Polynucleotide constructs and related viral vectors and methods

Abstract

Provided herein are polycistronic constructs useful for co-expression of synthetic cytokine receptor complexes and chimeric antigen receptor systems, as well as vectors (such as viral vectors) comprising the polycistronic constructs, cells comprising the polycistronic constructs, and methods of using the polycistronic constructs.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 422,920, filed on November 4, 2022, U.S. Provisional Application No. 63 / 449,289, filed on March 1, 2023, and U.S. Provisional Application No. 63 / 466,714, filed on May 15, 2023, all of which are entitled “POLYNUCLEOTIDE CONSTRUCT AND RELATED VIRAL VECTORS AND METHODS,” the contents of which are incorporated herein by reference in their entirety. References to electronic sequence listings

[0002] The contents of the electronic sequence listing (260132000940SEQLIST.xml; size 246,342 bytes; and creation date: October 31, 2023) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure provides polycistronic constructs for co-expressing synthetic cytokine receptor complexes and chimeric antigen receptor systems, as well as vectors (such as viral vectors) comprising the polycistronic constructs, cells comprising the polycistronic constructs, and methods of using the polycistronic constructs. Background Art

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

[0005] Provided herein is a multicistronic construct comprising, in 5' to 3' order, (a) a first expression cassette comprising a nucleotide sequence encoding FRB, (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine γ chain polypeptide, (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine β chain polypeptide, and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each expression cassette is separated by a cleavage site sequence.

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

[0007] In some of any 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 of any embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide include the nucleotide sequence of SEQ ID NO: 15.

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

[0009] In some of any of the 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 of any of the embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some of any of the 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 of any of the embodiments, the FRB comprises the amino acid sequence of SEQ ID NO: 14.

[0010] In some of any of the 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 embodiments, the second expression cassette further comprises a nucleotide sequence encoding FKBP12. In some of any 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 embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. In some of any embodiments, 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. In some of any embodiments, the 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 that comprises the sequence of SEQ ID NO: 54, 57, or 128.

[0014] In some of any embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB). In some of any embodiments, the nucleotide 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. In some of any embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. In some of any embodiments, 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. In some of any embodiments, the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62.

[0015] In some of any embodiments, the third expression cassette further 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. In some of any embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21. In some of any embodiments, 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. In some of any embodiments, the 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 any embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 55.

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

[0018] In some of any 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 of any embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. In some of any 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 of any embodiments, the 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 of any embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, and the intracellular domain comprises a costimulatory signaling domain and a primary activation signaling domain, such as a CD3 ζ signaling domain. In some of any embodiments, the extracellular antigen binding domain and the transmembrane domain are separated by a spacer sequence (such as comprising a hinge domain). In some embodiments, the extracellular antigen binding domain comprises an scFv.

[0022] In some of any embodiments, the CAR includes a scFv domain. In some embodiments, the scFv domain includes anti-fluorescein isothiocyanate (FITC) E2.

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

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

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

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

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

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

[0029] In some of any 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 embodiments, the CD8 alpha hinge comprises the nucleotide sequence of SEQ ID NO: 38. In some of any 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 embodiments, the CD8 alpha hinge comprises the amino acid sequence of SEQ ID NO: 39 or 115.

[0030] In some of any embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises CD8 or CD28. In some embodiments, the CD8 transmembrane domain comprises a CD8 alpha transmembrane domain.

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

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

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

[0034] In some of any embodiments, the 4-1BB intracellular 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: 42 or 69. In some of any embodiments, the 4-1BB intracellular domain comprises a nucleotide sequence of SEQ ID NO: 42 or 69. In some of any embodiments, the 4-1BB intracellular 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: 43. In some of any embodiments, the 4-1BB intracellular domain comprises an amino acid sequence of SEQ ID NO: 43.

[0035] In some of any embodiments, the CD3 zeta intracellular 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: 46, 70, 100, or 118. In some of any embodiments, the CD3 zeta intracellular domain comprises a nucleotide 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 embodiments, the CD3 zeta intracellular domain 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 any of the embodiments, each cleavage site sequence comprises a 2A cleavable linker sequence.

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

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

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

[0041] In some of any of the 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 of any of the embodiments, the nucleotide sequence encoding the P2A cleavable linker is shown in SEQ ID NO: 17, 25, 52, or 58.

[0043] In some of any of the 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 any of the embodiments, the P2A cleavable linker comprises the sequence shown in SEQ ID NO:18.

[0045] In some of any of the embodiments, at least one 2A 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 of any of the embodiments, the nucleotide sequence encoding the T2A cleavable linker is shown in SEQ ID NO:9.

[0047] In some of any of the 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 any of the embodiments, the T2A cleavable linker comprises the sequence shown in SEQ ID NO:10.

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

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

[0051] In some of any of the 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 any embodiments, the nucleotide sequence encoding the furin cleavage site sequence comprises the sequence shown in SEQ ID NO: 7. In some of any embodiments, the furin cleavage site sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

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

[0054] In some of any of the embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A).

[0055] 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.

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

[0057] In some of any of the 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 any of the embodiments, the cleavage site sequence comprises the amino acid sequence of SEQ ID NO:6.

[0059] In some of any of the embodiments, the first expression cassette and the second expression cassette are separated by furinT2A, 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 any 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 any embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 1.

[0061] In some of any 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 any embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0062] In some of any 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 any embodiments, the construct comprises the nucleotide sequence of SEQ ID NO: 48.

[0063] In some of any 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 any embodiments, the construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49.

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

[0065] In some of any 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.

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

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

[0068] In some aspects, a method of transducing cells is provided herein, comprising contacting a target cell with any polycistronic construct disclosed herein. In some aspects, a method of transducing cells is provided herein, comprising contacting a target cell with any of the viral vectors disclosed herein. In some of any embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC).

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

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

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

[0072] In some aspects, the present invention provides a method for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell. In some aspects, the present invention provides a method for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell, the method 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. In 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 any of the embodiments, the method is performed ex vivo or in vitro.

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

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

[0078] In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

[0079] In some aspects, the present invention provides a method of administering any of the cells disclosed herein to a subject. In some embodiments, the chimeric antigen receptor is capable of being targeted to an antigen associated with a disease or condition in the subject, and the subject suffers from 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, there is provided herein a method for administering any one of the viral vectors disclosed herein to a subject. In some of any embodiments, the method treats the subject's disease or illness. In some of any embodiments, the disease or illness can be treated by a chimeric antigen receptor (CAR) encoded by the polycistronic construct. In some of any embodiments, the CAR is an anti-FITC CAR, and CAR is targeted to the cell of the disease or illness by administering a bifunctional ligand, the bifunctional ligand comprising FITC and a ligand specifically binding to a molecule expressed on the cell of the disease or illness. In some of any embodiments, the disease or illness 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 any embodiments, the chimeric antigen receptor can be targeted to an antigen associated with a disease or condition in a subject, and the subject has a disease or condition that can be treated by the chimeric antigen receptor. In some of any embodiments, the disease or condition is cancer.

[0082] In some of any embodiments, the CAR is a CAR targeting ligand, and the ligand can be bound to an antigen on the surface of a cell associated with a disease or illness. In some of any embodiments, the CAR of any embodiment provided is an anti-FITC CAR for FITC, and the ligand is a bifunctional ligand consisting of FITC and a binding molecule, and the binding molecule can bind to a surface molecule or receptor on the target cell. In some of any embodiments, the method further includes administering a bifunctional ligand to label the subject's cancer cells, wherein the bifunctional ligand specifically binds to molecules expressed on tumors. In some of any embodiments, the bifunctional ligand is FITC-folic acid. In some embodiments, the cancer is osteosarcoma. In some of any embodiments, the bifunctional ligand includes 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 embodiments, the method further includes administering a non-physiological ligand to the subject. In some of any embodiments, the non-physiological ligand is capable of binding to a synthetic cytokine receptor and inducing gamma cytokine signaling in a cell. In some of any embodiments, the non-physiological ligand. In some of any embodiments, the non-physiological ligand binds to a synthetic cytokine receptor, and the synthetic cytokine receptor binds to a synthetic gamma chain polypeptide and a synthetic cytokine beta chain polypeptide encoded by a polycistronic construct. In some of any embodiments, the non-physiological ligand includes an analog of rapamycin or rapamycin. In some of any embodiments, the binding of the non-physiological ligand to the synthetic cytokine receptor stimulates intracellular cytokine signals in cells that are transduced to express a synthetic cytokine receptor. In some embodiments, the binding of the non-physiological ligand to the synthetic cytokine receptor promotes the proliferation of cells that are transduced to express a synthetic cytokine receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1A Lentiviral particles are shown that are surface-engineered to engage and activate T cells to deliver a payload containing free FKBP12-rapamycin binder (FRB), rapamycin-activated cytokine receptor (RACR), and a chimeric antigen receptor (CAR) that binds a tumor tag (TagCAR). Rapamycin, FRB, and RACR drive the expansion of transduced TagCAR T cells while also inhibiting tumor proliferation and immune responses to the lentiviral particles.

[0085] Figure 1B Demonstrating how the TagCAR T system targets tumor cells. Bispecific tumor tags contain a universal tag antigen on one end and an exchangeable ligand that binds to a tumor or tumor microenvironment-associated antigen on the other end. Once a T cell expresses the TagCAR, it can bind to a universal tag (e.g., FITC-folate).

[0086] Figures 2A-2B8 polycistronic constructs are shown. The polynucleotide of TagCAR encodes a CAR having the following composition in the order from N-terminus to C-terminus: scFv (such as anti-FITC E2), hinge (spacer), transmembrane domain, intracellular domain with costimulatory signaling domain and CD3ζ signaling domain (Z). These constructs differ in hinge (spacer) domain (IgG4 hinge (IgG4H) or CD8α hinge (CD8H)); transmembrane domain (CD28 TM or CD8 TM) and costimulatory domain (41BB costimulatory domain or CD28 costimulatory domain). The construct is also different in the placement of TagCAR, appearing to be located at the front end of the construct transgenic sequence or at the end of the construct transgenic sequence. The various polynucleotide compositions of the construct are separated by a 2A cleavage site sequence.

