e6 TagCAR T cells with FITC-folate. Tumor volumes were measured using calipers. [Figure 13A] Figure 13A shows the timeline of the in vivo mouse breast cancer model. NSG MHCI / IIDKO mice were injected with FRα+ MDA-MB-231 cells 14 days before the start of the experiment. On the first day of the experiment (D0), mice were humanized with PBMCs and administered lentiviral particles. Mice were injected subcutaneously with FITC-folate twice weekly for 7 weeks. Blood was collected weekly for flow cytometry. Tumor volume was measured using a caliper. [Figure 13B] Figure 13B shows the detection of circulating TagCAR T cells by flow cytometry. The left panel depicts the percentage of TagCAR+ per CD3+ T cell on day 7. The right panel depicts the total number of CD3+ / TagCAR+ T cells per μL of blood on day 7. Mice were injected with (i) no vector and FITC-folate; (ii) 100e6 transfection units (TU) of TagCAR vector and FITC-folate; (iii) 25e6 TU of TagCAR vector and FITC-folate; (iv) 100e6 TU of TagCAR vector and FITC-folate. [Figure 13C] Figure 13C shows a graph depicting tumor volume in mice over a 7-week period. Tumor volume was measured using calipers. [Figure 14A]Figure 14A shows a graph depicting the tumor volume of mice over 25 days. Mice were injected with (i) FITC-folic acid alone; (ii) 5.0e6 transfection units (TU) of TagCAR vector without FITC-folic acid; (iii) 0.2e6 TU of TagCAR vector together with FITC-folic acid; (iv) 1.0e6 TU of TagCAR vector together with FITC-folic acid; (v) 5.0e6 TU of TagCAR vector together with FITC-folic acid. Tumor volume was measured using a caliper. [Figure 14B] Figure 14B shows circulating CD3 TagCAR T cells per μL of blood as detected by flow cytometry. Mice were injected with (i) FITC-folate alone (triangles); (ii) 5.0e6 TU of TagCAR vector alone; (iii) 0.2e6 TU of TagCAR vector with FITC-folate; (iv) 1.0e6 TU of TagCAR vector with FITC-folate; (v) 5.0e6 TU of TagCAR vector with FITC-folate. DETAILED DESCRIPTION OF THE INVENTION

[0085] Detailed Description The present disclosure generally relates to a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least two synthetic receptors, and its use method. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a synthetic cytokine receptor, a synthetic chimeric antigen receptor (CAR), and a freely diffusible FRB, wherein the cytokine receptor therein is responsive to rapamycin binding. Advantageously, the FRB reduces the inhibitory effect of rapamycin on mTOR in cells engineered to express the polycistronic construct provided herein. The expression of the freely diffusible FRB can promote consistent activation and proliferation of engineered cells.

[0086] The present disclosure also provides that the 5' to 3' order of a polycistronic construct is important for the expression of a polypeptide encoded by the construct. In some embodiments, the 5' to 3' order of a polynucleotide construct improves the expression of the encoded polypeptide. In some embodiments, the polycistronic constructs provided herein include a nucleotide encoding FRB at the 5' end, which improves FRB expression and correlates with enhanced protection against rapamycin-mediated immunosuppression. Moreover, it has been surprisingly found that CAR expression is sufficiently high to mediate antigen-specific killing by cells in which the polynucleotide construct is expressed, even when the CAR is located at the 3' end of the construct.

[0087] In some aspects, provided herein is a polycistronic construct comprising four expression cassettes separated by cleavage site sequences. In some embodiments, the four expression cassettes comprise, in 5' to 3' order, a first expression cassette comprising a nucleotide sequence encoding an FRB, a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide, a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide, and a fourth expression cassette comprising a nucleotide sequence encoding a CAR.

[0088] In some aspects, provided herein is a viral vector comprising any one of the polycistronic constructs disclosed herein.

[0089] In some aspects, provided herein is a cell comprising any of the viral vectors disclosed herein.

[0090] In some aspects, provided herein are methods of transducing cells, comprising contacting a target cell with any one of the viral vectors disclosed herein.

[0091] In some aspects, provided herein are methods of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell, comprising contacting the target cell with any one of the viral vectors disclosed herein.

[0092] In some aspects, provided herein are cells produced by any of the methods disclosed herein.

[0093] In some aspects, provided herein are methods of administering any of the cells disclosed herein to a subject. In some aspects, provided herein are methods of administering any of the viral vectors disclosed herein to a subject.

[0094] 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. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0095] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0096] I. Polycistronic Constructs Provided herein are polycistronic constructs that encode one or more separate proteins. In some embodiments, the polycistronic constructs comprise one, two, three, or four expression cassettes, each encoding a separate protein. In some embodiments, the polycistronic constructs comprise four expression cassettes, each encoding a separate protein. In some embodiments, the expression cassettes are separated by a cleavable linker.

[0097] In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding an FRB. In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a synthetic cytokine polypeptide. In some embodiments, the synthetic cytokine polypeptide comprises a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide. In some embodiments, the synthetic cytokine gamma chain comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises an FRB. In some embodiments, the synthetic cytokine beta chain comprises interleukin-2 receptor subunit beta (IL2RB). In some embodiments, the synthetic cytokine gamma chain further comprises FKBP12. In other embodiments, the synthetic cytokine gamma chain comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises FKBP12. In some embodiments, the synthetic cytokine beta chain comprises interleukin 2 receptor subunit beta (IL2RB). In some embodiments, the synthetic cytokine beta chain further comprises FRB.

[0098] In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding an FRB, a synthetic cytokine polypeptide, and a CAR.

[0099] In some embodiments, the polycistronic construct comprises, in 5' to 3' order, a nucleotide sequence encoding an FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises, in 5' to 3' order, a first nucleotide sequence encoding FRB:IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises, in 5' to 3' order, a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding FRB:IL2RB.

[0100] In one aspect, provided herein is a polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding an FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

[0101] A. Cytosolic FRB In some embodiments, the expression cassette of the polycistronic construct encodes an FRB domain, which is an approximately 270 base pair (bp) domain derived from the mTOR protein kinase, which can be expressed in the cytosol as a freely diffusible, soluble protein.

[0102] In some embodiments, the first expression cassette in the polycistronic construct comprises a nucleotide sequence encoding FRB. In some embodiments, when FRB is expressed, it is a freely diffusible, soluble protein.

[0103] In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO:3, 13, or 50. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID No:3, 13, or 50.

[0104] In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO:4, 14, or 51. In some embodiments, the FRB consists of the amino acid sequence of SEQ ID NO:4, 14, or 51.

[0105] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to a ligand or a complex that includes a ligand.

[0106] Advantageously, cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalogs).

[0107] B. Synthetic Cytokine Receptors In some embodiments, the expression cassette of the polycistronic construct encodes a synthetic cytokine receptor. The synthetic cytokine receptor of the present disclosure comprises a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain. The dimerization domains controllably dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0108] The synthetic cytokine receptor can comprise a transmembrane receptor protein comprising a synthetic gamma chain polypeptide and a synthetic beta chain polypeptide, provided as a first transmembrane receptor and a second transmembrane receptor. The synthetic 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 can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2RG intracellular domain). The synthetic 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 can 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).

[0109] In some embodiments, the polycistronic 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 expression cassettes. In some embodiments, the polynucleotide constructs provided herein comprise one expression cassette encoding the IL2RG chain of the synthetic cytokine receptor and a second expression cassette encoding the IL2RB chain of the synthetic cytokine receptor.

[0110] In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. Those of skill in the art are familiar with signal peptides that can provide a signal for transport of a nascent protein within a cell. Any of a wide variety of signal peptides can be used.

[0111] 1. Intracellular domain In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

[0112] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.

[0113] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Therefore, when referred to herein, IL2RB can also mean IL15RB. That is, the terms are used interchangeably in this disclosure.

[0114] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.

[0115] In some embodiments, the synthetic beta chain comprises the interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

[0116] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.

[0117] In some embodiments, the synthetic beta chain comprises the interleukin-21 receptor subunit beta (IL21RB) intracellular domain.

[0118] 2. Dimerization Domain The dimerization domain may be a heterodimerization domain, including but not limited to the 12 kD sized FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB) domains, which are known in the art to dimerize in the presence of rapamycin or a rapalog.

[0119] Alternatively, the first and second dimerization domains may be 12 kD-sized FK506 binding protein (FKBP) and calcineurin domains, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0120] In some embodiments, the dimerization domain is a homodimerization domain selected from the following: i) 12 kD size FK506 binding protein (FKBP); ii) Cyclophilin A (CypA); or iii)iii) Gyrase B (CyrB); where the corresponding non-physiological ligands are, respectively: i) FK1012, AP1510, AP1903, or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or its analogues is.

[0121] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a cyclophilin domain.

[0122] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0123] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0124] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0125] 3. Transmembrane domain The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0126] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain.

[0127] In some embodiments, one or more additional contiguous amino acids of the ectodomain immediately adjacent to the TM domain of the cytokine receptor can also be included as part of the polypeptide sequence of the synthetic cytokine receptor chain. In some embodiments, 1 to 20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the synthetic cytokine receptor chain. The portion of the ectodomain can 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 immediately adjacent to (e.g., N-terminal to) the TM sequence.

[0128] In some embodiments, the synthetic cytokine receptor is capable of binding 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, wherein binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in a cell, e.g., via the JAK / STAT pathway.

[0129] 4. Exemplary Synthetic Cytokine Receptors The synthetic cytokine receptors of the present disclosure comprise a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain that controllably dimerizes in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0130] The synthetic 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 can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2G intracellular domain). In some embodiments, the synthetic gamma chain polypeptide comprises an FRB:IL2RG fusion protein. In some embodiments, the synthetic gamma chain polypeptide comprises an FKBP12:IL2RG fusion protein.

[0131] The synthetic 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 can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N-terminal or C-terminal to the IL-2RB or IL-7RB intracellular domain). In some embodiments, the synthetic beta chain polypeptide comprises an FKBP12:IL2RB fusion protein. In some embodiments, the synthetic beta chain polypeptide comprises an FKBP12:IL2RB fusion protein.

[0132] In some embodiments, the second expression cassette comprises a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide, wherein the synthetic cytokine gamma chain polypeptide is an FRB:IL2RG fusion protein. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 80% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 85% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 90% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 95% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 96% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 97% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 98% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 99% identical to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO:15.In some embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide consists of the nucleotide sequence of SEQ ID NO: 15.

[0133] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, IL2RG consists of the amino acid sequence of SEQ ID NO:16.

[0134] In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO:13.

[0135] In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID NO:13.

[0136] In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB consists of the amino acid sequence of SEQ ID NO:14.

[0137] In some embodiments, the second expression cassette is codon optimized.

[0138] In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:11. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:11.

[0139] In some embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the second expression cassette consists of the nucleotide sequence of SEQ ID NO: 11.

[0140] In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO:12. In some embodiments, the second expression cassette encodes an amino acid sequence that consists of the sequence of SEQ ID NO:12.

[0141] In some embodiments, the second expression cassette comprises nucleotides encoding a synthetic cytokine gamma chain polypeptide, wherein the synthetic cytokine gamma chain polypeptide is an FKBP12:IL2RG fusion protein. In some embodiments, the second expression cassette comprises a nucleotide sequence encoding FKBP12. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 85% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 90% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 95% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 96% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 97% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 98% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 99% identical to the nucleotide sequence of SEQ ID NO:21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO:21 or 55.In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. In some embodiments, the nucleotide sequence encoding FKBP12 consists of the nucleotide sequence of SEQ ID NO: 21 or 55.

[0142] In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 consists of the amino acid sequence of SEQ ID NO:22.

[0143] In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette comprises a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO:53 or 56. In some embodiments, the second expression cassette consists of a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO:53 or 56.

[0144] In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that comprises the amino acid sequence of SEQ ID NO:54, 57, or 128. In some embodiments, the second expression cassette encodes an amino acid sequence that consists of the amino acid sequence of SEQ ID NO:54, 57, or 128.

[0145] In some embodiments, the third expression cassette comprises nucleotides encoding a synthetic cytokine beta chain polypeptide, wherein the synthetic cytokine beta chain polypeptide is an FKBP12:IL2RB fusion protein. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 80% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 85% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 90% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 95% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 96% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 97% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 98% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 99% identical to the nucleotide sequence of SEQ ID NO:23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO:23 or 61.In some embodiments, the nucleotide sequence encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotide sequence encoding the synthetic cytokine beta chain polypeptide consists of the nucleotide sequence of SEQ ID NO: 23 or 61.

[0146] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB).

[0147] In some embodiments, IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB comprises the amino acid sequence of SEQ ID NO:24 or 62. In some embodiments, IL2RB consists of the amino acid sequence of SEQ ID NO:24 or 62.

[0148] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

[0149] In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 85% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 90% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 95% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 96% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 97% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 98% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 99% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence encoding FKBP12 consists of the nucleotide sequence of SEQ ID NO:21.

[0150] In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 80% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 85% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 90% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 95% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 96% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 97% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 98% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 99% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO:55. In some embodiments, the nucleotide sequence encoding FKBP12 consists of the nucleotide sequence of SEQ ID NO:55.

[0151] In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, FKBP12 consists of the amino acid sequence of SEQ ID NO:22.

[0152] In some embodiments, the third expression cassette is codon optimized.

[0153] In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO:19. In some embodiments, the third expression cassette consists of the nucleotide sequence of SEQ ID NO:19.

[0154] In some embodiments, the third expression cassette comprises nucleotides encoding a synthetic cytokine beta chain polypeptide, wherein the synthetic cytokine beta chain polypeptide is an FRB:IL2RB fusion protein. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 95% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 96% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 97% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO:59. In some embodiments, the third expression cassette consists of the nucleotide sequence of SEQ ID NO:59.

[0155] In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO:60 or 129. In some embodiments, the third expression cassette encodes an amino acid sequence that consists of the sequence of SEQ ID NO:60 or 129.

[0156] In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that comprises the sequence of SEQ ID NO:60. In some embodiments, the third expression cassette encodes an amino acid sequence that consists of the sequence of SEQ ID NO:60.

[0157] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 80% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 85% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 90% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 95% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 96% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 97% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 98% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 99% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO:13. In some embodiments, the nucleotide sequence encoding FRB consists of the nucleotide sequence of SEQ ID NO:13.

[0158] In some embodiments, the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the FRB consists of the amino acid sequence of SEQ ID NO:14.

[0159] C. Chimeric Antigen Receptor In some aspects, the expression cassette of the polycistronic construct encodes a chimeric antigen receptor.