[0087] Figures 3A-3B The multiplicity of infection (MOI) of 2 or 10 is shown. Figures 2A-2B Activation of CD8+ and CD4+ T cell populations cultured with the polynucleotide constructs in on day 3 and transduction on day 7.

[0088] Figure 3C Shows when FRB is located Figures 2A-2B The first or second position of the depicted transgene is FRB expression in T cells.

[0089] Figure 4 Shown are the percentages 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 IL-2 and rapamycin.

[0090] Figures 5A-5B For display Figures 2A-2B Tumor cell killing assay of PBMCs transduced with the constructs disclosed in and treated with IL-2 alone or both IL-2 and rapamycin (rapa).

[0091] Figure 6A CD19 chimeric antigen receptor (CAR) and TagCAR expression is shown in PBMCs transduced with lentivirus encoding Construct V or Construct C.2 polynucleotides.

[0092] Figure 6B The number of CD19-CAR+ (left panel) or FITC-folate+ (right panel) PBMCs over 11 days is shown. PBMCs were transduced with lentivirus encoding Construct V or Construct C.2 polynucleotides.

[0093] Figure 7AThe orientation of the polynucleotide constructs is shown from N to C terminus. In construct C.2, FRB is encoded immediately adjacent to IL2Rβ. In construct C.2U, FRB is encoded immediately adjacent to IL2Rγ.

[0094] Figure 7B Shown are the percentages of CD25 T cells in five donors 3 days after transfection with lentivirus encoding Construct V or Construct C.2 polynucleotides.

[0095] Figure 7C Shown are the percentages 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.

[0096] Figure 7D Shown are TagCAR MFI in T cells on day 7. PBMCs were transduced with lentivirus encoding Construct C.2 or Construct C.2U polynucleotides.

[0097] Figure 7E Immunoblots depicting the expression of FRB:IL2Rβ and FRB:IL2Rγ in T cells are shown. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. FRB was detected using a rabbit pAb against FRB. Cells were harvested on day 8 for Western blotting.

[0098] Figure 7F Immunoblots depicting the expression of FKBP12:IL2Rβ and FKBP12:IL2Rγ in T cells are shown. PBMCs were transduced with lentivirus encoding construct C.2 or construct C.2U polynucleotides. FKBP12 was detected using a mouse mAb against FKBP12. Cells were harvested on day 8 for Western blotting.

[0099] Figure 8A Shown are the total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 over 14 days in one donor. Tag-CAR+ T cells were treated with IL-2 (250 U / mL) or rapamycin (10 nM).

[0100] Figure 8B Shown are the total Tag-CAR+ T cells transduced with construct C.2U or construct C.2 over 12 days in one donor. Tag-CAR+ T cells were treated with IL-2 (250 U / mL), rapamycin (10 nM), rapamycin and IL-2, or AP21967 (50 nM).

[0101] Figure 9A Activation of CD8+ and CD4+ T cell populations cultured with construct C.2 at a multiplicity of infection (MOI) of 2 or 10 on day 3 and transduction on day 7 are shown.

[0102] Figure 9B Representative flow cytometric plots of TagCAR-expressing CD8+ T cells transduced with construct C.2 at day 7 post-transduction are shown.

[0103] Figure 10A Figure 2: Timeline of in vitro assays for measuring T cell activation and TagCAR T cell abundance. On day 0, PBMCs were transduced with lentiviral particles. On day 3, IL-2 or IL-2 and rapamycin were added to measure T cell activation. On days 7, 11, and 14, TagCAR T cell abundance was measured.

[0104] Figure 10B Figure 2: Enrichment (left) and expansion (right) of TagCAR T cells over 2 weeks. Enrichment was measured by flow cytometry. Expansion was measured by flow cytometry using counting beads.

[0105] Figure 11A Figures depicting tumor cell growth 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 are shown. Breast cancer cells not incubated with TagCAR T cells, FITC-folate or rapamycin are indicated by arrows. Tumor cells were reintroduced at 72 hours, 144 hours and 216 hours (i.e., tumor cell re-challenge).

[0106] Figure 11B Show depicts a graph of T cell proliferation after incubation of breast cancer cells (MDA-MB-231 or MDA) with rapamycin, TagCAR T cells and FITC-folic acid, or TagCAR T cells, FITC-folic acid and rapamycin (rapa). Breast cancer cells incubated without TagCAR T cells, FITC-folic acid or rapamycin are indicated by arrows. Tumor cells were reintroduced at 72 hours, 144 hours and 216 hours (i.e., tumor cells were challenged again).

[0107] Figure 12A Timeline of the in vivo mouse model of breast cancer is shown. 14 days before the start of the experiment, NSG MHCI / II DKO Mice were injected with FRα+MDA-MB-231 cells. On the first day of the experiment (D0), mice were infused with ex vivo generated TagCAR T cells. Mice were subcutaneously injected with FITC-folic acid twice weekly for four weeks. Blood was collected weekly for flow cytometry.

[0108] Figure 12BFigure 3 shows a graph depicting tumor volume in mice over 4 weeks. Mice were injected with: (i) 10e6 T cells not transduced with lentiviral particles (mock T cells), FITC-folate; (ii) 10e6 TagCAR T cells, no FITC-folate; (iii) 5e6 TagCAR T cells, with FITC-folate; and (iv) 10e6 TagCAR T cells, with FITC-folate. Tumor volume was measured using a caliper.

[0109] Figure 13A Timeline of the in vivo mouse model of breast cancer is shown. 14 days before the start of the experiment, NSG MHCI / II DKO Mice were injected with FRα+MDA-MB-231 cells. On the first day of the experiment (D0), mice were humanized with PBMCs and administered lentiviral particles. Mice were subcutaneously injected with FITC-folic acid twice weekly for 7 weeks. Blood was collected weekly for flow cytometry. Tumor volume was measured using a caliper.

[0110] Figure 13B Detection of circulating TagCAR T cells by flow cytometry is shown. The left graph depicts the percentage of each TagCAR+ / CD3+ T cell at day 7. The right graph depicts the total number of CD3+ / TagCAR+ T cells per μL of blood at day 7. Mice were injected with: (i) no vector and FITC-folic acid; (ii) 100e6 transfection units (TU) of TagCAR vector and FITC-folic acid; (iii) 25e6 TU TagCAR vector and FITC-folic acid; and (iv) 100e6 TU TagCAR vector and FITC-folic acid.

[0111] Figure 13C Shown is a graph depicting tumor volume in mice over 7 weeks. Tumor volume was measured using calipers.

[0112] Figure 14A Figure 2 shows a graph depicting tumor volume in mice over 25 days. Mice were injected with: (i) FITC-folate alone; (ii) 5.0e6 transfection units (TU) of TagCAR vector without FITC-folate; (iii) 0.2e6 TU of TagCAR vector with FITC-folate; (iv) 1.0e6 TU of TagCAR vector with FITC-folate; and (v) 5.0e6 TU of TagCAR vector with FITC-folate. Tumor volume was measured using a caliper.

[0113] Figure 14BCirculating CD3 TagCAR T cells per μL of blood are shown as detected by flow cytometry. Mice were injected with: (i) FITC-folic acid only (triangles); (ii) 5.0e6 TU TagCAR vector only; (iii) 0.2e6 TU TagCAR vector with FITC-folic acid; (iv) 1.0e6 TU TagCAR vector with FITC-folic acid; and (v) 5.0e6 TU TagCAR vector with FITC-folic acid. DETAILED DESCRIPTION

[0114] The present disclosure generally relates to a kind of polynucleotide construct and the method using the polynucleotide construct, the polynucleotide construct comprises a continuous polynucleotide sequence encoding at least two synthetic receptors.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 is responsive to rapamycin binding. Advantageously, in cells engineered to express the polynucleotide construct provided herein, FRB reduces the inhibitory effect of rapamycin on mTOR. The expression of freely diffusible FRB can promote the sustained activation and proliferation of engineered cells.

[0115] The present disclosure also provides that the 5' to 3' sequence of the polycistronic construct is crucial for the expression of the polypeptide encoded by the construct. In some embodiments, the 5' to 3' sequence of the polynucleotide construct improves the expression of the encoded polypeptide. In some embodiments, the polycistronic construct provided herein comprises nucleotides encoding FRB at the 5' end, which improves FRB expression and is associated with enhanced protection against rapamycin-mediated immunosuppression. In addition, surprisingly, the CAR expression - even when located at the 3' end of the construct - is high enough to mediate antigen-directed killing of cells expressing the polynucleotide construct.

[0116] In some aspects, the present invention provides a polycistronic construct comprising four expression cassettes separated by a cleavage site sequence. In some embodiments, the four expression cassettes comprise, in 5' to 3' order: a first expression cassette comprising a nucleotide sequence encoding FRB, a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine γ chain polypeptide, a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine β chain polypeptide, and a fourth expression cassette comprising a nucleotide sequence encoding a CAR.

[0117] In some aspects, provided herein is a viral vector comprising any of the polycistronic constructs disclosed herein.

[0118] In some aspects, provided herein is a cell comprising any of the viral vectors disclosed herein.

[0119] In some aspects, provided herein is a method of transducing a cell, comprising contacting a target cell with any one of the viral vectors disclosed herein.

[0120] In some aspects, provided herein is a method 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.

[0121] In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

[0122] In some aspects, provided herein is a method of administering any of the cells disclosed herein to a subject. In some aspects, provided herein is a method of administering any of the viral vectors disclosed herein to a subject.

[0123] All publications, including patent documents, scientific articles, and databases, mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. In the event of a conflict or inconsistency between definitions set forth herein and definitions in patents, applications, published applications, and other publications cited herein, the definitions set forth herein shall control.

[0124] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Polycistronic Constructs

[0125] Provided herein are polycistronic constructs encoding one or more individual proteins. In some embodiments, the polycistronic construct comprises one, two, three, or four expression cassettes, each encoding an individual protein. In some embodiments, the polycistronic construct comprises four expression cassettes, each encoding an individual protein. In some embodiments, the expression cassettes are separated by cleavable linkers.

[0126] In some embodiments, provided herein are polycistronic constructs comprising a nucleotide sequence encoding FRB. In some embodiments, provided herein are polycistronic constructs comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, provided herein are polycistronic constructs comprising a nucleotide sequence encoding a synthetic cytokine polypeptide. In some embodiments, the synthetic cytokine polypeptide comprises a synthetic cytokine γ chain polypeptide and a synthetic cytokine β chain polypeptide. In some embodiments, the synthetic cytokine γ chain comprises interleukin 2 receptor subunit γ (IL2RG). In some embodiments, the synthetic cytokine γ chain further comprises FRB. In some embodiments, the synthetic cytokine β chain comprises interleukin 2 receptor subunit β (IL2RB). In some embodiments, the synthetic cytokine γ chain further comprises FKBP12. In other embodiments, the synthetic cytokine γ chain comprises interleukin 2 receptor subunit γ (IL2RG). In some embodiments, the synthetic cytokine γ 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.

[0127] In some embodiments, the polycistronic constructs provided herein comprise nucleotide sequences encoding a FRB, a synthetic cytokine polypeptide, and a CAR.

[0128] In some embodiments, the polycistronic construct comprises, in 5' to 3' order, a nucleotide sequence encoding 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.

[0129] In one aspect, provided herein is a multicistronic construct comprising, in 5' to 3' order, (a) a first expression cassette comprising a nucleotide sequence encoding FRB, (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine γ chain polypeptide, (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine β chain polypeptide, and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each expression cassette is separated by a nucleotide sequence encoding a cleavage site sequence. A. Cytoplasmic FRB

[0130] In some embodiments, the expression cassette of the polycistronic construct encodes a FRB domain. The FRB domain is an approximately 270 base pair (bp) domain derived from the mTOR protein kinase. It can be expressed in the cytoplasm as a freely diffusible soluble protein.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0135] Advantageously, cytoplasmic FRBs confer resistance to the immunosuppressive effects of non-physiological ligands, such as rapamycin or rapamycin analogs. B. Synthetic cytokine receptors

[0136] 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 comprising a dimerization domain. In the presence of a non-physiological ligand, the dimerization domain can controllably dimerize, thereby activating signal transduction of the synthetic cytokine receptor.

[0137] The synthetic cytokine receptor may include a transmembrane receptor protein, and the transmembrane receptor protein includes a synthetic γ chain polypeptide and a synthetic β chain polypeptide, such as provided as a first transmembrane receptor and a second transmembrane receptor. The synthetic γ chain polypeptide includes a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit γ (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to transmembrane domain) or intracellular (C-terminal to transmembrane domain and N-terminal or C-terminal to IL-2G intracellular domain). The synthetic β chain polypeptide includes a second dimerization domain, a second transmembrane domain and an intracellular domain, and the intracellular domain is selected from interleukin-2 receptor subunit β (IL-2RB) intracellular domain, interleukin-7 receptor subunit β (IL-7RB) intracellular domain or interleukin-21 receptor subunit β (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).

[0138] 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 a synthetic cytokine receptor and a second expression cassette encoding the IL2RB chain of a synthetic cytokine receptor.

[0139] 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 skilled in the art are readily familiar with signal peptides that can provide a signal for transport of nascent proteins within cells. Any of a variety of signal peptides can be used. 1. Intracellular domain

[0140] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

[0141] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-2RB intracellular domain, and a second dimerization domain.

[0142] In some embodiments, the synthetic beta chain comprises the intracellular domain of interleukin-2 receptor subunit beta (IL2RB). IL2RB is also known as IL15RB or CD122. Thus, when referred to herein, IL2RB may also refer to IL15RB. That is, in this disclosure, these terms can be used interchangeably.

[0143] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-7RB intracellular domain, and a second dimerization domain.

[0144] In some embodiments, the synthetic beta chain comprises the interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

[0145] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising the IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising the IL-21RB intracellular domain, and a second dimerization domain.

[0146] In some embodiments, the synthetic beta chain comprises the interleukin-2 receptor subunit beta (IL21RB) intracellular domain. 2. Dimerization domain

[0147] The dimerization domain may be a heterologous dimerization domain, including but not limited to the 12 kD FK506 binding protein (FKBP) and the FKBP12 rapamycin binding (FRB) domain, which are known in the art to dimerize in the presence of rapamycin or a rapamycin analog.

[0148] Alternatively, the first dimerization domain and the second dimerization domain may be the 12 kD FK506 binding protein (FKBP) and calcineurin domains, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0149] In some embodiments, the dimerization domain is a homodimerization domain selected from the group consisting of: i) 12 kD FK506 binding protein (FKBP); ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are: i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporine-A (CsA); or iii) Coumarin or an analogue thereof.

[0150] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0151] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0152] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0153] 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). 3. Transmembrane domain

[0154] The transmembrane domain is a sequence of a 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.

[0155] 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.

[0156] In some embodiments, one or more additional contiguous amino acids of the extracellular domain directly adjacent to the TM domain of the cytokine receptor may also be included as part of the polypeptide sequence of the synthetic cytokine receptor chain. In some embodiments, 1-20 contiguous amino acids of the extracellular domain 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 extracellular domain can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids that are directly adjacent to the TM sequence (e.g., located at its N-terminus).

[0157] In some embodiments, the synthetic cytokine receptor is capable of being bound by a non-physiological ligand, rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to a 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 cell signaling in the cell, such as via the JAK / STAT pathway. 4. Exemplary Synthetic Cytokine Receptors

[0158] The synthetic cytokine receptors of the present disclosure comprise a synthetic gamma chain and a synthetic beta chain, each comprising a dimerization domain. In the presence of a non-physiological ligand, the dimerization domain can controllably dimerize, thereby activating the signaling of the synthetic cytokine receptor.

[0159] 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.

[0160] The synthesized β chain polypeptide comprises a second dimerization domain, a second transmembrane domain and an intracellular domain, and the intracellular domain is selected from interleukin-2 receptor subunit β (IL-2RB) intracellular domain, interleukin-7 receptor subunit β (IL-7RB) intracellular domain or interleukin-21 receptor subunit β (IL-21RB) intracellular domain. The dimerization domain can be extracellular (N-terminal to transmembrane domain) or intracellular (C-terminal to transmembrane domain and N-terminal or C-terminal to IL-2RB or IL-7RB intracellular domain). In some embodiments, the synthesized β chain polypeptide comprises FKBP12:IL2RB fusion protein. In some embodiments, the synthesized β chain polypeptide comprises FRB:IL2RB fusion protein.

[0161] 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 a FRB:IL2RG fusion protein. In some 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 embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 80% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 85% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 90% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is 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 nucleotides encoding the synthetic cytokine gamma chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide consist of the nucleotide sequence of SEQ ID NO: 15.

[0162] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, the IL2RG comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the IL2RG comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL2RG consists of the amino acid sequence of SEQ ID NO: 16.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] In some embodiments, the second expression cassette is codon optimized.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] In some embodiments, the second expression cassette comprises nucleotides encoding a synthetic cytokine gamma chain polypeptide, wherein the synthetic cytokine gamma chain polypeptide is a 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.

[0171] In some embodiments, 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. In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the FKBP12 consists of the amino acid sequence of SEQ ID NO: 22.

[0172] 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 comprising the nucleotide sequence of SEQ ID NO: 53 or 56. In some embodiments, the second expression cassette consists of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 53 or 56.

[0173] 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.

[0174] In some embodiments, the third expression cassette comprises nucleotides encoding a synthetic cytokine β chain polypeptide, wherein the synthetic cytokine β chain polypeptide is a FKBP12:IL2RB fusion protein. In some embodiments, the nucleotides encoding the synthetic cytokine β 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 β 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 β 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 β 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 β 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 β 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 β 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 β 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 β 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 β chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine β chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 23 or 61. In some embodiments, the nucleotides encoding the synthetic cytokine β chain polypeptide consist of the nucleotide sequence of SEQ ID NO: 23 or 61.

[0175] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB).

[0176] In some embodiments, 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. In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62. In some embodiments, the IL2RB consists of the amino acid sequence of SEQ ID NO: 24 or 62.

[0177] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

[0178] 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.

[0179] 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.

[0180] In some embodiments, 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. In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the FKBP12 consists of the amino acid sequence of SEQ ID NO: 22.

[0181] In some embodiments, the third expression cassette is codon-optimized.

[0182] 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.

[0183] In some embodiments, the third expression cassette comprises a nucleotide encoding a synthetic cytokine beta chain polypeptide, wherein the synthetic cytokine beta chain polypeptide is a FRB:IL2RB fusion protein. 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: 59. 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: 59. 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: 59. 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: 59. 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: 59. 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: 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.

[0184] 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 second expression cassette encodes an amino acid sequence that consists of the sequence of SEQ ID NO: 60 or 129.

[0185] 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.

[0186] 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.

[0187] 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. C. Chimeric Antigen Receptor

[0188] In some embodiments, the expression cassette of the polycistronic construct encodes a chimeric antigen receptor. 1. CAR constructs and encoding nucleotides

[0189] In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain and / or an activation signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises an activation signaling domain. In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a costimulatory signaling domain and an activation signaling domain.

[0190] 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, etc.

[0191] In some embodiments, the costimulatory signaling domain is used to enhance the proliferation and survival of lymphocytes when the CAR is bound to the targeted portion. The characteristics of the costimulatory signaling domain are limited to the ability to enhance cell proliferation and survival activation when the targeted portion is bound by the CAR. Suitable costimulatory signaling domains include, but are not limited to: CD28 (see, e.g., Alvarez-Vallina, L. et al., Eur J Immunol. 1996. 26 (10): 2304-9); CD137 (4-1BB), a member of the tumor necrosis factor receptor family (TNF) (see, e.g., Imai, C. et al., Leukemia. 2004. 18: 676–84); and CD134 (OX40), a member of the TNFR receptor superfamily (see, e.g., Latza, U. et al., Eur. J. Immunol. 1994. 24: 677). It will be understood by those skilled in the art that sequence variants of these costimulatory signaling domains can be used, wherein 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.

[0192] In some embodiments of the present invention, the CAR construct includes two costimulatory signaling domains. Although a specific combination includes all possible variants of the four indicated domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2) CD28+CD134 (OX40).

[0193] In some embodiments, the activation signaling domain is used to activate cells when the CAR binds to a targeted portion. The properties of the activation signaling domain are limited to the ability to induce activation of selected cells when the targeted portion is bound by the CAR. 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. It will be understood by those skilled in the art that sequence variants of these indicated activation signaling domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domain from which they are modeled. These 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.