[0160] 1. CAR construct and coding nucleotides In some embodiments, the CAR construct contains an extracellular binding moiety, 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 intracellular signaling domain comprising an extracellular binding moiety, a transmembrane domain, and a costimulatory signaling domain. In some embodiments, the CAR construct contains an intracellular signaling domain comprising an extracellular binding moiety, a transmembrane domain, and an activation signaling domain. In some embodiments, the CAR construct contains an extracellular binding moiety, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and an activation signaling domain.

[0161] In any of the embodiments described herein, the binding portion of the CAR can be, for example, a single chain fragment variable region (scFv), Fab, Fv, Fc, or (Fab')2 fragment of an antibody.

[0162] In some embodiments, the costimulatory signaling domain plays a role in enhancing lymphocyte proliferation and survival when CAR binds to the targeted moiety. The identity of the costimulatory signaling domain is limited only in that it has the ability to enhance cell proliferation and survival activation when CAR binds to the targeted moiety. Suitable costimulatory signaling domains include, but are not limited to, CD28 (see, for example, 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, for example, Imai, C. et al., Leukemia. 2004.18:676-84); and CD134 (OX40), a member of the TNFR superfamily of receptors (see, for example, Latza, U. et al., Eur. J. Immunol. 1994.24:677). Those skilled in the art 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 from 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.

[0163] In some embodiments of the invention, the CAR construct comprises two costimulatory signaling domains. Specific combinations include all possible variations of the four described domains, but specific examples include 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

[0164] In some embodiments, the activation signaling domain serves to activate cells when the CAR binds to the targeted moiety. The identity of the activation signaling domain is limited only by its ability to induce activation of the selected cell when the CAR binds to the targeted moiety. Suitable activation signaling domains include CD3 zeta chain and Fc receptor gamma. In some embodiments, the signaling domain is the signaling domain of NKG2C or NKp44. Those skilled in the art will understand that sequence variants of these described activation signaling domains can be used without adversely affecting the present invention, and in this case, the variants have the same or similar activity as the domains they are modeled after. 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.

[0165] In some embodiments, CARs may contain additional elements, such as a signal peptide to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein remains as an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows for strong binding to the targeted moiety.

[0166] In some embodiments, the nucleotide sequence 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, e.g., a single intracellular domain that provides both costimulatory and activation signals to the 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 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the nucleotide sequence encodes a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0167] Exemplary CAR constructs suitable for the provided polycistronic constructs are provided below: (1) scFv-CD8 TM -4-1BB IC -CD3ζs (see, e.g., Liu E, Tong Y, Dotti G, et al., Leukemia. 2018;32: 520-531); (2) scFv-CD28 TM+IC-CD3ζs (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 S A. 2008; 105: 17481-17486; and Chu J, Deng Y, Benson DM et al., Leukemia. 2014; 28: 917-927); (3) scFv-DAP12 TM+IC (See, e.g., Muller N, Michen S, Tietze S et al., J Immunother. 2015;38: 197-210); (4) scFv-CD8 TM -2B4 IC -CD3ζs (see, e.g., Xu Y, Liu Q, Zhong M et al., J Hematol Oncol. 2019; 12: 49); (5) scFv-2B4 TM+IC CD3ζs (see, e.g., Altvater B, Landmeier S, Pscherer S et al., Clin Cancer Res. 2009; 15: 4857-4866); (6) scFv-CD28 TM+IC -4-1BB IC -CD3ζs (see, e.g., Kloss S, Oberschmidt O, Morgan M et al., Hum Gene Ther. 2017; 28: 897-913); (7) scFv-CD16 TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (8)scFv-NKp44 TM -DAP10 IC-CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (9)scFv-NKp46 TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (10)scFv-NKG2D TM -2B4 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (11)scFv-NKG2D TM -4-1BB IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (12)scFv-NKG2D TM -2B4 IC -DAP12 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (13)scFv-NKG2D TM -2B4 IC -DAP10 IC -CD3ζs (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); (14)scFv-NKG2D TM -4-1BB IC -2B4 ICCD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192); and (15)scFv-NKG2D TM -CD3ζS (see, e.g., Li Y, Hermanson DL, Moriarity BS, Kaufman DS, Cell Stem Cell. 2018; 23: 181-192).

[0168] a. CAR extracellular domain In some embodiments, the binding portion of the CAR can be directed to any antigen that one desires to target, such as by its overexpression on a cell or its association with a disease or condition such as cancer.

[0169] In some embodiments, the binding portion of the CAR is specific for a tumor antigen. The choice of antigen-binding domain depends on the particular cancer type to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid (plysialic acid) acid), PLAC1, RU1, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mutated 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 mutants, Ras mutants, 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 cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG(TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include 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 Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, 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 27.29\BCAA, 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, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related 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 targeting 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 comprises binding domains targeting two or more antigens on the same target protein, for example, two epitopes in BCMA.

[0170] Those skilled in the art are familiar with CARs directed against a variety of tumor antigens. Any one of these CARs can be used as a CAR. Numerous CARs have been incorporated into FDA-approved products, including, but not limited to, anti-CD19 and anti-BCMA CAR T cells such as tisagenlecleucel (Kymriah), axicabtagene ciloreucel (Yescarta), brexcabtagene outrucel (Tecartus), lisocabtagene maraleucel (Breyanzi), idecabtagene vicrueucel (Abecma), or siltacabtagene outrueucel (Carvykti). It is within the skill of those skilled in the art to create similar constructs for specific targeting of desired tumor antigens.

[0171] In some embodiments, the binding portion of CAR can be directed to a universal antigen to target a wide variety of tumors, without the need to prepare separate CAR constructs.The targeting portion recognized by CAR can also remain constant.In some embodiments, a ligand can be administered to a subject to enable interaction with target cells and interaction with the binding portion of CAR.Only the ligand portion of the small conjugate molecule needs to be changed to allow the system to target cancer cells of different identities.Exemplary CAR systems are described in the following paragraphs.

[0172] In some embodiments, the CAR is an anti-CD19 CAR, and the extracellular binding domain of the CD19 CAR is specific for CD19, e.g., human CD19. In some embodiments, the extracellular domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), comprising the heavy chain variable region (VH) and light chain variable region (VL) of FMC63 connected by a linker. FMC63 and derived scFvs are 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 herein by reference. Exemplary anti-CD19 CARs are shown in Table 1, along with their various portions, including the extracellular domain.

[0173] In some embodiments, the CAR is an anti-CD20 CAR, and the extracellular binding domain of the CD20 CAR is specific for CD20, e.g., human CD20. In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific for CD20, including, for example, Leu16, 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 CAR can comprise or consist of the VH, VL, and / or one or more CDRs of any of the antibodies. Exemplary anti-CD20 CARs are shown in Tables 2 and 3, along with their various portions, including their extracellular domains.

[0174] 1) Universal Car Conventionally, CAR is produced by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding transmembrane and intracellular domain, and then transducing the polynucleotide and corresponding VH or VL into cells as needed.Many variations of CAR are known in the art, and the present disclosure contemplates the use of any known variations.In addition, VL / VH pairs and scFvs for numerous haptens are known in the art, or can be routinely produced by conventional methods.Therefore, the present disclosure contemplates the use of any known hapten binding domain.

[0175] 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.

[0176] An exemplary anti-FITC CAR is shown in Table 4, along with its various moieties.

[0177] 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).

[0178] In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL comprises the nucleotide sequence of SEQ ID NO:30 or 65. In some embodiments, the scFv VL consists of the nucleotide sequence of SEQ ID NO:30 or 65.

[0179] In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, the scFv VL consists of the amino acid sequence of SEQ ID NO:31.

[0180] In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH comprises the nucleotide sequence of SEQ ID NO:34 or 67. In some embodiments, the scFv VH consists of the nucleotide sequence of SEQ ID NO:34 or 67.

[0181] In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the scFv VH consists of the amino acid sequence of SEQ ID NO:35.

[0182] In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker comprises the nucleotide sequence of SEQ ID NO:32 or 66. In some embodiments, the scFv linker consists of the nucleotide sequence of SEQ ID NO:32 or 66.

[0183] In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv linker consists of the amino acid sequence of SEQ ID NO:33.

[0184] In some embodiments, the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv comprises the nucleotide sequence of SEQ ID NO:28 or 64. In some embodiments, the scFv consists of the nucleotide sequence of SEQ ID NO:28 or 64.

[0185] In some embodiments, the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO:29.

[0186] Various methods for targeting CARs and CAR-expressing cells have been described in the art, including, for example, US 2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, a fluorescein or fluorescein isothiocyanate (FITC) moiety can be conjugated to an agent that binds to a desired target cell (such as a cancer cell), thereby allowing a CAR expressing an anti-fluorescein / FITC chimeric antigen receptor to selectively target the target cell labeled by the conjugate. Alternatively, other haptens recognized by the CAR can be used instead of fluorescein / FITC. CARs can be produced using various scFv sequences known in the art or scFv sequences produced by conventional routine methods. Additional exemplary scFv sequences for fluorescein / FITC and other haptens are provided, for example, in WO 2021 / 076788, the disclosure of which is incorporated herein by reference.

[0187] In one aspect, the present disclosure provides an illustration of this conjugate molecule / CAR system.

[0188] In some embodiments, the CAR system of the present disclosure utilizes a conjugate molecule as a bridge between CAR-expressing cells and targeted cancer cells. The conjugate molecule is a conjugate comprising a hapten and a cell targeting moiety, such as any suitable tumor cell-specific ligand. Exemplary haptens that CAR can recognize and bind to include low molecular weight organic molecules such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, as well as fluorescein and its derivatives (including FITC (fluorescein isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives), knottins, centrins, and DARPins. Suitable cell-targeting moieties that can themselves act as haptens for CAR include knottins (see Kolmar H. et al., The FEBS Journal. 2008. 275(11): 26684-90), centirins, and DARPins (see Reichert, JM MAbs 2009. 1(3): 190-209).

[0189] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a ligand that PSMA-positive human prostate cancer cells bind with nanomolar affinity (K D =14 nM; see Kularatne, SA et al., Mol Pharm. 2009. 6(3):780-9). In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, which is incorporated herein by reference.

[0190] In some embodiments, the cell targeting moiety is a CCK2R ligand, i.e., a ligand that is bound by CCK2R-positive cancer cells (e.g., thyroid, lung, pancreatic, ovarian, brain, stomach, gastrointestinal stromal, and colon cancers; see Wayua, C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0191] In some embodiments, the cell targeting moiety is folate, folic acid or an analog thereof, i.e., a ligand that is bound by folate receptors on cancer cells, including ovarian, cervical, endometrial, lung, kidney, brain, breast, colon, and head and neck cancers (see Sega, EI et al., Cancer Metastasis Rev. 2008. 27(4):655-64).

[0192] In some embodiments, the cell targeting moiety is an NK-1R ligand. Receptors for NK-1R ligands are found, for example, on colon and pancreatic cancers. In some embodiments, NK-1R ligands can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0193] In some embodiments, the cell targeting moiety can be a peptide ligand, for example, the ligand can be a peptide ligand that is an endogenous ligand for the NK1 receptor. In some embodiments, the small conjugate molecule ligand can be a regulatory peptide belonging to the tachykinin family that targets 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).

[0194] In some embodiments, the cell targeting moiety is a CAIX ligand. Receptors for CAIX ligands are found, for example, on kidney, ovarian, vulvar, and breast cancers. CAIX ligands may also be referred to herein as CA9.

[0195] In some embodiments, the cell targeting moiety is a ligand of gamma glutamyl transpeptidase, which is overexpressed in, for example, ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0196] In some embodiments, the cell targeting moiety is a CCK2R ligand. Receptors for CCK2R ligands are found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon, among others.

[0197] In some embodiments, the cell targeting moiety is a PSMA ligand.

[0198] In some embodiments, the cell targeting moiety is a FAP ligand.

[0199] In one embodiment, the cell targeting moiety can 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 can have a mass of about 1 to about 10,000 daltons, about 1 to about 9000 daltons, about 1 to about 8,000 daltons, about 1 to about 7000 daltons, about 1 to about 6000 daltons, about 1 to about 5000 daltons, about 1 to about 4500 daltons, about 1 to about 4000 daltons, about 1 to about 3500 daltons, about 1 to about 3000 daltons, about 1 to about 2500 daltons, about 1 to about 2000 daltons, about 1 to about 1500 daltons, about 1 to about 1000 daltons, or about 1 to about 500 daltons.

[0200] In one exemplary embodiment, the linkage in the conjugates described herein can be a direct linkage (e.g., reaction between an isothiocyanate group of FITC and a free amine group of a small molecule ligand), or the linkage can be via an intermediate linker. In one embodiment, the intermediate linker, if present, can be any biocompatible linker known in the art, e.g., a bivalent linker. In one exemplary embodiment, the bivalent linker can comprise from about 1 to about 30 carbon atoms. In another exemplary embodiment, the bivalent linker can comprise from about 2 to about 20 carbon atoms. In other embodiments, lower molecular weight bivalent linkers (i.e., those having an approximate molecular weight of from about 30 to about 300 Da) are used. In another embodiment, suitable linker lengths 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.

[0201] In some embodiments, haptens and cell-targeting moieties can be directly conjugated via a method such as the reaction between the isothiocyanate group of FITC and the free amine group of a small ligand (e.g., folic acid, DUPA, and CCK2R ligand). However, the use of a linking domain to connect the two molecules can only be useful if 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) non-natural peptidoglycans; 6) polyvinylpyrrolidone; and 7) Pluronic F-127. Suitable linker lengths 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.

[0202] In some embodiments, the linker can be a bivalent linker that can include one or more spacers.

[0203] An exemplary conjugate of the present disclosure is FITC-folate. TIFF2025538152000001.tif38128

[0204] An exemplary conjugate of the present disclosure is FITC-CA9. TIFF2025538152000002.tif30128

[0205] Exemplary conjugates of the present disclosure include the following molecules: FITC-(PEG) 12 -Folic acid, FITC-(PEG) 20 -Folic acid, FITC-(PEG) 108 -Folic acid, FITC-DUPA, FITC-(PEG) 12 -DUPA, FITC-CCK2R ligand, FITC-(PEG) 12 -CCK2R ligand, FITC-(PEG) 11 -NK1R ligand and FITC-(PEG)2-CA9.

[0206] The binding affinity between a ligand and a cancer cell receptor can vary, and in some cases lower affinity binding (such as about 1 μM) may be preferred, but the binding affinity between a ligand and a cancer cell receptor is generally at least about 100 μM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0207] Examples of conjugates and methods of making the same 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.

[0208] b. Spacer (e.g., hinge domain) 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 CD8 or IgG. In some embodiments, the CD8 hinge comprises a CD8α 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 IgG1 hinge. In some embodiments, the hinge domain comprises a PD1 hinge. In some embodiments, the hinge domain comprises a CD28 hinge.

[0209] In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge comprises the nucleotide sequence of SEQ ID NO:38 or 114. In some embodiments, the CD8 alpha hinge consists of the nucleotide sequence of SEQ ID NO:38 or 114.

[0210] In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8 alpha hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge comprises the amino acid sequence of SEQ ID NO:39 or 115. In some embodiments, the CD8α hinge consists of the amino acid sequence of SEQ ID NO:39 or 115.

[0211] In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge comprises the nucleotide sequence of SEQ ID NO:123. In some embodiments, the CD8 hinge consists of the nucleotide sequence of SEQ ID NO:123.

[0212] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO:119. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO:119.

[0213] In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge comprises the amino acid sequence of SEQ ID NO:120. In some embodiments, the modified IgG4 hinge consists of the amino acid sequence of SEQ ID NO:120.

[0214] In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge comprises the amino acid sequence of SEQ ID NO:122. In some embodiments, the IgG1 hinge consists of the amino acid sequence of SEQ ID NO:122.

[0215] In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge comprises the amino acid sequence of SEQ ID NO:121. In some embodiments, the PD1 hinge consists of the amino acid sequence of SEQ ID NO:121.

[0216] In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge comprises the amino acid sequence of SEQ ID NO:124. In some embodiments, the CD28 hinge consists of the amino acid sequence of SEQ ID NO:124.

[0217] c. Transmembrane domain In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises CD8 or 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 a CD8α transmembrane domain.

[0218] In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain comprises the nucleotide sequence of SEQ ID NO:40. In some embodiments, the transmembrane domain consists of the nucleotide sequence of SEQ ID NO:40.

[0219] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the transmembrane domain consists of the amino acid sequence of SEQ ID NO:41.

[0220] d. Intracellular domain (i.e., endodomain) In some embodiments, the CAR comprises an endodomain. In some embodiments, the endodomain comprises a costimulatory molecule. In some embodiments, the endodomain comprises 4-1BB, CD3ζ, and / or CD28. In some embodiments, the endodomain comprises 4-1BB. In some embodiments, the endodomain comprises CD3ζ. In some embodiments, the endodomain comprises CD28.

[0221] In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain comprises the nucleotide sequence of SEQ ID NO:42 or 69. In some embodiments, the 4-1BB endodomain consists of the nucleotide sequence of SEQ ID NO:42 or 69.

[0222] In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the 4-1BB endodomain consists of the amino acid sequence of SEQ ID NO:43.

[0223] In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3 zeta endodomain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118.In some embodiments, the CD3 zeta endodomain consists of the nucleotide sequence of SEQ ID NO:46, 70, 100, or 118.

[0224] In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the CD3 zeta endodomain consists of the amino acid sequence of SEQ ID NO:47.

[0225] 2. Exemplary CAR Polynucleotides In some embodiments, the CAR is an anti-CD19 CAR, and in these embodiments, the polycistronic vector comprises a fourth expression cassette containing a nucleotide sequence encoding the CD19 CAR. In some embodiments, the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds to CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth expression cassette encodes an anti-CD19 CAR having the characteristics shown in Table 1.

[0226] Table 1. Exemplary sequences of anti-CD19 CARs and components TIFF2025538152000003.tif87166

[0227] In some embodiments, the CAR is an anti-CD20 CAR, and in these embodiments, the polycistronic vector comprises a fourth expression cassette containing a nucleotide sequence encoding the CD20 CAR. In some embodiments, the CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds to CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth expression cassette encodes an anti-CD20 CAR having the characteristics set forth in Table 2 (anti-CD20 CAR with Flag) or Table 3 (anti-CD20 CAR without Flag).

[0228] Table 2. Exemplary sequences of anti-CD20 CARs and components (with Flag) TIFF2025538152000004.tif94166

[0229] Table 3. Exemplary sequences of anti-CD20 CAR and components (without Flag) TIFF2025538152000005.tif87166

[0230] In some embodiments, the fourth expression cassette encodes a CAR having the characteristics shown in Table 4. In some embodiments, the CAR is an anti-FITC CAR.

[0231] Table 4. Exemplary sequences of anti-FITC CARs and components TIFF2025538152000006.tif102166

[0232] In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette comprises the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82. In some embodiments, the fourth expression cassette consists of the nucleotide sequence of SEQ ID NO:26, 63, 71, or 82.In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:27, 72 or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence consisting of the sequence of SEQ ID NO:27, 72, or 127.

[0233] In a further aspect, an exemplary nucleotide sequence encoding a CAR can comprise SEQ ID NO:71, and an exemplary CAR amino acid sequence can comprise SEQ ID NO:72.

[0234] An exemplary nucleotide insert can include SEQ ID NO:73.

[0235] In some embodiments, the CAR can be encoded by a nucleic acid sequence that encodes a signal peptide to signal the transport of the CAR within the cell. It is understood that the signal peptide is usually removed from the protein.

[0236] An exemplary CAR amino acid sequence without the signal peptide can include SEQ ID NO:74.

[0237] An exemplary CAR amino acid sequence signal peptide can comprise SEQ ID NO:75. In various embodiments, a CAR-expressing cell is provided comprising the nucleic acid of SEQ ID NO:71 or 73. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO:72 is contemplated. In some embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO:74 is contemplated. In some embodiments, a vector comprising SEQ ID NO:71 or 73 is contemplated. In some embodiments, a lentiviral vector comprising SEQ ID NO:71 or 73 is contemplated. In some embodiments, SEQ ID NO:72 can comprise or consist of a human or humanized amino acid sequence. In some embodiments, SEQ ID NO:74 can comprise or consist of a human or humanized amino acid sequence.

[0238] In some embodiments, variant nucleic acid or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74 are contemplated.

[0239] Although the affinity with which a CAR expressed by a lymphocyte binds to a targeted moiety can vary, and in some cases low affinity binding (such as about 50 nM) may be preferred, the binding affinity of the CAR for the targeted ligand is generally at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM, or 10 fM, and even more preferably at least about 100 fM.

[0240] D. Cleavable Linkers As provided herein, the expression cassettes of a polycistronic construct can be separated by a linker. In some aspects, the linker comprises a site for cleavage, thereby making it a cleavable linker.

[0241] The use of cleavage sites in the design of polycistronic constructs can achieve co-expression of multiple genes. In some embodiments, the cleavage site comprises a self-cleavage site. In some embodiments, the self-cleavage site comprises a 2A site. 2A peptides are a class of 18-22 amino acid long peptides that can induce ribosome skipping during translation, resulting in the loss of a peptide bond between a glycine and a proline residue, allowing proteolytic enzymes to recognize the 2A site. The most commonly used 2A peptides in molecular biology include T2A, P2A, E2A, and F2A.

[0242] In some embodiments, the polycistronic 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.

[0243] In some embodiments, in addition to the 2A site, the cleavable linker may also contain 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.

[0244] In some embodiments, the polycistronic constructs provided herein comprise a T2A, P2A, E2A, or F2A cleavage site within the cleavable linker. In some embodiments, the polycistronic construct comprises a T2A cleavage site within the cleavable linker. In some embodiments, the polycistronic construct comprises a P2A cleavage site within the cleavable linker. In some embodiments, the polycistronic construct comprises a furin cleavage site within the cleavable linker. In some embodiments, the polycistronic construct comprises a T2A cleavage site and a furin cleavage site within the cleavable linker.

[0245] In some embodiments, the polycistronic constructs provided herein comprise at least one, at least two, or at least three 2A-cleavable linker sequences. In some embodiments, the polycistronic constructs herein comprise a T2A cleavage site and a P2A, E2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise a P2A cleavage site and a T2A, E2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise an E2A cleavage site and a P2A, T2A, or F2A cleavage site. In some embodiments, the polycistronic constructs herein comprise an F2A cleavage site and a P2A, E2A, or T2A cleavage site.

[0246] In some embodiments, the polycistronic constructs provided herein comprise a 2A cleavable linker sequence. In some embodiments, each nucleotide sequence encoding the 2A cleavable linker sequence is different. In some embodiments, the 2A cleavable linkers are independently T2A, P2A, E2A, or F2A cleavage sites. In some embodiments, the 2A cleavable linkers are independently P2A or T2A.

[0247] In some embodiments, the P2A cleavable linker is P2A, and the nucleotide sequence encoding the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52, or 58. In some embodiments, the nucleotide sequence encoding the P2A cleavable linker is set forth in SEQ ID NO: 17, 25, 52, or 58. In some embodiments, the P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. In some embodiments, the P2A cleavable linker comprises the sequence set forth in SEQ ID NO: 18.

[0248] In some embodiments, at least one T2A cleavable linker is T2A, and the nucleotide sequence encoding the T2A cleavable linker comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9. In some embodiments, the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO:9. In some embodiments, the T2A cleavable linker comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10. In some embodiments, the T2A cleavable linker comprises a sequence set forth in SEQ ID NO:10.

[0249] In some embodiments, at least one of the cleavage site sequences comprises a furin cleavage site sequence. In some embodiments, the furin cleavage site sequence is located between the first expression cassette and the second expression cassette. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7. In some embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence set forth in SEQ ID NO:7. In some embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8. In some embodiments, the furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO:8.

[0250] In some embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furin T2A). In some embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:5. In some embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO:5.

[0251] In some embodiments, the cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6. In some embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO:6.

[0252] In some embodiments, the first expression cassette and the second expression cassette are separated by Furin T2A, the second expression cassette and the third expression cassette are separated by P2A, and the third expression cassette and the fourth expression cassette are separated by P2A.

[0253] E. Exemplary Polycistronic Constructs In some embodiments, the polycistronic constructs provided herein comprise the features set forth in Table 5, Table 6, or Table 7.

[0254] Table 5. Exemplary sequences of RACR constructs TIFF2025538152000007.tif57166

[0255] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.

[0256] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:125. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:125.

[0257] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:1. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:1.

[0258] Table 6. Exemplary sequences of RACR constructs TIFF2025538152000008.tif52166

[0259] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:48.

[0260] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:126. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:126.

[0261] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:48. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:48.

[0262] Table 7. Exemplary sequences of RACR constructs TIFF2025538152000009.tif53166

[0263] In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:103. In some embodiments, the construct encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:103.

[0264] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:102. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:102.

[0265] In some embodiments, the construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 96% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct comprises the nucleotide sequence of SEQ ID NO:101. In some embodiments, the construct consists of the nucleotide sequence of SEQ ID NO:101.

[0266] II. Nucleic Acid Vectors In some embodiments, the polycistronic construct 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. Nucleic acid vectors can also have operational functions, such as cloning or shuttle vectors. The structure of a vector can include any desired form that is feasible to manufacture 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. Nucleic acid vectors can be composed of, for example, DNA or RNA and can contain, partially or completely, nucleotide derivatives, analogs, and mimetics. Such nucleic acid vectors can be obtained from natural sources, recombinantly produced, or chemically synthesized.

[0267] Non-limiting examples of vector systems of the present disclosure include retroviruses, lentiviruses, foamy viruses, and Sleeping Beauty transposons.

[0268] A. Retroviral Vectors Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to adjust its life cycle, as seen during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). Retroviral vectors have been created by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0269] Exemplary 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. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these vectors are configured to carry essential sequences for selecting cells containing the vector, incorporating foreign nucleic acid into lentiviral particles, and introducing nucleic acid into target cells.

[0270] A commonly used lentiviral vector system is the so-called third-generation system. Third-generation lentiviral vector systems contain four plasmids. The "transfer plasmid" encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system. The transfer plasmid generally contains one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to disable replication of the resulting vector. For example, the transfer plasmid lacks genetic elements necessary for the production of infectious particles in host cells. In addition, the transfer plasmid is designed to lack the 3' LTR, which allows the virus to become "self-inactivating" (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 contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that aid in packaging, reverse transcription, and integration of the proviral genome into the cell after contact of the cell with the retroviral particle.

[0271] Third-generation systems also typically contain two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is a CMV promoter. Third-generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev as an additional safety feature; this is an improvement over the single packaging plasmid of the so-called second-generation system. While safer, third-generation systems can be more cumbersome to use and result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0272] Many retroviral vector systems rely on the use of "packaging cell lines." Generally, packaging cell lines are cell lines that are capable of producing infectious retroviral particles when a transfer plasmid, a packaging plasmid, and an envelope plasmid are introduced into the cells. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, packaging cell lines are adapted for high-efficiency packaging of retroviral vector systems into retroviral particles.

[0273] As used herein, the term "retroviral vector" or "lentiviral vector" refers to a nucleic acid encoding a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to target cells.Retroviral and lentiviral particles generally contain an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope with an Env protein embedded therein, and other accessory proteins, including integrase, protease, and matrix protein.As used herein, the terms "retroviral particle" and "lentiviral particle" refer to a viral particle that contains an envelope, has one or more characteristics of a lentivirus, and is capable of invading target host cells. 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 containing one or more gag structural polypeptides, containing a retroviral or lentiviral envelope containing one or more env-encoded glycoproteins, containing a genome containing one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase, or integrase, or containing a genome encoding a regulatory activity such as Tat or Rev. The transfer plasmid may contain a cPPT sequence as described in U.S. Patent No. 8,093,042.

[0274] The efficiency of the system is an important concern in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), or viral titer as infectious units per milliliter (IU / mL). For example, titer can be evaluated 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 evaluated in a continuously dividing cultured cell line, no stimulation is required, and therefore the measured titer is not affected by the surface manipulation of retroviral particles. 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).

[0275] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adaptor. In some embodiments, the sequence encoding the receptor that specifically binds to the gating adaptor is operably linked to a promoter. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and an MND promoter.

[0276] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a tagged protein.

[0277] In some embodiments, each retroviral particle 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 a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

[0278] In some embodiments, the retroviral particle contains a cell surface receptor that binds to a surface marker on a target host cell, thereby enabling transduction of the host cell. In some embodiments, the cell surface receptor is a T cell surface receptor. The viral vector can contain a heterologous viral envelope glycoprotein that provides a pseudotyped viral vector. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or is an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein from the Kocal strain or a functional variant thereof.

[0279] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), but many other viral proteins have also been used to pseudotype lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0280] In some embodiments, pseudotyping the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specialized 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 selected from the group consisting of human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibbon ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphotropic retrovirus (Ampho) gp70, 10A1 gp70, and the like. MLV(10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon leukemia virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D The polypeptides are full-length polypeptides, functional fragments, homologs, or functional variants of HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipura virus and Pili virus CNV-G and PRV-G.

[0281] 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), Candipra vesiculovirus (CHPV), Cocarveciclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocarvirus G protein.