[0194] In some embodiments, the CAR may include additional elements, such as a signal peptide that ensures the proper export of the fusion protein to the cell surface, a transmembrane domain that ensures the fusion protein is retained as an integral membrane protein, and a hinge domain that confers flexibility to the recognition region and allows it to strongly bind to the targeted molecule.

[0195] In some embodiments, the nucleotide sequence encoding CAR comprises 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 or an activation signaling domain. In some embodiments, the costimulatory and activation signaling domains are single domains, such as a single intracellular domain that provides both costimulatory and activation signals to cells. In some embodiments, the intracellular signaling domain comprises 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 ζ signaling domain. In some embodiments, the nucleotide sequence encodes an extracellular domain, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ζ 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 ζ 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 co-stimulatory domain, and a CD3zeta signaling domain.

[0196] Illustrative 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., Liu E, Tong Y, Dotti G et al., J Hematol Oncol. 2019; 12-49); (5)scFv-2B4 TM+IC -CD3ζs (see 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, MoriarityBS 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 IC -CD3ζS (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). a.CAR extracellular domain

[0197] In some embodiments, the binding portion of the CAR can be directed against any antigen that is desired to be targeted, such as an antigen because it is overexpressed on a cell or is associated with a disease or disorder (such as cancer).

[0198] In some embodiments, the binding portion of the CAR is specific for a tumor antigen. The choice of the antigen binding domain will depend on the specific type of cancer to be treated.Tumor antigens are well known in the art and include, for example, glioma-associated antigen, 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, α-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, PLAC1, RU1, RU2(AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, legumin, 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 (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, O-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin.Non-limiting examples of tumor antigens include the following: differentiation antigens, such as tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens (such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5); overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as 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, β-catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-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, SDCCAG1 6, 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 a binding domain targeting two or more antigens (in any combination) as disclosed herein. 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, such as two epitopes in BCMA.

[0199] Those skilled in the art are familiar with CARs for different tumor antigens. Any of these CARs can be used as CARs. A large number of CARs have been incorporated into products approved by the FDA, including but not limited to anti-CD19 and anti-BCMA CAR T cells, such as tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta), brexucabtagene autoleucel (Tecartus), lisocabtagene maraleucel (Breyanzi), idecabtagene vicleucel (Abecma) or ciltacabtagene autoleucel (Carvykti). It is within the level of those skilled in the art to generate similar constructs for specifically targeting the desired tumor antigen.

[0200] In some embodiments, the binding portion of the CAR can be directed against a universal antigen to target a wide range of tumors without the need to prepare a separate CAR construct. The targeted portion recognized by the CAR can also remain constant. In some embodiments, a ligand can be administered to the subject to allow interaction with the target cell, as well as interaction with the binding portion of the CAR. Only the ligand portion of the small conjugated molecule needs to be changed to allow the system to target cancer cells of different characteristics. An exemplary CAR system is described in the next section.

[0201] In some embodiments, the CAR is an anti-CD19 CAR, and the extracellular binding domain of the CD19 CAR is specific to CD19 (for example, human CD19). In some embodiments, the extracellular domain of the CD19 CAR includes the scFv derived from FMC63 monoclonal antibody (FMC63), which includes the heavy chain variable region (VH) and light chain variable region (VL) of the FMC63 connected by a joint. FMC63 and its derived scFv are described in Nicholson et al., Mol.Immun.34 (16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 2133337, each of which is incorporated herein by reference in its entirety. Exemplary anti-CD19 CAR is shown in Table 1, and its different parts include extracellular domain.

[0202] 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 may include VH, VL, and / or one or more CDRs of any one of the antibodies, or consist of. Exemplary anti-CD20 CARs are shown in Tables 2 and 3, and their different portions include their extracellular domains. 1) Universal CAR

[0203] Conventionally, if desired, CAR is generated by fusing a polynucleotide encoding VL, VH or scFv to the 5' end of a polynucleotide encoding a transmembrane and intracellular domain, and transducing cells with the polynucleotide and the corresponding VH or VL. In the art, there are a large number of mutants of CAR well known in the art, and the present disclosure contemplates the use of any known mutants. In addition, countless VL / VH pairs and scFvs of haptens are known in the art or can be conventionally generated by conventional methods. Thus, the present disclosure contemplates the use of any known hapten binding domain.

[0204] In some embodiments, the CAR is an anti-FITC CAR, and the ligand is composed of a fluorescein or fluorescein isothiocyanate (FITC) portion conjugated to a medicament combined with a desired target cell (such as a cancer cell). Exemplary ligands are described below. In some embodiments, the ligand is FITC-folic acid.

[0205] Exemplary anti-FITC CARs and their different parts are shown in Table 4.

[0206] 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 joint and a heavy chain variable domain (VH).

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] Various methods for targeting CAR and expressing cells of CAR have been described in the art, including, for example, described in US2020 / 0123224, the disclosure of which is incorporated herein by reference. For example, fluorescein or fluorescein isothiocyanate (FITC) moieties can be conjugated to agents that bind to desired target cells (such as cancer cells), and thereby express anti-fluorescein / FITC chimeric antigen receptor CARs that can be selectively targeted to target cells labeled by conjugates. In different cases, other haptens identified by CAR can be used to replace fluorescein / FITC. The CAR can be generated using a variety of scFv sequences known in the art or scFv sequences generated by conventional methods. For example, further exemplary scFv sequences for fluorescein / FITC and other haptens are provided in WO2021 / 076788, the disclosure of which is incorporated herein by reference.

[0216] In one embodiment, the present disclosure provides a description of such a conjugated molecule / CAR system.

[0217] In some embodiments, the CAR system of the present disclosure utilizes conjugated molecules as a bridge between cells expressing CAR and targeted cancer cells. The conjugated molecules are conjugates comprising haptens and cell targeting moieties (such as any suitable tumor cell specific ligands). Illustrative haptens that can be recognized and combined by CAR include small molecular weight organic molecules, such as DNP (2,4-dinitrophenol), TNP (2,4,6-trinitrophenol), biotin and digoxin, and fluorescein and its derivatives, including FITC (fluorescein isothiocyanate), NHS-fluorescein, pentafluorophenyl ester (PFP) and tetrafluorophenyl ester (TFP) derivatives, knottin (knottin), centyrin and DARPin. Cell targeting moieties that are themselves suitable as haptens for CARs include desmin (see Kolmar H. et al., The FEBS Journal. 2008. 275(11):26684-90), centyrins, and DARPins (see Reichert, JMM Abs 2009. 1(3):190-209).

[0218] In some embodiments, the cell targeting moiety is DUPA (DUPA-(99m)Tc), a ligand bound by PSMA-positive human prostate cancer cells with nanomolar affinity (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 molecule, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0219] In some embodiments, the cell targeting moiety is a CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., thyroid, lung, pancreatic, ovarian, brain, gastric, gastrointestinal stromal tumors, and colon cancer; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).

[0220] In some embodiments, the cell targeting moiety is folate, folic acid, or an analog thereof, a ligand bound by a folate receptor 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.

[0221] In some embodiments, the cell targeting moiety is an NK-1R ligand. For example, receptors for NK-1R ligands are found in cancers of the colon and pancreas. In some embodiments, the NK-1R ligand can be synthesized according to the methods disclosed in International Patent Application No. PCT / US2015 / 044229, which is incorporated herein by reference.

[0222] 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 conjugated 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 (neuromediatin K) (see Hennig et al., International Journal of Cancer: 61, 786-792).

[0223] In some embodiments, the cell targeting moiety is a CAIX ligand. For example, receptors for the CAIX ligand are found in renal cancer, ovarian cancer, vulvar cancer, and breast cancer. The CAIX ligand may also be referred to herein as CA9.

[0224] In some embodiments, the cell targeting moiety is a ligand for gamma glutamyl transpeptidase, which is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia.

[0225] In some embodiments, the cell targeting moiety is a CCK2R ligand, a receptor for which is found in cancers such as thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal tumors, and colon cancer.

[0226] In some embodiments, the cell targeting moiety is a PSMA ligand.

[0227] In some embodiments, the cell targeting moiety is a FAP ligand.

[0228] 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 8000 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.

[0229] In an exemplary embodiment, the bonding in the conjugates described herein can be direct bonding (e.g., the reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the bonding can be through an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In an illustrative embodiment, the divalent linker can contain about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can contain about 2 to about 20 carbon atoms. In other embodiments, divalent linkers with lower molecular weights (i.e., those with an approximate molecular weight of about 30 to 300 Da) are employed. In another embodiment, suitable linker lengths include, but are not limited to, those 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.

[0230] In some embodiments, the hapten and cell targeting moiety can be directly conjugated by reacting the isothiocyanate group of FITC with the free amine groups of small ligands (such as folic acid, DUPA, and CCK2R ligands). However, the use of a linker domain to connect the two molecules may be helpful because it can provide flexibility and stability. Examples of suitable linker domains include: 1) polyethylene glycol (PEG); 2) polyproline; 3) hydrophilic amino acids; 4) carbohydrates; 5) non-natural peptidoglycans; 6) polyvinylpyrrolidone; 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.

[0231] In some embodiments, the linker can be a bivalent linker, which can include one or more spacers.

[0232] An illustrative conjugate of the present disclosure is FITC-folic acid An illustrative conjugate of the present disclosure is FITC-CA9

[0233] Illustrative 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.

[0234] Although the affinity with which a ligand binds to a cancer cell receptor can vary, and in some cases low affinity binding (such as about 1 μM) may be preferred, the ligand typically binds to a cancer cell receptor with an affinity of 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.

[0235] Examples of conjugates and methods of making the same are provided in U.S. Patent Application Nos. US2017 / 0290900, US2019 / 0091308, and US2020 / 0023009, all of which are incorporated herein by reference. b. Spacer (such as hinge domain)

[0236] 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.

[0237] In some embodiments, 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. In some embodiments, the CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α 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 CD8α hinge comprises the nucleotide sequence of SEQ ID NO: 38 or 114. In some embodiments, the CD8α hinge consists of the nucleotide sequence of SEQ ID NO: 38 or 114.

[0238] 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 CD8 alpha 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 consists of the amino acid sequence of SEQ ID NO: 39 or 115.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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. c. Transmembrane domain

[0245] 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 CD8 alpha transmembrane domain.

[0246] 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.