[0282] 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), Candipra vesiculovirus (CHPV), Cocarveciclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocarvirus G protein. The present disclosure further provides various retroviral vectors, including, but not limited to, gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, or a gamma-retroviral vector. In some embodiments, the viral vector comprises a VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises a cocal G protein or a functional variant thereof.

[0283] In some embodiments, provided herein is a viral vector comprising any of the polycistronic 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 cell activators. In some embodiments, the one or more surface T cell activators comprise CD58, anti-CD3, or CD80.

[0284] III. Virus particles In some embodiments, provided herein are viral particles encapsulating the polycistronic constructs disclosed herein. In some embodiments, any of the polycistronic constructs can be provided as a payload in the production of viral particles. Also provided herein are viral particles, e.g., lentiviral vectors, incorporating any of the provided polycistronic constructs for delivering components of the rapamycin-activated cytokine receptor (RACR) system, including FRB, synthetic cytokine receptors, and CARs, to target cells. In further embodiments, the viral particles can be engineered to express one or more surface T cell activators. In some embodiments, the one or more surface T cell activators include T cell surface receptors. In some embodiments, the T cell surface receptors include CD58, anti-CD3, or CD80.

[0285] As is well known in the art, viral particles are tools that allow or facilitate the transfer of entities from one environment to another. According to the present disclosure and as an example, some viral particles used in recombinant DNA technology can transfer entities such as DNA segments into host cells. Examples of vectors used in recombinant DNA technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" refers to a construct that can be expressed in vivo or in vitro / ex vivo.

[0286] A. Retroviral particles In some embodiments, the viral particles comprise retroviral particles. In some embodiments, the present disclosure provides methods for preparing viral formulations. In some embodiments, the virus is a retrovirus. Many different retroviruses have been identified. Examples of retroviruses include, but are not limited to, murine leukemia virus (MLV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin et al., 1997, "Retroviruses", Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus, pp 758-763.

[0287] Retroviruses include lentiviruses, gamma-retroviruses, and alpha-retroviruses, each of which can be used to deliver polynucleotides to cells using methods known in the art. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to adjust its life cycle, as seen during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). Retroviral vectors have been created by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0288] The lentiviral vectors of the present disclosure can be derived from or can be derived from any suitable lentivirus. Recombinant retroviral vector particles are capable of transducing recipient cells with a nucleotide of interest (NOI). After entering the cell, the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the recipient cell's DNA. In some embodiments of the present disclosure, at least a portion of one or more protein coding regions essential for replication can be removed from the virus. This renders the viral vector replication-deficient. Portions of the viral genome can also be replaced by an NOI to generate a vector containing an NOI capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0289] Exemplary 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. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, these vectors are configured to carry essential sequences for selecting cells containing the vector, incorporating foreign nucleic acid into lentiviral particles, and introducing nucleic acid into target cells.

[0290] A commonly used lentiviral vector system is the so-called third-generation system. Third-generation lentiviral vector systems contain four plasmids. The "transfer plasmid" encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system. The transfer plasmid generally contains one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is generally designed to disable replication of the resulting vector. For example, the transfer plasmid lacks genetic elements necessary for the production of infectious particles in host cells. In addition, the transfer plasmid is designed to lack the 3' LTR, which allows the virus to become "self-inactivating" (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 contain a 3' untranslated region (UTR) and a 5' UTR. The UTR contains retroviral regulatory elements that aid in packaging, reverse transcription, and integration of the proviral genome into the cell after contact of the cell with the retroviral particle.

[0291] Third-generation systems also typically contain two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In an exemplary third-generation system, the Env gene is VSV-G and the promoter is a CMV promoter. Third-generation systems use two packaging plasmids, one encoding gag and pol, and the other encoding rev as an additional safety feature; this is an improvement over the single packaging plasmid of the so-called second-generation system. While safer, third-generation systems can be more cumbersome to use and result in lower virus titers due to the addition of an additional plasmid. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0292] Many retroviral vector systems rely on the use of "packaging cell lines." Generally, packaging cell lines are cell lines that are capable of producing infectious retroviral particles when a transfer plasmid, a packaging plasmid, and an envelope plasmid are introduced into the cells. Various methods for introducing plasmids into cells can be used, including transfection or electroporation. In some cases, packaging cell lines are adapted for high-efficiency packaging of retroviral vector systems into retroviral particles.

[0293] As used herein, the term "retroviral vector" or "lentiviral vector" refers to a nucleic acid encoding a retroviral or lentiviral cis nucleic acid sequence required for genome packaging and one or more polynucleotide sequences to be delivered to target cells.Retroviral and lentiviral particles generally contain an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope with an Env protein embedded therein, and other accessory proteins, including integrase, protease, and matrix protein.As used herein, the terms "retroviral particle" and "lentiviral particle" refer to a viral particle that contains an envelope, has one or more characteristics of a lentivirus, and is capable of invading target host cells. 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 containing one or more gag structural polypeptides, containing a retroviral or lentiviral envelope containing one or more env-encoded glycoproteins, containing a genome containing one or more retroviral or lentiviral cis-acting sequences that function in replication, proviral integration, or transcription, containing a genome encoding a retroviral or lentiviral protease, reverse transcriptase, or integrase, or containing a genome encoding a regulatory activity such as Tat or Rev. The transfer plasmid may contain a cPPT sequence as described in U.S. Patent No. 8,093,042.

[0294] The efficiency of the system is an important concern in vector engineering. The efficiency of retroviral or lentiviral vector systems can be evaluated by various methods known in the art, including measuring vector copy number (VCN) or vector genome (vg) by quantitative polymerase chain reaction (qPCR), or viral titer as infectious units per milliliter (IU / mL). For example, titer can be evaluated 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 evaluated in a continuously dividing cultured cell line, no stimulation is required, and therefore the measured titer is not affected by the surface manipulation of retroviral particles. 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).

[0295] In some embodiments, the retroviral and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gating adaptor. In some embodiments, the sequence encoding the receptor that specifically binds to the gating adaptor is operably linked to a promoter. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and an MND promoter.

[0296] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a tagged protein.

[0297] In some embodiments, each retroviral particle 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 a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

[0298] In some embodiments, the retroviral particle contains a cell surface receptor that binds to a surface marker on a target host cell, thereby enabling transduction of the host cell. In some embodiments, the cell surface receptor is a T cell surface receptor. The viral vector can contain a heterologous viral envelope glycoprotein that provides a pseudotyped viral vector. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or is an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is the VSV G protein from the Kocal strain or a functional variant thereof.

[0299] In some embodiments, the viral envelope comprises a viral envelope protein. In some embodiments, the viral envelope protein is a VSV-G envelope protein, a measles virus envelope protein, a Nipah 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 include a mutation at position 47 (e.g., K47Q) and / or a mutation at position 354 (e.g., R354A).

[0300] In some embodiments, the viral envelope protein is a protein derived from the Kocal strain (Kocal glycoprotein). In some embodiments, the protein is a Kocal envelope protein containing a mutation at position 354 (R354). In some embodiments, the protein is a Kocal envelope protein containing a mutation at position 47 (K47). In some embodiments, the protein is a Kocal envelope variant containing an R354Q mutation. In some embodiments, the protein is a Kocal envelope variant containing a K47Q mutation. In some embodiments, this variant may be referred to as a "blinded" Kocal envelope. Exemplary Kocal envelope variants are provided, for example, in US 2020 / 0216502 A1, the entire contents of which are incorporated herein by reference.

[0301] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. The most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), but many other viral proteins have also been used to pseudotype lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure contemplates the substitution of various fusion glycoproteins. Notably, some fusion glycoproteins result in higher vector efficiency.

[0302] In some embodiments, pseudotyping the fusion glycoprotein or functional variant thereof facilitates targeted transduction of specialized cell types, including, but not limited to, innate lymphoid cells or NK cells. In some embodiments, the fusion glycoprotein or functional variant thereof is selected from the group consisting of human immunodeficiency virus (HIV) gp160, 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 leukemia virus (BaEV) gp70, measles virus (MV) H and F, Nipah virus (NiV) H and F, rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, baculovirus GP64, chikungunya virus (CHIKV) E1 and E2, Ross River virus (RRV) E1 and E2, Semliki Forest virus (SFV) E1 and E2, Sindbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza A, B, C, or D The polypeptides are full-length polypeptides, functional fragments, homologs, or functional variants of HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or Chandipura virus and Pili virus CNV-G and PRV-G.

[0303] 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), Candipra vesiculovirus (CHPV), Cocarveciclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocarvirus G protein.

[0304] 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), Candipra vesiculovirus (CHPV), Cocarveciclovirus (COCV), vesicular stomatitis Indiana virus (VSIV), Isfahan vesiculovirus (ISFV), Maraba vesiculovirus (MARAV), vesicular stomatitis New Jersey virus (VSNJV), or Bass-Congo virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is a cocarvirus G protein.

[0305] The present disclosure further provides various retroviral vectors, including, but not limited to, gamma-retroviral vectors, alpha-retroviral vectors, and lentiviral vectors. In some embodiments, the vector may be a viral vector, a retroviral vector, a lentiviral vector, or a gamma-retroviral vector. In some embodiments, the viral vector comprises a VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises a cocal G protein or a functional variant thereof.

[0306] B. Engineered viral envelopes In some embodiments, viral particles encapsulating the nucleotide vectors provided herein can comprise engineered viral envelopes. In some embodiments, the viral envelopes comprise transduction enhancers. In some embodiments, the viral envelopes comprise immune cell activating proteins. In some embodiments, the viral envelopes comprise costimulatory molecules. In some embodiments, the viral envelopes comprise immune cell activating proteins and costimulatory molecules.

[0307] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancer comprises a T cell activating receptor, an NK cell activating receptor, and / or a costimulatory molecule. In some embodiments, the one or more transduction enhancers comprise one or more of an anti-CD3 scFv, CD86, CD80, and / or CD58. In some embodiments, the transduction enhancer comprises at least an anti-CD3 scFv and CD58. In some embodiments, the transduction enhancer comprises at least an anti-CD3 scFv and CD80. In some embodiments, the transduction enhancer comprises at least an anti-CD3 scFv and CD86. In some embodiments, the transduction enhancer comprises at least an anti-CD3 scFv, CD80, and CD58. In some embodiments, the transduction enhancer comprises at least an anti-CD3 scFv, CD86, and CD58.

[0308] In some embodiments, the viral particle comprises a cell surface receptor that binds to a ligand on a target host cell, thereby enabling transduction of the host cell. In some embodiments, the cell surface receptor is a T cell surface receptor. In some embodiments, the viral particle comprises a heterologous viral envelope glycoprotein that results in a pseudotyped viral particle. For example, the viral envelope glycoprotein can be derived from RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), or is an amphotropic envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is the VSV G protein (cocal glycoprotein) from the cocal strain or a functional variant thereof.

[0309] In some embodiments, the virus envelope comprises more than one polypeptide on its surface.In some embodiments, the more than one polypeptide binds to target immune cells and replicates immune synapse.In some embodiments, the virus envelope comprises immune cell activation protein, costimulatory molecule and adhesion molecule, wherein the immune cell activation protein, costimulatory molecule and adhesion molecule bind to target immune cells respectively.

[0310] 1. Immune cell activators In some embodiments, the transduction enhancer comprises a mitogenic stimulus that is incorporated into the retroviral or lentiviral capsid so that the virus both activates and transduces T cells. This eliminates the need for additional vectors and mitogens. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more costimulatory molecules that become incorporated into the retrovirus upon budding from the producer / packaging cell membrane. In some embodiments, the transduction enhancer is expressed as a separate cell surface molecule on the producer cell, rather than as part of the viral envelope glycoprotein.

[0311] In some embodiments, the viral vectors described herein comprise a mitogenic transduction enhancer in the viral envelope. In some embodiments, the mitogenic transduction enhancer is derived from the host cell during retroviral vector production. In some embodiments, the mitogenic transduction enhancer is produced by the packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer can be incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer. In some embodiments, the mitogenic enhancer is an antibody or fragment thereof. In some embodiments, the mitogenic enhancer is a single domain antibody, e.g., a camelid antibody. In some embodiments, the mitogenic enhancer is an scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0312] In some embodiments, the transduction enhancer is host cell-derived. The term "host cell-derived" indicates that the mitogenic transduction enhancer is derived from a host cell as described above and is not produced as a fusion or chimera from one of the viral genes, such as gag, which encodes a major structural protein, or env, which encodes an envelope protein.

[0313] Envelope proteins are formed by two subunits: a transmembrane (TM) subunit that anchors the protein in the lipid membrane and a surface (SU) subunit that binds to cellular receptors. In some embodiments, the mitogenic transduction enhancer derived from the packaging cells of the present invention does not comprise a surface envelope subunit (SU).

[0314] In some embodiments, the mitogenic transduction enhancer has the structure: MS-TM, where M is the mitogenic domain; S is an optional spacer domain, and TM is the transmembrane domain.

[0315] The mitogenic domain is part of a mitogenic transduction enhancer that causes T cell activation. It can directly or indirectly bind to or otherwise interact with T cells, leading to T cell activation. In some embodiments, the mitogenic domain binds to T cell surface antigens, such as CD3, CD28, CD134, and CD137.

[0316] CD3 is a T cell coreceptor. It is a protein complex composed of four separate chains. In mammals, this complex contains the CD3y chain, the CD35 chain, and two CD3e chains. These chains associate with the T cell receptor (TCR) and the z chain to generate an activation signal in T lymphocytes. The TCR, z chain, and CD3 molecule together constitute the TCR complex. In some embodiments, a mitogenic domain is attached to the CD3e chain.

[0317] In some embodiments, the mitogenic domain comprises all or a portion of an antibody or other molecule that specifically binds to a T cell surface antigen. In some embodiments, the antibody activates TCR or CD28. In some embodiments, the antibody binds to TCR, CD3, or CD28. Examples of such antibodies include OKT3, 15E8, and TGN1412. Other suitable antibodies include: Anti-CD28:CD28.2, 10F3 Anti-CD3 / TCR: UCHT1, YTH12.5, TR66.

[0318] In some embodiments, the mitogenic domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0319] In some embodiments, the mitogenic domain comprises all or part of a costimulatory molecule, such as OX40L and 41BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41BBL.

[0320] OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3e chain. It is used clinically to reduce acute rejection in organ transplant patients. It was the first monoclonal antibody approved for clinical use in humans.

[0321] In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the immune cell activation protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activation protein provides signal 1 for T cell activation.

[0322] In some embodiments, the immune cell activating protein specifically binds to CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80. In some embodiments, the immune cell activating protein specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activating protein specifically binds to CD3γ, CD3δ, CD3ε, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCR α chain, TCR β chain, TCR γ chain, or TCR δ chain. In some embodiments, the immune cell activating protein specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activating protein specifically binds to CD3.

[0323] In some embodiments, the immune cell activating protein is an antibody or antigen-binding fragment thereof that specifically binds to a receptor on an immune cell, ie, CD28, CD2, CD3, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, or NKp80. In some embodiments, the immune cell activating protein is an antibody or antigen-binding fragment thereof that specifically binds to CD28, CD2, CD3γ, CD3δ, CD3ε, CD4, CD8, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, OX40, 4-1BB, CD40L, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCR α chain, TCR β chain, TCR γ chain, or TCR δ chain. In some embodiments, the immune cell activating protein is an antibody or antigen-binding fragment thereof that specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activating protein is an antibody or antigen-binding fragment thereof that specifically binds to CD3.

[0324] Antibodies that target the polypeptides described herein are known to those skilled in the art. Methods for producing antibodies are known to those skilled in the art.

[0325] In some embodiments, the viral envelope comprises an anti-CD3ε antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD3ε antibody or an antigen-binding fragment thereof is coupled to a transmembrane domain. An exemplary anti-CD3ε antibody is OKT3. OKT3, also known as muromonab-CD3, is a monoclonal antibody that targets the CD3ε chain.

[0326] In some embodiments, the viral envelope comprises a single chain Fv fragment (scFv) of an anti-CD3 antibody.

[0327] 2. Co-stimulatory molecules In some embodiments, the viral envelope comprises at least one costimulatory molecule. In some embodiments, the costimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, the costimulatory molecule provides signal 2 for cell activation.

[0328] As used herein, the term "costimulatory molecule" refers to a molecule capable of generating a costimulatory signal for T cells. Lymphocytes, such as T cells and natural killer (NK) cells, typically require several signals and interactions with antigen-presenting cells (APCs) for optimal priming to acquire full effector function. For T cells, these include signaling via the T cell receptor (TCR), costimulatory molecules (such as CD28 and CD2), cytokines, and various adhesion molecules necessary to ensure sufficient time for proper synapse formation and signal transduction. NK cells require similar types of stimuli but may rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, appropriate costimulation, in addition to TCR stimulation, is particularly important for effective priming; numerous studies have shown that TCR stimulation alone can lead to functional anergy and unresponsiveness. Costimulatory signals enhance T cell and NK cell function by enhancing cellular metabolism, cytokine production, differentiation, and long-term persistence. Costimulation is an important factor in cell proliferation, differentiation, and survival. In some embodiments, costimulatory molecules include, but are not limited to, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, costimulatory molecules include binding agents that bind to any of the costimulatory molecules described herein, such as, but not limited to, scFvs, antibodies, single-domain antibodies, antibody fragments, and nanobodies.In some embodiments, these binding agents may include anti-CD28, anti-CD2, anti-CD45, anti-CD4, anti-CD5, anti-CD8, anti-CD9, anti-CD16, anti-CD22, anti-CD33, anti-CD37, anti-CD64, anti-CD80, anti-CD86, anti-CD137, anti-CD154, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCR alpha chain, anti-TCR beta chain, anti-TCR gamma chain, and anti-TCR delta chain agents.

[0329] In some embodiments, the costimulatory molecule is a ligand for CD28. CD28 is one of the proteins expressed on T cells that provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins (particularly IL-6). In some embodiments, the costimulatory molecule is an antibody that binds to CD28, or a fragment thereof. Examples of such antibodies include 15E8 and TGN1412. Other suitable antibodies include CD28.2 and 10F3.

[0330] In some embodiments, the costimulatory molecule is CD86. CD86, also known as B7-2, is a ligand for CD28. In some embodiments, the ligand for CD28 is CD86. In some embodiments, the costimulatory molecule is CD80. CD80 is an additional ligand for CD28. In some embodiments, the ligand for CD28 is CD80. In some embodiments, the ligand for CD28 is an anti-CD28 antibody or anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or anti-CD28 scFv is coupled to a transmembrane domain for surface display of the viral envelope.

[0331] In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the costimulatory molecule is a CD86 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:76.

[0332] In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the costimulatory molecule is a CD80 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:77.

[0333] In some embodiments, the CD86 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the CD86 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:78.

[0334] In some embodiments, the CD80 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 79. In some embodiments, the CD80 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79.

[0335] CD134, also known as OX40, is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary costimulatory molecule expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed following their activation. In some embodiments, the viral particle comprises a ligand for OX40 or a functional fragment thereof coupled to its native or heterologous transmembrane domain.

[0336] CD134, also known as OX40, is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 can promote cell division and survival. OX40 is a secondary costimulatory molecule expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed following their activation. In some embodiments, the viral particle comprises a ligand for OX40 or a functional fragment thereof coupled to its native or heterologous transmembrane domain.

[0337] CD137, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. CD137 is expressed on activated T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells in the vascular wall at sites of inflammation. The most well-characterized activity of CD137 is its costimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytotoxic activity. In some embodiments, the viral particle contains a ligand for 4-1BB or a functional fragment thereof coupled to its native or heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signaling factor that acts as a ligand for 4-1BB, a costimulatory receptor molecule in T lymphocytes. In addition to promoting T lymphocyte proliferation, 4-1BBL has been shown to reactivate anergic T lymphocytes.

[0338] Viral particles comprising one or more activating or costimulatory molecules may be produced by engineering packaging cell lines by methods provided by WO 2016 / 139463; or by expression of T cell activating or costimulatory molecules from a polycistronic helper vector as described in International Patent Application Publication No. WO 2020 / 106992 A1, both of which are incorporated herein by reference in their entireties.

[0339] 3. Adhesion molecules In some embodiments, the viral particle comprises an adhesion molecule. As used herein, the term "adhesion molecule" refers to a subset of cell surface molecules involved in cell binding to other cells. Adhesion cells may help form more stable interactions between immune cells, such as immune synapses. An immune synapse is a stable adhesive junction between polarized immune effector cells and antigen-bearing cells. In some embodiments, adhesion molecules may provide costimulatory signals to target cells. In some embodiments, adhesion molecules include, but are not limited to, CD58, HHLA2, ICAM-1, OX40L, 4-1BBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6. In some embodiments, adhesion molecules include binding agents that bind to any of the adhesion molecules or costimulatory molecules described herein, such as, but not limited to, scFvs, antibodies, single-domain antibodies, antibody fragments, and nanobodies. In some embodiments, these binding agents may include anti-CD28, anti-CD2, anti-CD28H, anti-LFA-1, anti-OX40, anti-4-1BB, anti-CD40L, anti-DNAM-1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly-9, anti-CD84, anti-Ly108, anti-NKG2D, anti-NKp46, anti-NKp44, anti-NKp30, anti-CD244, anti-NKp80, anti-TCR alpha chain, anti-TCR beta chain, anti-TCR gamma chain, and anti-TCR delta chain agents.

[0340] In some embodiments, the adhesion molecule binds to CD2. CD2, also known as T11, LFA-2, and erythrocyte rosette receptor, is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to fulfilling its adhesive function, CD2 engagement provides a costimulatory signal that can enhance activation and effector function. In some embodiments, the lentiviral particle comprises a molecule that binds to CD2. In some embodiments, the lentiviral particle comprises an antibody, single domain antibody, antibody fragment, and / or nanobody specific to CD2. In some embodiments, the lentiviral particle comprises CD58, or a functional portion thereof, that binds to CD2.

[0341] In some embodiments, the adhesion molecule is CD58. In some embodiments, the costimulatory molecule is a CD58 polypeptide comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the costimulatory molecule is a CD58 polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 80.

[0342] In some embodiments, the CD58 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 81. In some embodiments, the CD58 polypeptide is encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:81.

[0343] 4. Additional non-viral proteins In some embodiments, the viral particle comprises at least one non-viral protein. In some embodiments, the viral particle comprises at least one non-viral protein in addition to those described above.

[0344] In some embodiments, the viral particle comprises a targeting ligand. In some embodiments, the viral particle comprises CD19 or a functional fragment thereof coupled to its native or heterologous transmembrane domain. In some embodiments, CD19 acts as a ligand for blinatumomab, thus providing an adapter for coupling the particle to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand may be useful for coupling appropriately surface-engineered lentiviral particles to T cells using a multispecific antibody comprising a binding moiety for the particle surface ligand. In some embodiments, the multispecific antibody is a bispecific antibody, e.g., a bispecific T cell engager (BiTE).

[0345] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine may be selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, and any combination thereof. If the non-viral protein used is a soluble protein (such as an scFv or cytokine), the protein may be anchored to the surface of the viral particle by fusion to a transmembrane domain, such as the transmembrane domain of CD8. Alternatively, the protein may be indirectly anchored to the lentiviral particle by using a transmembrane protein engineered to bind to the soluble protein. The further inclusion of one or more cytoplasmic residues may increase the stability of the fusion protein.

[0346] Mitogenic transduction enhancers and / or cytokine-based transduction enhancers may contain a "spacer sequence" connecting the antigen-binding domain and the transmembrane domain. A flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding. As used herein, the term "coupled to" refers to chemical linkage, direct C-terminal to N-terminal fusion of two proteins; chemical linkage in a non-peptide space; chemical linkage in a polypeptide space; and C-terminal to N-terminal fusion of two proteins via a polypeptide spacer, for example, a peptide bond to a spacer sequence.

[0347] The spacer sequence may comprise, for example, an lgG1 Fc region, an lgG1 hinge, or a human or mouse CD8 stalk. Alternatively, the spacer may comprise an alternative linker sequence having similar length and / or domain spacing characteristics to the lgG1 Fc region, lgG1 hinge, or CD8 stalk. The human lgG1 spacer may be altered to remove the Fc binding motif. In some embodiments, the spacer sequence may be derived from a human protein.

[0348] In some embodiments, the spacer sequence comprises a CD8-derived hinge.

[0349] In some embodiments, the spacer sequence comprises a "short" hinge, which is described as a hinge region that contains fewer nucleotides compared to CAR hinge regions known in the art.

[0350] The transmembrane domain is a membrane-spanning mitogenic transduction enhancer and / or cytokine-based transduction enhancer sequence. The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28. In some embodiments, the transmembrane domain is derived from a human protein.

[0351] The viral particles of the present invention may contain a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from the host cell during viral particle production. In some embodiments, the cytokine-based transduction enhancer is produced by the host cell and expressed on the cell surface. When the nascent viral particle buds from the host cell membrane, the cytokine-based transduction enhancer may be incorporated into the viral envelope as part of the packaging cell-derived lipid bilayer.

[0352] The cytokine-based transduction enhancer may comprise a cytokine domain and a transmembrane domain. The cytokine-based transduction enhancer may have the structure CS-TM, where C is the cytokine domain, S is an optional spacer domain (e.g., a spacer sequence), and TM is the transmembrane domain. The spacer domain and the transmembrane domain are as defined above.

[0353] The cytokine domain can include a T cell activating cytokine, for example, from IL2, IL7, and IL15, or a functional fragment thereof. As used herein, a "functional fragment" of a cytokine is a fragment of a polypeptide that retains the ability to bind to its particular receptor and activate T cells.

[0354] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and certain B cells. IL2 is a lymphokine that induces proliferation of responding T cells. It is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity. Solution NMR suggests that the structure of IL2 contains a bundle of four helices (designated A–D) flanked by two shorter helices and several poorly defined loops. Residues in helix A and in the loop region between helices A and B are important for receptor binding.

[0355] IV. METHODS AND USES THEREOF In some embodiments, methods are provided herein that use the polycistronic constructs or nucleotide vectors disclosed herein. In some embodiments, the provided methods deliver the polycistronic constructs to cells for expression of the provided systems, including cytosolic FRB, synthetic cytokine receptors, and CARs. In some embodiments, the polycistronic constructs are contained within viral vectors, and the viral vectors are used to transduce target cells. In some embodiments, the provided methods are performed ex vivo or in vitro to engineer target cells with the polycistronic constructs. In some embodiments, the engineered cells are administered to a subject. In some embodiments, the provided methods are performed in vivo, and a viral vector containing the polycistronic construct is introduced into a subject for in vivo targeted delivery of the polycistronic vector to target cells, such as T cells.

[0356] A. Methods for Transducing Cells In some embodiments, provided herein are methods for transducing cells, comprising contacting the target cells with a particle, e.g., a viral vector, containing any of the polycistronic constructs provided herein. In some embodiments, the target cells comprise stem cells. In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs). In some embodiments, the target cells comprise progenitor cells. In some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the target cells comprise T cells. In some embodiments, the T cells comprise CD4+ or CD8+ T cells. In some embodiments, the method further comprises contacting the target cells with (i) a guide RNA (gRNA) that targets a target site in the endogenous gene and (ii) an RNA-guided endonuclease, thereby inserting the nucleotide sequence into the endogenous gene.

[0357] In some aspects, the polynucleotides described herein can be delivered to cells in vivo. In some embodiments, polynucleotides encoding elements of the polycistronic constructs disclosed herein are directly administered to a subject via administration of particles containing the polynucleotides. In some embodiments, the particles are viral particles. In some embodiments, the viral particles comprise anti-CD3 scFv and cocal glycoprotein, enabling in vivo delivery of polynucleotides to cells. In some embodiments, the polynucleotides encode cytosolic FRB, synthetic cytokine receptors, and chimeric antigen receptors (CARs). The polynucleotides disclosed herein can be administered to a subject to enable in vivo production of various construct components (e.g., FRB, synthetic cytokine receptors, and CARs). In some embodiments, administration of such polynucleotides produces effects in vivo similar to direct ex vivo administration of engineered cells expressing FRB, synthetic cytokine receptors, and CARs. In some embodiments, administration of such polynucleotides improves the in vivo transduction efficiency of particles. In some embodiments, the polynucleotide is mRNA.

[0358] The polynucleotides described herein can also be delivered to cells ex vivo. The viral particles described herein can be used ex vivo, either in traditional cell manufacturing processes or in ex vivo or bedside processes, such as those described in International Patent Application Publication No. WO 2022 / 072885 A1. In one aspect, the present disclosure provides an ex vivo method of transducing target cells, comprising contacting the target cells with a particle according to the present disclosure. In some aspects, the particles described herein can be used to transduce cells that have not previously been activated. For example, the particles described herein can be useful for transducing cells that have not previously been contacted with cell activation beads or activation reagents (e.g., Dynabeads, or other reagents comprising anti-CD3 and / or anti-CD28 antibodies or binding fragments thereof).