[0247] 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. c. Intracellular domain (i.e., intracellular domain)

[0248] In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the intracellular domain comprises a co-stimulatory molecule. In some embodiments, the intracellular domain comprises 4-1BB, CD3ζ and / or CD28. In some embodiments, the intracellular domain comprises 4-1BB. In some embodiments, the intracellular domain comprises CD3ζ. In some embodiments, the intracellular domain comprises CD28.

[0249] In some embodiments, the 4-1BB intracellular 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: 42 or 69. In some embodiments, the 4-1BB intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain comprises the nucleotide sequence of SEQ ID NO: 42 or 69. In some embodiments, the 4-1BB intracellular domain consists of the nucleotide sequence of SEQ ID NO: 42 or 69.

[0250] In some embodiments, the 4-1BB intracellular 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: 43. In some embodiments, the 4-1BB intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the 4-1BB intracellular domain consists of the amino acid sequence of SEQ ID NO: 43.

[0251] In some embodiments, the CD3 zeta intracellular 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: 46, 70, 100, or 118. In some embodiments, the CD3 zeta intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100, or 118. In some embodiments, the CD3ζ intracellular domain consists of the nucleotide sequence of SEQ ID NO:46, 70, 100 or 118.

[0252] In some embodiments, the CD3 zeta intracellular 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: 47. In some embodiments, the CD3 zeta intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain 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 intracellular domain comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the CD3 zeta intracellular domain consists of the amino acid sequence of SEQ ID NO: 47. 2. Exemplary CAR polynucleotides

[0253] In some embodiments, the CAR is an anti-CD19 CAR, and in these embodiments, the multicistronic vector comprises a fourth expression cassette comprising a nucleotide sequence encoding a 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 co-stimulatory 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.

[0254] In some embodiments, the CAR is an anti-CD20 CAR, and in these embodiments, the multicistronic vector comprises a fourth expression cassette containing a nucleotide sequence encoding 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 co-stimulatory domain, and an intracellular activation signaling domain. In some embodiments, the fourth expression cassette encodes an anti-CD20 CAR having the characteristics shown in Table 2 (anti-CD20 CAR with a tag) or Table 3 (anti-CD20 CAR without a tag).

[0255] 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.

[0256] In some of any of the 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 of any of the 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid sequence of SEQ ID NO: 27, 72, or 127. In some embodiments, the fourth expression cassette encodes an amino acid sequence that comprises 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.

[0257] In a further embodiment, an illustrative nucleotide sequence encoding a CAR may comprise SEQ ID NO:71, and an illustrative CAR amino acid sequence may comprise SEQ ID NO:72.

[0258] An illustrative nucleotide insertion may comprise SEQ ID NO:73.

[0259] In some embodiments, the CAR can be encoded by a nucleic acid sequence encoding a signal peptide, which is used to indicate the transport of the CAR in the cell. It should be understood that the signal peptide is usually removed from the protein.

[0260] An illustrative CAR amino acid sequence without the signal peptide can comprise SEQ ID NO:74.

[0261] An illustrative CAR amino acid sequence signal peptide may comprise SEQ ID NO: 75. In various embodiments, a CAR-expressing cell comprising a nucleic acid of SEQ ID NO: 71 or 73 is provided. 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 may comprise or consist of a human or humanized amino acid sequence. In some embodiments, SEQ ID NO: 74 may comprise or consist of a human or humanized amino acid sequence.

[0262] In some embodiments, mutant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74 are encompassed.

[0263] Although the affinity of a CAR expressed by a lymphocyte 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 to the targeted ligand will typically be at least about 100 nM, 1 pM, or 10 pM, preferably at least about 100 pM, 1 fM, or 10 fM, and more preferably at least about 100 fM. D. Cleavable Linker

[0264] As provided herein, the expression cassettes of the polycistronic construct can be separated by a linker. In some embodiments, the linker comprises a site for cleavage, making it a cleavable linker.

[0265] Cleavage sites can be used in the design of polycistronic constructs to 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 peptides 18-22 amino acids in length that can induce ribosome skipping during translation, resulting in the loss of the peptide bond between the glycine and proline residues, which allows proteolytic enzymes to recognize the 2A site. The most commonly used 2A peptides in molecular biology include T2A, P2A, E2A, and F2A.

[0266] 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 identical. In some embodiments, the one or more 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 identical. In some embodiments, the one or more cleavage sites are different.

[0267] In some embodiments, in addition to the 2A site, the cleavable linker may further comprise another cleavage site. In some embodiments, the additional cleavage site comprises a furin site. There are three known furin sites, including FC1, FC2, and FC3.

[0268] In some embodiments, the polycistronic constructs provided herein comprise a T2A, P2A, E2A, or F2A cleavage site in the cleavable linker. In some embodiments, the polycistronic constructs comprise a T2A cleavage site in the cleavable linker. In some embodiments, the polycistronic constructs comprise a P2A cleavage site in the cleavable linker. In some embodiments, the polycistronic constructs comprise a furin cleavage site in the cleavable linker. In some embodiments, the polycistronic constructs comprise a T2A cleavage site and a furin cleavage site in the cleavable linker.

[0269] 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.

[0270] 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 linker is independently a T2A, P2A, E2A, or F2A cleavage site. In some embodiments, the 2A cleavable linker is independently P2A or T2A.

[0271] In some embodiments, the 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. 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.

[0272] In some embodiments, at least one 2A 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. 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 that is 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 the sequence set forth in SEQ ID NO: 10.

[0273] In some embodiments, at least one cleavage site sequence 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 shown 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.

[0274] In some embodiments, the cleavage site sequence comprises a furin cleavage site sequence and a T2A cleavage sequence (furinT2A). In some embodiments, the nucleotide sequence encoding the cleavage site sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the cleavage site sequence comprises the nucleotide sequence of SEQ ID NO: 5.

[0275] 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.

[0276] In some embodiments, the first and second expression cassettes are separated by furinT2A, the second and third expression cassettes are separated by P2A, and the third and fourth expression cassettes are separated by P2A. E. Exemplary Polycistronic Constructs

[0277] In some embodiments, the polycistronic constructs provided herein comprise the features shown in Table 5, Table 6, or Table 7.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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. II. Nucleic Acid Vectors

[0287] In some embodiments, the polycistronic construct can be inserted into a nucleic acid vector. As used herein, the term "nucleic acid vector" refers to any nucleic acid having the function of carrying, accommodating or expressing a nucleic acid of interest. Nucleic acid vectors can have specialized functions, such as, for example, expression, packaging, pseudotyping, transduction or sequencing. For example, nucleic acid vectors can also have operational functions, such as cloning or shuttle vectors. The structure of the vector can include any feasible and suitable form for a specific application. Such forms include, for example, circular forms (such as plasmids and phages), and linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, or partially or entirely comprise nucleotide derivatives, analogs and analogs. Such nucleic acid vectors can be obtained from natural sources, recombinantly produced or chemically synthesized.

[0288] Non-limiting examples of vector systems of the present disclosure include retroviruses, lentiviruses, foamyviruses, and Sleeping Beauty transposons. A. Retroviral vectors

[0289] Retroviruses include lentiviruses, gammaretroviruses, and alpharetroviruses, each of which can be used to deliver polynucleotides into cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The higher level of complexity allows the virus to regulate its life cycle, such as during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Lentivirus vectors are generated by multiple attenuation of HIV virulence genes, for example, by deleting the genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0290] Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Typically, these vectors are configured to carry the necessary sequences for selecting cells containing the vector, for incorporating exogenous nucleic acid into lentiviral particles, and for transferring the nucleic acid into target cells.

[0291] Commonly used lentiviral vector systems are so-called third-generation systems. The third-generation lentiviral vector system includes four plasmids. A "transfer plasmid" encodes a polynucleotide sequence that is delivered to target cells by a lentiviral vector system. The transfer plasmid typically has one or more transgenic 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 typically designed to make the resulting vector replication defective. For example, the transfer plasmid lacks the genetic elements necessary to generate infectious particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, making the virus "self-inactivated" (SIN). See Dull et al. (1998) J.Virol.72:8463-71; Miyoshi et al. (1998) J.Virol.72:8150-57. Viral particles may also include a 3' untranslated region (UTR) and a 5'UTR. The UTRs contain retroviral regulatory elements that support the packaging, reverse transcription, and integration of the proviral genome into cells following contact of the retroviral particle with the cell.

[0292] The third generation system also typically includes two "packaging plasmids" and an "envelope plasmid". The "envelope plasmid" typically encodes the Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G and the promoter is the CMV promoter. The third generation system uses two packaging plasmids, one encoding gag and pol, and the other encoding rev as a further safety feature; it is an improvement on the single packaging plasmid of the so-called second generation system. Although safer, the third generation system can be more cumbersome to use and has lower viral titers due to the addition of the extra plasmid. Illustrative packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0293] Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line capable of producing infectious retroviral particles after the transfer plasmid, packaging plasmid, and envelope plasmid are introduced into the cell. A variety of methods can be used to introduce plasmids into cells, including transfection or electroporation. In some cases, packaging cell lines are adapted to efficiently package a retroviral vector system into retroviral particles.

[0294] As used herein, the term "retroviral vector" or "lentiviral vector" means a nucleic acid encoding the cis nucleic acid sequence of a retrovirus or lentivirus required for genome packaging, and one or more polynucleotide sequences to be delivered to a target cell. Retroviral particles and lentiviral particles typically include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope into which the Env protein is inserted, and other auxiliary proteins, including integrase, protease, and matrix protein. As used herein, the terms "retroviral particle" and "lentiviral particle" refer to viral particles that comprise an envelope, have one or more characteristics of a lentivirus, and are capable of invading a target host cell. Such properties include, for example, infection of non-dividing host cells, transduction of non-dividing host cells, infection or transduction of host immune cells, containing retroviral or lentiviral virions comprising one or more gag structural polypeptides, containing a retroviral or lentiviral envelope comprising one or more env-encoded glycoproteins, containing a genome comprising 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 comprise a cPPT sequence, as described in U.S. Patent No. 8,093,042.