[0359] In some embodiments, the present disclosure provides a method for delivering nucleic acids to cells in the ex vivo CAR T manufacturing process. Such methods typically involve the isolation of PBMCs from patients via leukapheresis. These cells are washed and optionally further purified through one or more selection steps to isolate specific T cell populations of interest. In some aspects, these may include CD4+ and / or CD8+ T cells. The washed and / or purified cells may optionally be activated and then transduced using a lentiviral vector. The activation step may involve contacting the cells with an exogenous activating agent, such as anti-CD3 and anti-CD28 antibodies bound to a substrate, or using unbound antibodies. Exemplary activating agents include anti-CD3 and anti-CD28-presenting beads and / or soluble polymers. After transduction, the cells may optionally be further washed and cultured until harvested. Methods for producing engineered cell therapies, including CAR T cells, are known in the art (see, e.g., Abou-el-Enein, M. et al. Blood Cancer Discov (2021), Vol 2(5): 408-422; Arcangeli, S. et al. Front. Immunol (19 Jun 2020), Vol. 11 (1217) 1-13; Ghassemi, S. et al. Nat Biomed Eng (Feb 2022), Vol 6(2): 118-128; Vormittag, P. et al. Curr Opin Biotechnol (Oct 2018), Vol. 54: 164-181; each of which is incorporated herein by reference). Exemplary methods of autologous CAR T manufacturing are disclosed in U.S. Patent Publication Nos. 2019 / 0269727, 2016 / 0122782, 2021 / 0163893, and 2017 / 0037369, each of which is incorporated herein in its entirety.

[0360] In some embodiments, the present disclosure provides methods for delivering nucleic acids to cells in an ex vivo closed-loop manufacturing process. In some embodiments, the ex vivo manufacturing process is an extracorporeal process. In exemplary embodiments, the lentiviral vectors disclosed herein enable delivery of nucleic acids to target cells in a closed-loop process. Exemplary methods for closed-loop and / or extracorporeal processes are disclosed in U.S. Patent Publication No. 2021 / 0244871 and WO2022072885, both of which are incorporated herein in their entireties. In some embodiments, lentiviral vectors as disclosed herein can be used to transduce cells ex vivo. For example, in an exemplary closed-loop manufacturing process, cells are obtained from a subject, washed, incubated and / or contacted with lentiviral particles, optionally washed again, and infused into the subject in a closed-loop system. In such embodiments, lentiviral particles as disclosed herein are useful without preactivating the cells and are capable of binding to cells with a short incubation and / or contacting step. In some embodiments, the incubation and / or contacting step is for approximately 1 hour or less. In some embodiments, the incubation and / or contacting step is for approximately 2 hours or less, approximately 3 hours or less, approximately 4 hours or less, or approximately 5 hours or less. In some embodiments, the incubation and / or contacting step is for less than 12 hours or less than 24 hours. In some embodiments, the nucleic acid is delivered to the cell by transduction with a lentiviral vector, such that the nucleic acid enters the cell ex vivo. In some embodiments, the nucleic acid is delivered to the cell by contacting the lentiviral vector with the cell surface. In such embodiments, the nucleic acid can enter the cell ex vivo or in vivo after the cell (complexed with the lentiviral vector) is infused back into the subject.

[0361] In some embodiments, the lentiviral vectors disclosed herein eliminate the need for an ex vivo activation step. In such embodiments, isolated cells can be directly transduced after leukapheresis, washing, or selection. It is envisioned that the surface engineering described herein allows the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein enable a shorter or abbreviated manufacturing process, reducing the time spent on ex vivo manufacturing by eliminating one or more unit operations (e.g., activation before transduction) and / or reducing the time required for cell culture after transduction.

[0362] B. Methods for expressing receptors In some embodiments, provided herein are methods for expressing chimeric antigen receptors and / or synthetic cytokine receptors in target cells. In some embodiments, the methods include contacting target cells with particles, such as viral vectors, containing any of the polycistronic constructs provided herein. In some embodiments, the contacting is carried out ex vivo or in vitro. In some embodiments, the contacting is carried out in vivo in a subject by administering a polynucleotide construct or a particle, such as a viral vector, containing the polynucleotide construct to the subject.

[0363] In some embodiments, the target cells comprise stem cells.

[0364] In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs).

[0365] In some embodiments, the target cells comprise progenitor cells.

[0366] In some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

[0367] In some embodiments, the target cells comprise T cells.

[0368] In some embodiments, the T cells comprise CD4+ or CD8+ T cells.

[0369] In some embodiments, the method is performed ex vivo or in vitro.

[0370] In some embodiments, the method is performed in vivo.

[0371] In some aspects, provided herein are cells produced by any of the methods provided herein.

[0372] C. Method of Administration Also provided herein are methods of administering any of the provided cells engineered with the provided polycistronic constructs to a subject. Also provided herein are methods of administering any of the provided particles, e.g., viral vectors, e.g., lentiviral vectors, to a subject. In some embodiments, the subject has a disease or condition, and the method of administration is for treating the disease or condition. In any of the subject methods, the cells or particles are administered as a pharmaceutical composition. In some embodiments, the composition is for use in treating a disease or condition. Also provided are uses of the provided compositions for treating a disease or condition in a subject. Such methods and uses include, for example, therapeutic methods and uses involving administering engineered cells or particles (e.g., viral vectors) or compositions containing same to a subject with a disease or condition. In some cases, the disease or condition is a tumor or cancer. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to achieve treatment of the disease or disorder. Uses include the use of the cells or pharmaceutical compositions thereof in such methods and treatments, as well as in the preparation of medicaments for performing such therapeutic methods. In some embodiments, the method thereby treats a disease or condition or disorder in a subject.

[0373] In some embodiments, engineered cells containing any of the provided polycistronic constructs encoding cytosolic FRB, synthetic cytokine receptor, and chimeric antigen receptor (CAR) can be administered to a subject to treat a disease or condition. In some embodiments, particles, such as viral vectors (e.g., lentiviral vectors), can be administered directly to a subject for in vivo targeted delivery of the polycistronic construct to target cells for in vivo production of various construct components (e.g., FRB, synthetic cytokine receptor, and CAR). The disclosed cells or particles (e.g., viral vectors) can be administered in a number of ways, depending on whether local or systemic treatment is desired.

[0374] In the case of adoptive cell therapy, methods for the administration of cells for adoptive cell therapy are known and may be used in conjunction with the provided methods and compositions.

[0375] Generally, administration can be topical, parenteral, or enteral. The compositions of the present disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physical breaching of the target tissue, and administration of the pharmaceutical composition through an incision in the tissue, thus generally resulting in direct administration to the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, intrasynovial injection or infusion; and kidney dialysis infusion techniques. In some embodiments, parenteral administration of the compositions of the present disclosure includes intravenous administration. In some embodiments, the viral particles are administered by intraperitoneal injection of the viral particles. In some embodiments, the viral particles are administered by intranodal injection, i.e., the viral particles can be administered via injection into a lymph node, for example, the inguinal lymph node. In some embodiments, the viral particles are administered by injection of the viral particles into the tumor site (i.e., intratumorally). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intra-arterially. In some embodiments, the viral particles are lentiviral particles.

[0376] Typically, pharmaceutical composition formulations suitable for parenteral administration generally contain the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9); however, for some applications, these may be more suitably formulated as sterile nonaqueous solutions or as dry forms for use in conjunction with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form or a liposomal preparation. Formulations for parenteral administration can be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted, and programmed release. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, solutions intended for subcutaneous administration include hyaluronidase.

[0377] The compositions of the present invention may additionally contain other accessory components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional substances useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers.However, when added, such substances should not excessively interfere with the biological activity of the components of the compositions of the present invention.The preparations are sterilized, and if desired, can be mixed with auxiliary substances that do not adversely interact with the nucleic acid of the preparation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, flavorings and / or aromatic substances.

[0378] The polycistronic construct of the present invention can be administered in an amount effective for treating or preventing a disease or condition, for example, a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodically evaluating the treated subject. For repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0379] In certain embodiments, in the context of injecting differentiated cells or transgenic differentiated cells according to the present disclosure, a subject is injected with about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, , about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases, a range of about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween, and / or such number of cells per kilogram of body weight of the subject. For example, in some embodiments, administration of cells or populations of cells is in the ... 3 ~about 10 9 The method may include administering individual cells.

[0380] In some aspects, provided herein are methods of administering any of the cells provided herein to a subject.

[0381] In some aspects, provided herein are methods of administering any of the viral vectors provided herein to a subject.

[0382] In some embodiments, the CAR encoded by the provided polycistronic constructs is targeted to an antigen associated with a disease or condition, and the method comprises administering cells (e.g., by adoptive cell therapy) or a viral vector, e.g., a lentiviral vector, to a subject having or suspected of having a disease or condition. In some embodiments, among others, provided methods are for treating a subject suffering from cancer, comprising administering to the subject provided cells engineered with any of the provided polycistronic constructs of the present disclosure, wherein the cancer is treated in the subject. In some embodiments, among others, provided methods are for treating a subject suffering from cancer, comprising administering to the subject provided viral vectors, e.g., lentiviral particles, incorporating any of the provided polycistronic constructs of the present disclosure, wherein the cancer is treated in the subject.

[0383] In some embodiments, the cancer is a solid tumor, such as melanoma, non-small cell lung cancer, or breast cancer. The methods of the present disclosure are useful in treating, but are not limited to, acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, cancer), fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, common germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / invasive breast cancer, pancreatic Islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary breast carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cell neck cancer, mixed glioma, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, ocular cancer, intraocular melanoma, oligodendroglioma, oral cancer, oral cavity cancercancer), oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, ovarian primary peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal tumor, pineoblastoma, pituitary tumor, primary central nervous system, prostate cancer, rectal cancer, renal cell cancer, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft-tissue sarcoma, uterus, maxillary sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer The term "cancer" may include treating any cancer, including uterine adenocarcinoma, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, undiagnosed carcinoma, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0384] In some embodiments, the CAR encoded by the provided polycistronic construct is a CAR that targets a ligand that can bind to an antigen on the surface of a cell associated with a disease or pathology.In some embodiments, the CAR of any of the provided embodiments is an anti-FITC CAR directed against FITC, and the ligand is a bifunctional ligand composed of FITC and a binding molecule that can bind to a surface molecule or receptor on target cells.In some embodiments, the method further comprises administering a bifunctional ligand to tag cancer cells in a subject, wherein the bifunctional ligand specifically binds to a molecule expressed on tumors.In some embodiments, the bifunctional ligand is FITC-folate.In some embodiments, the cancer is osteosarcoma.

[0385] In some embodiments, the method further comprises administering a bifunctional ligand to tag cancer cells in the subject, wherein the bifunctional ligand specifically binds to a molecule expressed on the tumor. In some embodiments, the bifunctional ligand comprises FITC-folate.

[0386] In some of the optional embodiments, the method further comprises administering a non-physiological ligand to the subject. In some embodiments, the non-physiological ligand is capable of binding to a synthetic cytokine receptor and inducing gamma cytokine signaling in the cell. In some embodiments, the non-physiological ligand comprises rapamycin or a rapamycin analog.

[0387] 1. Non-physiological ligands In various embodiments of the compositions and methods of the present disclosure, the system includes a non-physiological ligand. Exemplary small molecules useful as ligands include, but are not limited to, rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (aMPOB), folic acid, rhodamine, acetazolamide, and CA9 ligand.

[0388] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0389] In some embodiments, the non-physiological ligand is a rapalog.

[0390] In some embodiments, the non-physiological ligand is rapamycin.

[0391] In some embodiments, the non-physiological ligand is AP21967.

[0392] In some embodiments, the non-physiological ligand is FK506.

[0393] In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporin-A (CsA). In some embodiments, the non-physiological ligand is coumermycin.

[0394] In some embodiments, the synthetic cytokine receptor complex is activated by folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, a rapalog (rapamycin analog), CD28 ligand, poly(His) tag, Strep-tag, FLAG-tag, VS-tag, Myc-tag, HA-tag, NE-tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0395] In some embodiments, the non-physiological ligand can be an inorganic or organic compound less than 1000 daltons.

[0396] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, rapalogs include variants of rapamycin that have one or more of the following modifications compared to rapamycin: demethylation, removal, or replacement of methoxy at C7, C42, and / or C29; removal, derivatization, or replacement of hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of ketone at C14, C24, and / or C30; replacement of the 6-membered pipecolic acid ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0397] Thus, in some embodiments, the rapalog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, Temsirolimus (CCI-779), C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-(S)-3-methylindolerapamycin (C16-iRap), AP21967 (A / C Heterodimerizer, Takara Bio®), mycophenolate sodium, benidipine hydrochloride, rapamine, AP23573 (Ridaforolimus), AP1903 (Rimiducid), or metabolites, derivatives and / or combinations thereof.

[0398] In some embodiments, the ligand comprises FK1012 (a semi-synthetic dimer of FK506), tacrolimus (FK506), FKCsA (a conjugate of FK506 and cyclosporine), rapamycin, coumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TMP-HTag (trimethoprim haloenzyme protein dimerizer), or ABT-737, or a functional derivative thereof.

[0399] In some embodiments, the non-physiological ligand has a concentration of 0 nM to 1000 nM, e.g., 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65.0 nM , 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or an amount within a range defined by any two of the foregoing amounts.

[0400] In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 100 nM.

[0401] In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 50 nM.

[0402] In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 100 nM.

[0403] In some embodiments, the non-physiological ligand is present or provided at 1 nM.

[0404] In some embodiments, the non-physiological ligand is present or provided at 10 nM.

[0405] In some embodiments, the non-physiological ligand is present or provided at 100 nM.

[0406] In some embodiments, the non-physiological ligand is present or provided at 1000 nM.

[0407] V. Definition Unless otherwise defined, all terminology, notation, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference beyond that commonly understood in the art.

[0408] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context indicates otherwise. The linkage "and / or" represents all possible combinations of one or more of the listed items.

[0409] The term "about," as used herein, refers to the normal error range for each value, which is well known to those skilled in the art. Reference herein to a value or parameter marked "about" encompasses (and describes) aspects directed to that value or parameter itself.

[0410] It is understood that aspects and embodiments of the invention described herein encompass "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0411] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur. For example, an optionally substituted group means that the group is either unsubstituted or substituted.

[0412] The term "composition" refers to any mixture of two or more products, substances, or compounds, including cells or antibodies. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. The preparation is generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

[0413] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0414] As used herein, combination refers to any relationship between or among two or more items.Combination can be two or more separate items, such as two compositions or two collections, or can be a mixture thereof, such as a single mixture of two or more items, or any variation thereof.The elements of combination are generally functionally related or related.

[0415] As used herein, a kit is a packaged combination that optionally includes other elements, e.g., additional agents, and instructions for using the combination or its elements for a purpose, including but not limited to, therapeutic use.

[0416] "Subject," as used herein, refers to the recipient of a polycistronic construct or other agent. This term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs, preferably humans.

[0417] "Treat," "treating," or "treatment," as used herein, refers to any type of action or administration that confers benefit on a subject with a disease or disorder, including improvement in the patient's condition (i.e., improvement, reduction, or amelioration of one or more symptoms, and partial or complete response to treatment).

[0418] The term "effective amount" refers to an amount effective to produce a desired biochemical, cellular, or physiological response. The term "therapeutically effective amount" refers to an amount, dosage, or dosing regimen of a therapy effective to produce a desired treatment effect. As used herein, an "individual" or "subject" is a mammal. "Mammals" for purposes of treatment include humans, domestic and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual or subject is a human.

[0419] "Polynucleotide," as used herein, refers to a biopolymer composed of two or more nucleotide monomers covalently linked in a chain via an ester bond between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar component of the next nucleotide. DNA and RNA are non-limiting examples of polynucleotides.

[0420] "Polypeptide," as used herein, refers to a polymer of amino acid residues joined in a chain by peptide bonds that forms part of (or the whole of) a protein.

[0421] Those skilled in the art will understand that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it should also be understood that those skilled in the art can, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide will be expressed.

[0422] Nucleic acids can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that contain synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for use as described herein, polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or life span of the polynucleotide of interest.

[0423] The term "variant" refers to a polynucleotide or polypeptide that has at least one substitution, insertion, or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or more functions of the reference polynucleotide or polypeptide.

[0424] As used herein, the term "sequence identity" or "identity" in reference to a polynucleotide or polypeptide sequence refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences match at each position in the alignment over the entire length of the reference sequence. The "percent identity" is calculated by dividing the number of matched positions in the optimal alignment by the length of the reference sequence, plus the sum of the lengths of any gaps in the reference sequence in the alignment. The optimal alignment is the alignment that results in the greatest percent identity. Aligning sequences to determine percent identity can be achieved by a number of well-known methods, including, for example, using mathematical algorithms, such as those in the BLAST suite or Clustal Omega sequence analysis program. Unless otherwise noted, the term "sequence identity" in the claims refers to sequence identity as calculated by BLAST version 2.12.0 using default parameters. And, unless otherwise noted, the alignment is an alignment of all or part of the polynucleotide or polypeptide sequence of interest over the entire length of the reference sequence.

[0425] As used herein, "small molecule" refers to an organic compound of low molecular weight (<1000 daltons). Small molecules can bind to specific biopolymers and have a wide variety of biological functions or uses, including but not limited to, acting as cell signaling molecules, drugs, secondary metabolites, or various other mechanisms of action.

[0426] The term "analog" with respect to a small molecule refers to a compound that has a similar structure and / or function to that of another compound but differs with respect to certain components. Analogs may differ in one or more atoms, functional groups, or substructures, which replace other atoms, groups, or substructures. Despite high structural and / or functional similarity, analogs may have different physical, chemical, physiochemical, biochemical, or pharmacological properties.

[0427] The term "rapalog" refers to a group of art-recognized analogs of rapamycin analogs that share structural and functional similarities with rapamycin. Certain rapalogs are known to share some, but not all, of the functional attributes of rapamycin. For example, some rapalogs promote dimerization but have substantially no immunosuppressive activity, making them suitable for use as non-physiological ligands (e.g., AP21967, AP23102, or iRAP).

[0428] An exemplary rapalog of the present disclosure is AP21967. TIFF2025538152000010.tif44128

[0429] An exemplary rapalog of the present disclosure is AP23102. TIFF2025538152000011.tif44128

[0430] An exemplary rapalog of the present disclosure is iRAP. TIFF2025538152000012.tif44128

[0431] The term "cell population" refers to a mixture of cells suspended in a solution, attached to a substrate, or stored in a container. The characteristics of a cell population as a whole can be studied by bulk measurement of a sample volume containing multiple cells. To mitigate the background fluorescence problems encountered in measuring bulk cell populations, flow cytometry methods can be used.

[0432] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide or that have undergone genetic editing to introduce, delete or modify a polynucleotide, or cells that have been transiently transduced with a polynucleotide to cause a stable phenotypic change in the cell.

[0433] VI. Illustrative Embodiments Among the embodiments provided are: 1. 1. A polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding an FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence. 2. 2. The polycistronic construct of embodiment 1, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50. 3. 3. The polycistronic construct of embodiment 1 or embodiment 2, wherein said nucleotide sequence encoding said FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13, or 50. 4. 4. The polycistronic construct of any of embodiments 1-3, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4, 14, or 51. 5. 5. The polycistronic construct of any of embodiments 1 to 4, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 4, 14, or 51. 6. 6. The polycistronic construct of any of embodiments 1-5, wherein the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:15. 7. 7. The polycistronic construct of any of embodiments 1 to 6, wherein the nucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NO:15. 8. 8. The polycistronic construct of any of embodiments 1-7, wherein said synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). 9. 9. The polycistronic construct of embodiment 8, wherein said IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16. 10. The polycistronic construct of embodiment 8 or embodiment 9, wherein said IL2RG comprises the amino acid sequence of SEQ ID NO:16. 11. 11. The polycistronic construct of any of aspects 1 to 10, wherein the second expression cassette further comprises a nucleotide sequence encoding FRB. 12. 12. The polycistronic construct of embodiment 11, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:13. 13.

[0023] Embodiment 13. The polycistronic construct of embodiment 11 or embodiment 12, wherein said nucleotide sequence encoding said FRB comprises the nucleotide sequence of SEQ ID NO:13. 14. 14. The polycistronic construct of any of embodiments 11-13, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14. 15. 15. The polycistronic construct of any of aspects 1-14, wherein said FRB comprises the amino acid sequence of SEQ ID NO:14. 16. 16. The polycistronic construct of any of aspects 1 to 15, wherein the second expression cassette is codon-optimized. 17. 17. The polycistronic construct of any of embodiments 1-16, wherein said second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:11. 18. 18. The polycistronic construct of any of aspects 1-17, wherein said second expression cassette comprises the nucleotide sequence of SEQ ID NO:11. 19. 19. The polycistronic construct of any of embodiments 1-18, wherein the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:12. 20. 20. The polycistronic construct of any of aspects 1 to 19, wherein the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:12. twenty one. 16. The polycistronic construct of any of aspects 1 to 15, wherein the second expression cassette further comprises a nucleotide sequence encoding FKBP12. twenty two. 22. The polycistronic construct of embodiment 21, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:21 or 55. twenty three. 23. The polycistronic construct of embodiment 21 or embodiment 22, wherein said nucleotide sequence encoding said FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. twenty four. 24. The polycistronic construct of any of embodiments 21 to 23, wherein said FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22. twenty five. 25. The polycistronic construct of any of aspects 21 to 24, wherein said FKBP12 comprises the amino acid sequence of SEQ ID NO:22. 26. 26. The polycistronic construct of any of embodiments 1-25, wherein the nucleotide sequence encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:23 or 61. 27. 27. The polycistronic construct of any of embodiments 1-26, wherein the nucleotide encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. 28. 28. The polycistronic construct of any of aspects 1-27, wherein said synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB). 29. 29. The polycistronic construct of embodiment 28, wherein said IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62. 30. 30. The polycistronic construct of embodiment 28 or embodiment 29, wherein said IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62. 31. 31. The polycistronic construct of any of aspects 1 to 30, wherein the third expression cassette further comprises a nucleotide sequence encoding FKBP12. 32. 32. The polycistronic construct of embodiment 31, wherein the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:21. 33. 33. The polycistronic construct of embodiment 31 or embodiment 32, wherein said nucleotide sequence encoding said FKBP12 comprises the nucleotide sequence of SEQ ID NO:21. 34. 34. The polycistronic construct of any of embodiments 31 to 33, wherein said FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:20. 35. 35. The polycistronic construct of any of aspects 31 to 34, wherein said FKBP12 comprises the amino acid sequence of SEQ ID NO:20. 36. 36. The polycistronic construct of any of aspects 1 to 35, wherein the third expression cassette is codon-optimized. 37. 37. The polycistronic construct of any of embodiments 1-36, wherein said third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:19. 38. 38. The polycistronic construct of any of aspects 1-37, wherein said third expression cassette comprises the nucleotide sequence of SEQ ID NO:19. 39. 39. The polycistronic construct of any of embodiments 1-38, wherein the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:20. 40. 40. The polycistronic construct of any of aspects 1 to 39, wherein the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:20. 41. 41. The polycistronic construct of any of aspects 1 to 40, wherein the third expression cassette further comprises a nucleotide sequence encoding FRB. 42. 42. The polycistronic construct of embodiment 41, wherein the nucleotide sequence encoding the FRB is a...

Claims

1. 1. A polycistronic construct comprising, in 5' to 3' order: (a) a first expression cassette comprising a nucleotide sequence encoding an FRB; (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

2. 2. The polycistronic construct of claim 1, wherein the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3, 13, or 50.

3. 3. The polycistronic construct of claim 1 or claim 2, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13, or 50.

4. 4. The polycistronic construct of any one of claims 1 to 3, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, 14, or 51.

5. The polycistronic construct of any one of claims 1 to 4, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 4, 14, or 51.

6. 6. The polycistronic construct of any one of claims 1 to 5, wherein the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

15.

7. 7. The polycistronic construct of any one of claims 1 to 6, wherein the nucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NO:

15.

8. 8. The polycistronic construct of any one of claims 1 to 7, wherein the synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG).

9. 9. The polycistronic construct of claim 8, wherein the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

16.

10. 10. The polycistronic construct of claim 8 or claim 9, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO:

16.

11. The polycistronic construct of any one of claims 1 to 10, wherein the second expression cassette further comprises a nucleotide sequence encoding FRB.

12. 12. The polycistronic construct of claim 11, wherein the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

13.

13. 13. The polycistronic construct of claim 11 or claim 12, wherein the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO:

13.

14. 14. The polycistronic construct of any one of claims 11 to 13, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

14.

15. 15. The polycistronic construct of any one of claims 1 to 14, wherein the FRB comprises the amino acid sequence of SEQ ID NO:

14.

16. 16. The polycistronic construct of any one of claims 1 to 15, wherein the second expression cassette is codon-optimized.

17. 17. The polycistronic construct of any one of claims 1 to 16, wherein the second expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

11.

18. 18. The polycistronic construct of any one of claims 1 to 17, wherein the second expression cassette comprises the nucleotide sequence of SEQ ID NO:

11.

19. 19. The polycistronic construct of any one of claims 1 to 18, wherein the second expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

12.

20. 20. The polycistronic construct of any one of claims 1 to 19, wherein the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:

12.

21. 16. The polycistronic construct of any one of claims 1 to 15, wherein the second expression cassette further comprises a nucleotide sequence encoding FKBP12.

22. 22. The polycistronic construct of claim 21, wherein the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 21 or 55.

23. 23. The polycistronic construct of claim 21 or claim 22, wherein the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55.

24. 24. The polycistronic construct of any one of claims 21 to 23, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

22.

25. 25. The polycistronic construct of any one of claims 21 to 24, wherein said FKBP12 comprises the amino acid sequence of SEQ ID NO:

22.

26. 26. The polycistronic construct of any one of claims 1 to 25, wherein the nucleotide sequence encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61.

27. 27. The polycistronic construct of any one of claims 1 to 26, wherein the nucleotide encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61.

28. 28. The polycistronic construct of any one of claims 1 to 27, wherein the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB).

29. 29. The polycistronic construct of claim 28, wherein the IL2RB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 or 62.

30. 30. The polycistronic construct of claim 28 or claim 29, wherein the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62.

31. 31. The polycistronic construct of any one of claims 1 to 30, wherein the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

32. 32. The polycistronic construct of claim 31, wherein the nucleotide sequence encoding FKBP12 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

21.

33. 33. The polycistronic construct of claim 31 or claim 32, wherein the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO:

21.

34. 34. The polycistronic construct of any one of claims 31 to 33, wherein the FKBP12 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

22.

35. 35. The polycistronic construct of any one of claims 31 to 34, wherein said FKBP12 comprises the amino acid sequence of SEQ ID NO:

22.

36. 36. The polycistronic construct of any one of claims 1 to 35, wherein the third expression cassette is codon-optimized.

37. 37. The polycistronic construct of any one of claims 1 to 36, wherein the third expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

19.

38. 38. The polycistronic construct of any one of claims 1 to 37, wherein the third expression cassette comprises the nucleotide sequence of SEQ ID NO:

19.

39. 39. The polycistronic construct of any one of claims 1 to 38, wherein the third expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

20.

40. 40. The polycistronic construct of any one of claims 1 to 39, wherein the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO:

20.

41. 41. The polycistronic construct of any one of claims 1 to 40, wherein the third expression cassette further comprises a nucleotide sequence encoding FRB.

42. 42. The polycistronic construct of claim 41, wherein the nucleotide sequence encoding FRB is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

13.

43. 43. The polycistronic construct of claim 41 or claim 42, wherein the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO:

13.

44. 44. The polycistronic construct of any one of claims 41 to 43, wherein the FRB comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

14.

45. 45. The polycistronic construct of any one of claims 1 to 44, wherein the FRB comprises the amino acid sequence of SEQ ID NO:

14.

46. 46. ​​The polycistronic construct of any one of claims 1 to 45, wherein the CAR comprises an scFv domain.

47. 47. The polycistronic construct of claim 46, wherein the scFv domain comprises anti-fluorescein isothiocyanate (FITC) E2.

48. 48. The polycistronic construct of claim 46 or claim 47, wherein the scFv domain comprises a light chain variable domain (VL), a linker, and a heavy chain variable domain (VH).

49. 49. The polycistronic construct of claim 48, wherein the scFv VL comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 30 or 65.

50. 50. The polycistronic construct of claim 48 or claim 49, wherein the scFv VL comprises the nucleotide sequence of SEQ ID NO: 30 or 65.

51. 51. The polycistronic construct of any one of claims 48 to 50, wherein the scFv VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

31.

52. 52. The polycistronic construct of any one of claims 48 to 51, wherein the scFv VL comprises the amino acid sequence of SEQ ID NO:

31.

53. 53. The polycistronic construct of any one of claims 48 to 52, wherein the scFv VH comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 34 or 67.

54. The polycistronic construct of any one of claims 48 to 53, wherein the scFv VH comprises the nucleotide sequence of SEQ ID NO: 34 or 67.

55. 55. The polycistronic construct of any one of claims 48 to 54, wherein the scFv VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

35.