[0295] The efficiency of the system is an important matter in vector engineering. The efficiency of retrovirus or slow virus vector system can be assessed by various methods known in the art, including such as by quantitative polymerase chain reaction (qPCR) or with the titer vector copy number (VCN) of the virus represented by every milliliter (mL) infectious unit (IU / mL) or the measurement of vector genome (vg). For example, titer can be assessed using the functional assay carried out on cultured tumor cell line HT1080, such as 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: described in 1237-1246 (2016). When titer is assessed on the cultured cell line of continuous division, it is not necessary to stimulate, and therefore the titer measured is not affected by the surface engineering 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).

[0296] In some embodiments, the retroviral particles and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gated adapter. In some embodiments, the sequence encoding the receptor that specifically binds to a gated adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0297] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a tag protein.

[0298] In some embodiments, each of the retroviral particles comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

[0299] In some embodiments, the retroviral particles comprise a cell surface receptor that binds to a surface marker on the target host cell, allowing host cell transduction. In some embodiments, the cell surface receptor is a T cell surface receptor. The viral vector may comprise a heterologous viral envelope glycoprotein that is given a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its mutants, VSV-G, gibbon ape leukemia virus (GALV), or an amphibious envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from the Cocal strain or a functional variant thereof.

[0300] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure encompasses the replacement of various fusion glycoproteins. It is worth noting that some fusion glycoproteins result in higher vector efficiency.

[0301] In some embodiments, pseudotyping of the fusion glycoprotein or its functional variant facilitates targeted transduction of specific cell types, including but not limited to innate lymphoid cells, cytotoxic innate lymphoid cells, or NK cells. In some embodiments, the fusion glycoprotein or its functional variant is human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibberish ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphibious retrovirus (Ampho) gp70, 10A1MLV (10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon simian 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 virus (EboZ) G, lymphocytic choriomenitis 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, Simbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza virus A, B, C or D HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or one or more full-length polypeptides, functional fragments, homologs or functional variants of Chandipura virus and Persian virus CNV-G and PRV-G.

[0302] In some embodiments, the fusion glycoprotein or a functional variant thereof is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of Alagoas vesicular stomatitis virus (VSAV), Carajas vesicular stomatitis virus (CJSV), Chandipura vesicular stomatitis virus (CHPV), Cocal vesicular stomatitis virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan vesicular stomatitis virus (ISFV), Maraba vesicular stomatitis virus (MARAV), New Jersey vesicular stomatitis virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is the Cocal virus G protein.

[0303] In some embodiments, the fusion glycoprotein or its functional variant is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of Alagoas vesicular stomatitis virus (VSAV), Caragas vesicular stomatitis virus (CJSV), Chandipura vesicular stomatitis virus (CHPV), Cocal vesicular stomatitis virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan vesicular stomatitis virus (ISFV), Maraba vesicular stomatitis virus (MARAV), New Jersey vesicular stomatitis virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or its functional variant is Cocal virus G protein. The present disclosure further provides various retroviral vectors, including but not limited to γ retroviral vectors, α retroviral vectors and lentiviral vectors. In some embodiments, the vector can be a viral vector, a retroviral vector, a lentiviral vector, a γ retroviral vector. In some embodiments, the viral vector comprises VSV G protein or its functional variant. In some embodiments, the viral vector comprises Cocal G protein or its functional variant.

[0304] In some aspects, provided herein are viral vectors comprising any of the polycistronic constructs disclosed 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. III. Virus Particles

[0305] In some embodiments, there is provided herein a kind of viral particle that is encapsulated with the polycistronic construct disclosed herein.In some embodiments, any one of the polycistronic constructs can be provided as a payload when generating viral particles.Also provided herein is a viral particle that is incorporated into any polycistronic construct provided, such as a lentiviral vector, for delivering the composition (including FRB, synthetic cytokine receptors and CAR) of rapamycin activated cytokine receptor (RACR) system to target cells.In a further embodiment, the viral particle 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.

[0306] As is well known in the art, viral particles are tools that allow or facilitate the transfer of entities from one environment to another. Consistent with the present disclosure, for example, some viral particles used in DNA recombinant technology allow the transfer of entities (such as DNA fragments) into host cells. Examples of vectors used in DNA recombinant technology include, but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses. The term "expression vector" means a construct capable of in vivo or in vitro / ex vivo expression. A. Retroviral particles

[0307] In some embodiments, the viral particles comprise retroviral particles. In some embodiments, the present disclosure provides a method for preparing a viral preparation. In some embodiments, the virus is a retrovirus. A large number of 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 (FBRMSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian medulloblastosis virus 29 (MC29) and avian erythrocytosis 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 Vamus pp 758-763.

[0308] Retroviruses include lentiviruses, gammaretroviruses, and alpharetroviruses, each of which can deliver polynucleotides into cells using methods known in the art. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The higher level of complexity allows the virus to regulate its life cycle, such as during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Lentivirus vectors are generated by multiple attenuation of HIV virulence genes, for example, by deleting the genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0309] The lentiviral vectors of the present disclosure may be derived from or may be derived from any suitable lentivirus. Recombinant retroviral vector particles are capable of transducing recipient cells with a nucleotide sequence of interest (NOI). Once inside the cell, the RNA genome in the vector particle is reverse transcribed into DNA and integrated into the DNA of the receiving cell. In some embodiments of the present disclosure, at least a portion of one or more protein coding regions important for replication may be removed from the virus. This renders the viral vector replication-defective. Portions of the viral genome may also be replaced by NOI to generate a vector comprising NOI that is capable of transducing a target non-dividing host cell and / or integrating its genome into the host genome.

[0310] Illustrative lentiviral vectors include those described in Naldini et al. (1996) Science 272:263-7; Zufferey et al. (1998) J. Virol. 72:9873-9880; Dull et al. (1998) J. Virol. 72:8463-8471; U.S. Patent No. 6,013,516; U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Typically, these vectors are configured to carry key sequences for selecting cells containing the vector, for incorporating exogenous nucleic acids into lentiviral particles, and for transferring the nucleic acids into target cells.

[0311] Commonly used lentiviral vector systems are so-called third-generation systems. The third-generation lentiviral vector system includes four plasmids. A "transfer plasmid" encodes a polynucleotide sequence that is delivered to target cells by a lentiviral vector system. The transfer plasmid typically has one or more transgenic 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 typically designed to make the resulting vector replication defective. For example, the transfer plasmid lacks the genetic elements necessary to generate infectious particles in the host cell. In addition, the transfer plasmid can be designed to lack 3'LTR, making the virus "self-inactivated" (SIN). See Dull et al. (1998) J.Virol.72:8463-71; Miyoshi et al. (1998) J.Virol.72:8150-57. Viral particles may also include a 3' untranslated region (UTR) and a 5'UTR. The UTRs contain retroviral regulatory elements that support the packaging, reverse transcription, and integration of the proviral genome into cells following contact of the retroviral particle with the cell.

[0312] The third generation system also typically includes two "packaging plasmids" and an "envelope plasmid". The "envelope plasmid" typically encodes the Env gene operably linked to a promoter. In an illustrative third generation system, the Env gene is VSV-G and the promoter is the CMV promoter. The third generation system uses two packaging plasmids, one encoding gag and pol, and the other encoding rev as a further safety feature; an improvement on the single packaging plasmid of the so-called second generation system. Although safer, the third generation system can be more cumbersome to use and result in lower viral titers due to the addition of the extra plasmid. Illustrative packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0313] Many retroviral vector systems rely on the use of a "packaging cell line." Generally, a packaging cell line is a cell line capable of producing infectious retroviral particles after the transfer plasmid, packaging plasmid, and envelope plasmid are introduced into the cell. A variety of methods can be used to introduce plasmids into cells, including transfection or electroporation. In some cases, packaging cell lines are adapted to efficiently package a retroviral vector system into retroviral particles.

[0314] As used herein, the term "retroviral vector" or "lentiviral vector" means a nucleic acid encoding the cis nucleic acid sequence of a retrovirus or lentivirus required for genome packaging, and one or more polynucleotide sequences to be delivered to a target cell. Retroviral particles and lentiviral particles typically include an RNA genome (derived from a transfer plasmid), a lipid bilayer envelope into which the Env protein is inserted, and other auxiliary proteins, including integrase, protease, and matrix protein. As used herein, the terms "retroviral particle" and "lentiviral particle" refer to viral particles that comprise an envelope, have one or more characteristics of a lentivirus, and are capable of invading a target host cell. Such properties include, for example, infection of non-dividing host cells, transduction of non-dividing host cells, infection or transduction of host immune cells, containing retroviral or lentiviral virions comprising one or more gag structural polypeptides, containing a retroviral or lentiviral envelope comprising one or more env-encoded glycoproteins, containing a genome comprising 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 comprise a cPPT sequence, as described in U.S. Patent No. 8,093,042.

[0315] The efficiency of the system is an important matter in vector engineering. The efficiency of retrovirus or slow virus vector system can be assessed by various methods known in the art, including such as by quantitative polymerase chain reaction (qPCR) or with the titer vector copy number (VCN) of the virus represented by every milliliter (mL) infectious unit (IU / mL) or the measurement of vector genome (vg). For example, titer can be assessed using the functional assay carried out on cultured tumor cell line HT1080, such as 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: described in 1237-1246 (2016). When titer is assessed on the cultured cell line of continuous division, it is not necessary to stimulate, and therefore the titer measured is not affected by the surface engineering 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).

[0316] In some embodiments, the retroviral particles and / or lentiviral particles of the present disclosure comprise a polynucleotide comprising a sequence encoding a receptor that specifically binds to a gated adapter. In some embodiments, the sequence encoding the receptor that specifically binds to a gated adapter is operably linked to a promoter. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0317] In some embodiments, the retroviral particle comprises a transduction enhancer. In some embodiments, the retroviral particle comprises a tag protein.

[0318] In some embodiments, each of the retroviral particles comprises a polynucleotide comprising, in 5' to 3' order: (i) a 5' long terminal repeat (LTR) or untranslated region (UTR), (ii) a promoter, (iii) a sequence encoding a receptor that specifically binds to a ligand, and (iv) a 3' LTR or UTR.

[0319] In some embodiments, the retroviral particles comprise a cell surface receptor that binds to a surface marker on the target host cell, allowing host cell transduction. In some embodiments, the cell surface receptor is a T cell surface receptor. The viral vector may comprise a heterologous viral envelope glycoprotein that is given a pseudotyped viral vector. For example, the viral envelope glycoprotein may be derived from RD114 or one of its mutants, VSV-G, gibbon ape leukemia virus (GALV), or an amphibious envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the cell surface receptor is a VSV G protein from the Cocal strain or a functional variant thereof.