56. 56. The polycistronic construct of any one of claims 48 to 55, wherein the scFv VH comprises the amino acid sequence of SEQ ID NO:

35.

57. 57. The polycistronic construct of any one of claims 48 to 56, wherein the scFv linker comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 32 or 66.

58. 58. The polycistronic construct of any one of claims 48 to 57, wherein the scFv linker comprises the nucleotide sequence of SEQ ID NO: 32 or 66.

59. 59. The polycistronic construct of any one of claims 48 to 58, wherein the scFv linker comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

33.

60. 60. The polycistronic construct of any one of claims 48 to 59, wherein the scFv linker comprises the amino acid sequence of SEQ ID NO:

33.

61. 61. The polycistronic construct of any one of claims 46 to 60, wherein the scFv comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 28 or 64.

62. 62. The polycistronic construct of any one of claims 46 to 61, wherein the scFv comprises the nucleotide sequence of SEQ ID NO: 28 or 64.

63. 63. The polycistronic construct of any one of claims 46 to 62, wherein the scFv comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

29.

64. 64. The polycistronic construct of any one of claims 46 to 63, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

29.

65. 65. The polycistronic construct of any one of claims 1 to 64, wherein the CAR comprises a hinge domain.

66. 66. The polycistronic construct of claim 65, wherein the hinge domain comprises a short hinge or a medium hinge domain.

67. 67. The polycistronic construct of claim 65 or claim 66, wherein the hinge domain comprises CD8 or IgG.

68. 68. The polycistronic construct of claim 67, wherein the CD8 hinge comprises a CD8α hinge.

69. 69. The polycistronic construct of claim 68, wherein the CD8α hinge comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 38 or 114.

70. 70. The polycistronic construct of claim 68 or claim 69, wherein the CD8α hinge comprises the nucleotide sequence of SEQ ID NO: 38 or 114.

71. 71. The polycistronic construct of any one of claims 68-70, wherein the CD8α hinge comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 115.

72. 72. The polycistronic construct of any one of claims 68 to 71, wherein the CD8α hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115.

73. 73. The polycistronic construct of any one of claims 1 to 72, wherein the CAR comprises a transmembrane domain.

74. 74. The polycistronic construct of claim 73, wherein the transmembrane domain comprises CD8 or CD28.

75. 75. The polycistronic construct of claim 74, wherein the CD8 transmembrane domain comprises a CD8α transmembrane domain.

76. 76. The polycistronic construct of any one of claims 73 to 75, wherein the transmembrane domain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

40.

77. 77. The polycistronic construct of any one of claims 73 to 76, wherein the transmembrane domain comprises the nucleotide sequence of SEQ ID NO:

40.

78. 78. The polycistronic construct of any one of claims 73 to 77, wherein the transmembrane domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

41.

79. 79. The polycistronic construct of any one of claims 73 to 78, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

41.

80. 80. The polycistronic construct of any one of claims 1 to 79, wherein the CAR comprises an endodomain.

81. 81. The polycistronic construct of claim 80, wherein the endodomain comprises a costimulatory molecule.

82. 82. The polycistronic construct of claim 80 or claim 81, wherein the endodomain comprises 4-1BB, CD3ζ and / or CD28.

83. 83. The polycistronic construct of claim 82, wherein the 4-1BB endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 42 or 69.

84. The polycistronic construct of claim 82 or claim 83, wherein the 4-1BB endodomain comprises the nucleotide sequence of SEQ ID NO: 42 or 69.

85. 85. The polycistronic construct of any one of claims 82 to 84, wherein the 4-1BB endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

43.

86. 86. The polycistronic construct of any one of claims 82 to 85, wherein the 4-1BB endodomain comprises the amino acid sequence of SEQ ID NO:

43.

87. 87. The polycistronic construct of any one of claims 82 to 86, wherein the CD3 zeta endodomain comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118.

88. The polycistronic construct of any one of claims 82 to 87, wherein the CD3ζ endodomain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118.

89. 89. The polycistronic construct of any one of claims 82 to 88, wherein the CD3 zeta endodomain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

47.

90. The polycistronic construct of any one of claims 82 to 89, wherein the CD3ζ endodomain comprises the amino acid sequence of SEQ ID NO:

47.

91. 91. The polycistronic construct of any one of claims 1 to 90, wherein the fourth expression cassette comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 26, 63, 71, or 82.

92. 92. The polycistronic construct of any one of claims 1 to 91, wherein the fourth expression cassette comprises the nucleotide sequence of SEQ ID NO: 26, 63, or 82.

93. 93. The polycistronic construct of any one of claims 1 to 92, wherein the fourth expression cassette encodes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 27, 72 or 127.

94. 94. The polycistronic construct of any one of claims 1 to 93, wherein the fourth expression cassette encodes the amino acid sequence of SEQ ID NO: 27, 72 or 127.

95. 2. The polycistronic construct of claim 1, wherein each of the cleavage site sequences comprises a 2A cleavable linker sequence.

96. 96. The polycistronic construct of claim 95, wherein each nucleotide encoding a 2A cleavable linker sequence is different.

97. 97. The polycistronic construct of claim 95 or claim 96, wherein said 2A cleavable linkers are independently a T2A, P2A, E2A or F2A cleavage site.

98. 98. The polycistronic construct of claim 95 or claim 97, wherein the 2A cleavable linker is independently P2A or T2A.

99. 99. The polycistronic construct of any one of claims 95-98, wherein at least one 2A cleavable linker is P2A, and the nucleotide sequence encoding said P2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, 25, 52, or 58.

100. 100. The polycistronic construct of claim 99, wherein the nucleotide sequence encoding the P2A cleavable linker is set forth in SEQ ID NO: 17, 25, 52, or 58.

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

18.

102. 102. The polycistronic construct of claim 101, wherein the P2A cleavable linker comprises the sequence shown in SEQ ID NO:

18.

103. 103. The polycistronic construct of any one of claims 95-102, wherein at least one T2A cleavable linker is T2A, and the nucleotide sequence encoding said T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

9.

104. 104. The polycistronic construct of any one of claims 95 to 103, wherein the nucleotide sequence encoding the T2A cleavable linker is set forth in SEQ ID NO:

9.

105. The polycistronic construct of any one of claims 97, 98, 103 and 104, wherein the T2A cleavable linker comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

10.

106. 106. The polycistronic construct of any one of claims 95 to 105, wherein the T2A cleavable linker comprises the sequence shown in SEQ ID NO:

10.

107. 107. The polycistronic construct of any one of claims 1 to 106, wherein at least one of said cleavage site sequences comprises a furin cleavage site sequence.

108. 108. The polycistronic construct of claim 107, wherein the furin cleavage site sequence is located between the first expression cassette and the second expression cassette.

109. 109. The polycistronic construct of claim 107 or claim 108, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

7.

110. 110. The polycistronic construct of any one of claims 107 to 109, wherein the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence shown in SEQ ID NO:

7.

111. 111. The polycistronic construct of any one of claims 107-110, wherein said furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

8.

112. 112. The polycistronic construct of any one of claims 106 to 111, wherein said furin cleavage site sequence comprises the amino acid sequence of SEQ ID NO:

8.

113. 113. The polycistronic construct of any one of claims 1 to 112, wherein the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furin T2A).

114. 114. The polycistronic construct of any one of claims 1 to 113, wherein the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

5.

115. 115. The polycistronic construct of any one of claims 1 to 114, wherein the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO:

5.

116. 116. The polycistronic construct of any one of claims 1 to 115, wherein said cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

6.

117. 117. The polycistronic construct of any one of claims 1 to 116, wherein the cleavage site sequence comprises the amino acid sequence of SEQ ID NO:

6.

118. 110. The polycistronic construct of any one of claims 1 to 109, wherein the first and second expression cassettes are separated by Furin T2A, the second and third expression cassettes are separated by P2A, and the third and fourth expression cassettes are separated by P2A.

119. 119. The polycistronic construct of any one of claims 1 to 118, wherein said construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

1.

120. 120. The polycistronic construct of any one of claims 1 to 119, wherein said construct comprises the nucleotide sequence of SEQ ID NO:

1.

121. 121. The polycistronic construct of any one of claims 1 to 120, wherein said construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

2.

122. 122. The polycistronic construct of any one of claims 1 to 121, wherein said construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

2.

123. 119. The polycistronic construct of any one of claims 1 to 118, wherein said construct comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:

48.

124. 124. The polycistronic construct of any one of claims 1 to 118 or 123, wherein said construct comprises the nucleotide sequence of SEQ ID NO:

48.

125. 125. The polycistronic construct of any one of claims 1-118, 123, or 124, wherein said construct encodes a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

49.

126. 126. The polycistronic construct of any one of claims 1 to 118 or 123 to 125, wherein said construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

49.

127. 127. A viral vector comprising the polycistronic construct of any one of claims 1 to 126.

128. The viral vector of claim 127, which is a lentiviral vector.

129. 129. The viral vector of claim 127 or claim 128, further comprising one or more surface T cell activators.

130. 130. The viral vector of claim 129, wherein said one or more surface T cell activators comprise CD58, anti-CD3, or CD80.

131. A cell comprising the viral vector of any one of claims 127 to 130.

132. 132. The cell of claim 131, comprising a stem or progenitor cell.

133. 133. The cell of claim 132, wherein the stem cells comprise induced pluripotent stem cells (iPSCs).

134. The cell of claim 131 , wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

135. The cell of claim 131, comprising a T cell.

136. 132. The cell of claim 131, comprising a cytotoxic innate lymphoid (CIL) cell.

137. 132. The cell of claim 131, comprising a natural killer (NK) cell.

138. 127. A method of transducing a cell, comprising contacting a target cell with any of the polycistronic constructs of any one of claims 1 to 126.

139. 131. A method of transducing a cell, comprising contacting a target cell with the viral vector of any one of claims 127 to 130.

140. 140. The method of claim 138 or claim 139, wherein the target cells comprise stem cells.

141. 141. The method of claim 140, wherein said stem cells comprise induced pluripotent stem cells (iPSCs).

142. 140. The method of claim 138 or claim 139, wherein the target cells comprise progenitor cells.

143. 143. The method of claim 142, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

144. 140. The method of claim 138 or claim 139, wherein the target cells comprise T cells.

145. The method of claim 144, wherein the T cells comprise CD4+ or CD8+ T cells.

146. 146. The method of any one of claims 138-145, further comprising contacting a target cell with (i) a guide RNA (gRNA) that targets a target site in the endogenous gene, and (ii) an RNA-guided endonuclease, thereby inserting said nucleotide sequence into the endogenous gene.

147. A method for expressing chimeric antigen receptors and / or synthetic cytokine receptors in target cells.

148. 131. A method of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell, the method comprising contacting said target cell with the viral vector of any one of claims 127-130.

149. 149. The method of claim 147 or claim 148, wherein the target cells comprise stem cells.

150. 150. The method of claim 149, wherein said stem cells comprise induced pluripotent stem cells (iPSCs).

151. 149. The method of claim 147 or claim 148, wherein the target cells comprise progenitor cells.

152. 152. The method of claim 151, wherein the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

153. 149. The method of claim 147 or claim 148, wherein the target cells comprise T cells.

154. The method of claim 153, wherein the T cells comprise CD4+ or CD8+ T cells.

155. The method of any one of claims 138-154, which is carried out ex vivo or in vitro.

156. The method of any one of claims 138-154, which is carried out in vivo.

157. 127. A method of transducing a T cell, comprising contacting said T cell with a viral vector comprising one or more T cell activators and the polycistronic construct of any one of claims 1 to 126, wherein said one or more T cell activators bind to a receptor on said T cell.

158. 127. A method of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a T cell, said method comprising contacting said T cell with a viral vector comprising one or more T cell activators and the polycistronic construct of any one of claims 1 to 126, wherein said one or more T cell activators bind to a receptor on said T cell.

159. 127. A method of delivering a payload to a T cell, comprising contacting the T cell with a viral vector comprising one or more T cell activators and the polycistronic construct of any one of claims 1-126, wherein the one or more T cell activators bind to a receptor on the T cell.

160. The method of any one of claims 157-159, wherein said T cells comprise CD4+ or CD8+ T cells.

161. The method of any one of claims 157-160, which is carried out ex vivo or in vitro.

162. The method of any one of claims 157-160, which is carried out in vivo.

163. The method of any one of claims 157-162, wherein said one or more T cell activators comprise CD58, anti-CD3, or CD80.

164. 164. The method of any one of claims 157-163, wherein said viral vector comprises a lentiviral vector.

165. 165. A cell produced by the method of any one of claims 138-164.

166. A method of administering the cells of claim 165 to a subject.

167. A method of administering to a subject the viral vector of any one of claims 127 to 130.

168. 168. The method of claim 166 or claim 167, wherein the method treats a disease or condition in the subject.

169. The method of claim 168, wherein said disease or condition is treatable by a chimeric antigen receptor (CAR) encoded by said polycistronic construct.

170. The method of claim 169, wherein said CAR is an anti-FITC CAR, and said CAR is targeted to cells of said disease or condition by administering a bifunctional ligand comprising FITC and a ligand that specifically binds to a molecule expressed on cells of said disease or condition.

171. 171. The method of any one of claims 168-170, wherein the disease or condition is cancer.

172. 172. The method of claim 171, wherein the cancer is a solid tumor.

173. The method of any one of claims 170 to 172, wherein the cell is a cancer cell.

174. 168. The method of claim 166 or claim 167, further comprising administering to the subject a bifunctional ligand to tag cancer cells in the subject, wherein the bifunctional ligand specifically binds to a molecule expressed on a tumor.

175. The method of claim 174, wherein the bifunctional ligand comprises a fluorescein isothiocyanate (FITC) moiety, and the chimeric antigen receptor (CAR) encoded by the polycistronic construct is an anti-FITC CAR.

176. 175. The method of any one of claims 167-174, wherein said bifunctional ligand comprises FITC-folate.

177. 177. The method of any one of claims 167-176, further comprising administering to said subject a non-physiological ligand, optionally wherein said non-physiological ligand binds to a synthetic cytokine receptor comprised of a synthetic gamma chain polypeptide and a synthetic cytokine beta chain polypeptide encoded by a polycistronic construct.

178. 178. The method of claim 177, wherein the non-physiological ligand comprises rapamycin or a rapamycin analog.

179. 179. The method of claim 177 or claim 178, wherein binding of said non-physiological ligand to said synthetic cytokine receptor stimulates an intracellular cytokine signal in a cell transduced to express said synthetic cytokine receptor.

180. 179. The method of claim 177 or claim 178, wherein binding of said non-physiological ligand to said synthetic cytokine receptor promotes proliferation of cells transduced to express said synthetic cytokine receptor.

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