[0320] 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 comprise mutations at positions 47 (e.g., K47Q) and / or 354 (e.g., R354A).

[0321] In some embodiments, the viral envelope protein is a protein (Cocal glycoprotein) from a Cocal strain. In some embodiments, the protein is a Cocal envelope protein comprising a mutation at position 354 (R354). In some embodiments, the protein is a Cocal envelope protein comprising a mutation at position 47 (K47). In some embodiments, the protein is a Cocal envelope variant comprising an R354Q mutation. In some embodiments, the protein is a Cocal envelope variant comprising a K47Q mutation. In some embodiments, the variant may be referred to as a "blinding" Cocal envelope. Illustrative Cocal envelope variants such as those provided in US2020 / 0216502A1 are incorporated herein by reference in their entirety.

[0322] Various fusion glycoproteins can be used to pseudotype lentiviral vectors. Although the most commonly used example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used for pseudotyping of lentiviral vectors. See Joglekar et al. Human Gene Therapy Methods 28:291-301 (2017). The present disclosure encompasses the replacement of various fusion glycoproteins. It is worth noting that some fusion glycoproteins result in higher vector efficiency.

[0323] In some embodiments, pseudotyping of the fusion glycoprotein or its functional variant facilitates targeted transduction of specific cell types, including but not limited to innate lymphoid cells or NK cells. In some embodiments, the fusion glycoprotein or its functional variant is human immunodeficiency virus (HIV) gp160, murine leukemia virus (MLV) gp70, gibberish ape leukemia virus (GALV) gp70, feline leukemia virus (RD114) gp70, amphibious retrovirus (Ampho) gp70, 10A1MLV (10A1) gp70, ecotropic retrovirus (Eco) gp70, baboon simian 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 virus (EboZ) G, lymphocytic choriomenitis 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, Simbis virus (SV) E1 and E2, Venezuelan equine encephalitis virus (VEEV) E1 and E2, Western equine encephalitis virus (WEEV) E1 and E2, influenza virus A, B, C or D HA, fowl plague virus (FPV) HA, vesicular stomatitis virus VSV-G, or one or more full-length polypeptides, functional fragments, homologs or functional variants of Chandipura virus and Persian virus CNV-G and PRV-G.

[0324] In some embodiments, the fusion glycoprotein or a functional variant thereof is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of Alagoas vesicular stomatitis virus (VSAV), Carajas vesicular stomatitis virus (CJSV), Chandipura vesicular stomatitis virus (CHPV), Cocal vesicular stomatitis virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan vesicular stomatitis virus (ISFV), Maraba vesicular stomatitis virus (MARAV), New Jersey vesicular stomatitis virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is the Cocal virus G protein.

[0325] In some embodiments, the fusion glycoprotein or a functional variant thereof is a full-length polypeptide, functional fragment, homologue or functional variant of the G protein of Alagoas vesicular stomatitis virus (VSAV), Carajas vesicular stomatitis virus (CJSV), Chandipura vesicular stomatitis virus (CHPV), Cocal vesicular stomatitis virus (COCV), Indiana vesicular stomatitis virus (VSIV), Isfahan vesicular stomatitis virus (ISFV), Maraba vesicular stomatitis virus (MARAV), New Jersey vesicular stomatitis virus (VSNJV), Bas-Congo virus (BASV). In some embodiments, the fusion glycoprotein or a functional variant thereof is the Cocal virus G protein.

[0326] 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 VSV G protein or a functional variant thereof. In some embodiments, the viral vector comprises Cocal G protein or a functional variant thereof. B. Engineered Viral Envelope

[0327] In some embodiments, the viral particles of the encapsulated nucleotide vectors provided herein may comprise an engineered viral envelope. In some embodiments, the viral envelope comprises a transduction enhancer. In some embodiments, the viral envelope comprises an immune cell activation protein. In some embodiments, the viral envelope comprises a costimulatory molecule. In some embodiments, the viral envelope comprises an immune cell activation protein and a costimulatory molecule.

[0328] In some embodiments, the viral envelope comprises one or more transduction enhancers. In some embodiments, the transduction enhancers include T cell activating receptors, NK cell activating receptors and / or co-stimulatory molecules. In some embodiments, the one or more transduction enhancers comprise one or more of anti-CD3 scFv, CD86, CD80 and / or CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD80. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv and CD86. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD80 and CD58. In some embodiments, the transduction enhancer comprises at least anti-CD3 scFv, CD86 and CD58.

[0329] In some embodiments, the viral particles comprise a cell surface receptor that binds to a ligand on a target host cell to allow transduction of the host cell. In some embodiments, the cell surface receptor is a T cell surface receptor. In some embodiments, the viral particles comprise a heterologous viral envelope glycoprotein that produces pseudotyped viral particles. For example, the viral envelope glycoprotein may be derived from RD114 or one of its mutants, VSV-G, gibbon ape leukemia virus (GALV), or an amphibious envelope, measles envelope, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a VSV G protein (Cocal glycoprotein) from the Cocal strain or a functional variant thereof.

[0330] In some embodiments, the viral envelope comprises one or more polypeptides on the surface. In some embodiments, the one or more polypeptides bind to target immune cells and replicate immune synapses. In some embodiments, the viral envelope comprises immune cell activation proteins, costimulatory molecules, and adhesion molecules, wherein the immune cell activation proteins, costimulatory molecules, and adhesion molecules each bind to target immune cells. 2. Immune cell activators

[0331] In some embodiments, the transduction enhancer comprises a mitogenic stimulator that is incorporated into the capsid of a retrovirus or lentivirus, thereby enabling the virus to activate and transduce T cells. This eliminates the need for the addition of a vector and mitogens. In some embodiments, the transduction enhancer comprises a mitogenic transmembrane protein and / or one or more co-stimulatory molecules that are incorporated into the retrovirus upon budding from the production cell membrane / packaging cell membrane. In some embodiments, the transduction enhancer is expressed on the production cell as a separate cell surface molecule, rather than as part of a viral envelope glycoprotein.

[0332] 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 a host cell during retroviral vector production. In some embodiments, the mitogenic transduction enhancer is manufactured by a packaging cell and expressed on the cell surface. When the nascent retroviral vector buds from the host cell membrane, the mitogenic transduction enhancer may be incorporated into the viral envelope as part of a lipid bilayer derived from the packaging cell. In some embodiments, the mitogenic enhancer is an antibody or a fragment thereof. In some embodiments, the mitogenic enhancer is a single domain antibody, such as a camelid antibody. In some embodiments, the mitogenic enhancer is a scFv. In some embodiments, the mitogenic enhancer is a nanobody.

[0333] In some embodiments, the transduction enhancer is derived from a host cell. The term "derived from a host cell" indicates that the mitogenic transduction enhancer is derived from a host cell as described above, rather than being produced by fusion or chimerism of one of the viral genes (such as gag encoding a major structural protein or env encoding an envelope protein).

[0334] The envelope protein consists of two subunits: a transmembrane subunit (TM) that anchors the protein to the lipid membrane and a surface subunit (SU) that binds to cell receptors. In some embodiments, the packaging cell-derived mitogenic transduction enhancer of the present invention does not contain a surface envelope subunit (SU).

[0335] In some embodiments, the mitogenic transduction enhancer has the structure: MS-TM, wherein M is a mitogenic domain; S is an optional spacer domain; and TM is a transmembrane domain.

[0336] The mitogenic structure is part of a mitogenic transduction enhancer that leads to 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.

[0337] CD3 is a T cell co-receptor. It is a protein complex composed of four different chains. In mammals, the complex contains the CD3γ chain, the CD35 chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the z chain to generate activation signals in T lymphocytes. The TCR, z chain, and CD3 molecule together constitute the TCR complex. In some embodiments, the mitogenic domain binds to the CD3ε chain.

[0338] In some embodiments, the mitogenic domain comprises all or part of an antibody or other molecule that specifically binds to a T cell surface antigen. In some embodiments, the antibody activates the TCR or CD28. In some embodiments, the antibody binds to the TCR, CD3, or CD28. Examples of such antibodies include: OKT3, 15E8, and TGN1412. Other suitable antibodies include:

[0339] Anti-CD28: CD28.2, 10F3

[0340] Anti-CD3 / TCR: UCHT1, YTH12.5, TR66

[0341] In some embodiments, the mitogenic domain comprises a binding domain from OKT3, 15E8, TGN1412, CD28.2, 10F3, UCHT1, YTH12.5, or TR66.

[0342] In some embodiments, the mitogenic domain comprises all or part of a co-stimulatory molecule, such as OX40L and 41BBL. For example, the mitogenic domain can comprise a binding domain from OX40L or 41BBL.

[0343] OKT3, also known as Muromonab-CD3, is a monoclonal antibody targeting the CD3 e chain. It is clinically used to reduce acute rejection in organ transplant patients. It is the first monoclonal antibody approved for clinical use in humans.

[0344] In some embodiments, the viral envelope comprises an immune cell activating protein. In some embodiments, the immune cell activating protein specifically binds to a receptor on an immune cell. In some embodiments, the immune cell activating protein provides a first signal for T cell activation.

[0345] In some embodiments, the immune cell activation 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 activation protein specifically binds to CD3γ, CD3δ, or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ, CD3ε, CD9, CD5, CD22, CD33, CD37, CD64, CD45, CD28H, LFA-1, DNAM-1, CD27, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, CD16, CD56, NKG2D, NKp46, NKp44, NKp30, CD244, NKp80, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein specifically binds to CD3.

[0346] 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. In some embodiments, the immune cell activating protein is an antibody or antigen binding fragment thereof that specifically binds to 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 activation 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, TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3γ, CD3δ or CD3ε. In some embodiments, the immune cell activation protein is an antibody or antigen binding fragment thereof that specifically binds to CD3.

[0347] Antibodies targeting the polypeptides described herein are known to those skilled in the art. Methods for generating antibodies are known to those skilled in the art.

[0348] 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 antigen-binding fragment thereof is conjugated to a transmembrane domain. An illustrative anti-CD3ε antibody is OKT3. OKT3, also known as Muromonab-CD3, is a monoclonal antibody that targets the CD3ε chain.

[0349] In some embodiments, the viral envelope comprises a single-chain Fv fragment (scFv) of an anti-CD3 antibody. 2. Co-stimulatory molecules

[0350] In some embodiments, the viral envelope comprises at least one co-stimulatory molecule. In some embodiments, the co-stimulatory molecule specifically binds to a receptor on an immune cell. In some embodiments, the co-stimulatory molecule provides a second signal for cell activation.

[0351] As used herein, the term "costimulatory molecules" refers to molecules that can generate co-stimulatory signals to T cells. Lymphocytes, such as T cells and natural killer (NK) cells, usually require some signals and interactions with antigen presenting cells (APCs) to obtain optimal activation in order to fully exert effector functions. For T cells, these include signals emitted by T cell receptors (TCRs), co-stimulatory molecules (such as CD28 and CD2), cytokines, and various adhesion molecules, which are necessary to obtain sufficient time for appropriate synapse formation and signal transduction. NK cells require similar types of stimulation, but can rely on different activating receptors, such as NKG2D, NKp46, and DNAM-1. For T cells, in addition to TCR stimulation, appropriate co-stimulation is particularly important for effective activation, and many studies have shown that TCR stimulation can only lead to functional hypersensitivity and anergy. Co-stimulatory signals enhance T and NK cell function by strengthening cell metabolism, cytokine production, differentiation, and long-term persistence. Co-stimulation 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 CD 154. In some embodiments, the costimulatory molecules include but are not limited to binding agents that bind to any of the costimulatory molecules described herein, such as scFv, antibodies, single domain antibodies, antibody fragments, 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-LFA1, anti-OX40, anti-41BB, anti-CD40L, anti-DNAM1, anti-CD27, anti-ICOS, anti-LIGHT, anti-GITR, anti-CD30, anti-SLAM, anti-Ly9, 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.

[0352] In some embodiments, the co-stimulatory molecule is a ligand for CD28. CD28 is one of the proteins expressed on T cells that provides the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 can also provide a potent signal for the production of various interleukins (especially IL-6). In some embodiments, the co-stimulatory molecule is an antibody or fragment thereof that binds to CD28. Examples of such antibodies include: 15E8 and TGN1412. Other suitable antibodies include: CD28.2 and 10F3.

[0353] 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 another 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 an anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or anti-CD28 scFv is coupled to a transmembrane domain for display on the surface of the viral envelope.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] CD134, also known as OX40, is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 promotes cell division and survival. OX40 is a secondary co-stimulatory molecule expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed upon activation. In some embodiments, the viral particle comprises an OX40 ligand or a functional fragment thereof coupled to the native or heterologous transmembrane domain of OX40.

[0359] CD134, also known as OX40, is a member of the TNFR superfamily of receptors expressed on activated T cells. OX40 promotes cell division and survival. OX40 is a secondary co-stimulatory molecule expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed on resting antigen-presenting cells but is expressed upon activation. In some embodiments, the viral particle comprises an OX40 ligand or a functional fragment thereof coupled to the native or heterologous transmembrane domain of OX40.

[0360] 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 is expressed on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The best feature of CD137 is its co-stimulatory activity for activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion survival, and cytolytic activity. In some embodiments, the viral particle comprises an OX40 ligand or a functional fragment thereof with 4-1BB, wherein the OX40 ligand is coupled to a natural transmembrane domain or a heterologous transmembrane domain. 4-1BBL is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transduction factor that acts as a ligand for 4-1BB (a co-stimulatory receptor molecule in T lymphocytes). 4-1BBL has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation.

[0361] Viral particles containing one or more activation or co-stimulatory molecules can be produced by engineering a packaging cell line by the methods provided in WO2016 / 139463; or by expressing T cell activation or co-stimulatory molecules through a polycistronic helper vector as described in International Patent Publication No. WO2020 / 106992A1, both of which are incorporated herein by reference in their entirety. 3. Adhesion molecules

[0362] In some embodiments, the viral particles include adhesion molecules. As used herein, the term "adhesion molecule" refers to a subset of cell surface molecules involved in the binding of cells to other cells. Adhesion cells can contribute to the formation of more stable interactions between immune cells, such as immune synapses. Immune synapses are stable adhesions between polarized immune effector cells and antigen-bearing cells. In some embodiments, the adhesion molecules can provide costimulatory signals to target cells. In some embodiments, the 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, the adhesion molecules include but are not limited to binding agents that can be bound to any adhesion molecule or costimulatory molecule described herein, such as scFv, antibodies, single domain antibodies, antibody fragments and nano antibodies. 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 α chain, anti-TCR β chain, anti-TCR γ chain and anti-TCR δ chain agents.

[0363] In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as T11, LFA-2, and erythrocyte rosette receptor, and is a surface protein expressed on T lymphocytes and NK cells. CD2 is the natural ligand for CD58. In addition to serving an adhesion function, engagement of CD2 also provides a co-stimulatory 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 for CD2. In some embodiments, the lentiviral particle comprises CD58, or a functional portion thereof, that binds to CD2.

[0364] 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.

[0365] 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. 4. Additional non-viral proteins

[0366] 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 proteins described above.

[0367] In some embodiments, the viral particles comprise a targeted ligand. In some embodiments, the viral particles comprise CD19 or a functional fragment thereof coupled to its native transmembrane domain or heterologous transmembrane domain. In some embodiments, CD19 can serve as a ligand for blinatumomab, thereby providing a linker for coupling the particles to T cells via the anti-CD3 portion of blinatumomab. In some embodiments, another type of particle surface ligand can be used to couple appropriately surface-engineered lentiviral particles to T cells using a multispecific antibody comprising a particle surface ligand binding portion. In some embodiments, the multispecific antibody is a bispecific antibody, such as a bispecific T cell engager (BiTE).

[0368] In some embodiments, the non-viral protein is a cytokine. In some embodiments, the cytokine can be selected from 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 scFv or cytokine), it can be tethered to the surface of the viral particle by fusion with a transmembrane domain (such as the transmembrane domain of CD8). Alternatively, it can also be indirectly tethered to the lentiviral particle by engineering a transmembrane protein to combine the use of a soluble protein. Further addition of one or more cytoplasmic residues can improve the stability of the fusion protein.

[0369] Mitogenic transduction enhancers and / or cytokine-based transduction enhancers may contain a "spacer sequence" to connect the antigen binding domain and the transmembrane domain. Flexible spacers allow the antigen binding domain to be oriented in different directions to facilitate binding. As used herein, the term "coupling" refers to chemical connection, fusion of two proteins directly from the C-terminus to the N-terminus; chemical connection with non-peptide space; chemical connection with polypeptide space; and fusion of two proteins from the C-terminus to the N-terminus via a peptide bond with a polypeptide spacer (such as a spacer sequence).

[0370] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human CD8 stalk or a mouse CD8 stalk. The spacer may alternatively comprise another linker sequence having similar length and / or domain spacing properties to the IgG1 Fc region, IgG1 hinge, or CD8 stalk. Modifying the human IgG1 spacer may remove the Fc binding motif. In some embodiments, the spacer may be derived from a human protein.

[0371] In some embodiments, the spacer sequence comprises a CD8-derived hinge.

[0372] In some embodiments, the spacer sequence comprises a "short" hinge. Relative to CAR hinge regions known in the art, the short hinge is described as a hinge region comprising fewer nucleotides.

[0373] The transmembrane domain is a transmembrane 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.

[0374] The viral particles of the present invention may comprise a cytokine-based transduction enhancer in the viral envelope. In some embodiments, the cytokine-based transduction enhancer is derived from host cells during viral particle production. In some embodiments, the cytokine-based transduction enhancer is produced by host cells 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.

[0375] A cytokine-based transduction enhancer can comprise a cytokine domain and a transmembrane domain. It can have the structure CS-TM, where C is the cytokine domain, S is an optional spacer domain (such as a spacer sequence), and TM is a transmembrane domain. The spacer domain and transmembrane domain are as defined above.

[0376] The cytokine domain may comprise a T cell activating cytokine, such as IL2, IL7 and IL15 or a functional fragment thereof. As used herein, a "functional fragment" of a cytokine refers to a fragment of a polypeptide that retains the ability to bind to a specific receptor and activate T cells.

[0377] IL2 is one of the factors secreted by T cells to regulate the growth and differentiation of T cells and some B cells. IL2 is a lymphokine that induces the proliferation of reactive T cells. It is secreted as a single glycosylated polypeptide, and its activity requires cleavage of a signal sequence. Solution NMR analysis reveals that the structure of IL2 consists of a bundle of four helices (termed AD), flanked by two shorter helices and several poorly defined loops. Residues in helix A and the loop region between helices A and B are important for receptor binding. IV. Methods and their uses

[0378] In some embodiments, provided herein is a method using a polycistronic construct or nucleotide vector disclosed herein. In some embodiments, the method provided delivers the polycistronic construct to a cell for expressing the system provided, the system including cytosolic FRB, synthetic cytokine receptors and CAR. In some embodiments, the polycistronic construct is contained in a viral vector, and the viral vector is used to transduce target cells. In some embodiments, the method provided can be carried out in vitro or in vitro to engineer target cells with a polycistronic construct. In some embodiments, the engineered cells are administered to the subject. In some embodiments, the method provided is carried out in vivo, and the viral vector comprising the polycistronic construct is introduced into a subject for targeting the polycistronic vector to target cells, such as T cells, in vivo. A. Methods of Transducing Cells

[0379] In some embodiments, a method for transducing cells is provided herein, comprising contacting a target cell with a particle (such as a viral vector) comprising any polycistronic construct provided herein. In some embodiments, the target cell comprises a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the target cell comprises a progenitor cell. In some embodiments, the progenitor cell comprises a peripheral blood mononuclear cell (PBMC). In some embodiments, the target cell comprises a T cell. In some embodiments, the T cell comprises a CD4+ or CD8+ T cell. In some embodiments, the method further comprises contacting the target cell with a guide RNA (gRNA) and (ii) RNA-guided endonuclease targeting a target site in an endogenous gene (i) to insert the nucleotide sequence into the endogenous gene.

[0380] In some aspects, the polynucleotides described herein can be delivered to cells in vivo. In some embodiments, via the administration of particles comprising the polynucleotides, the polynucleotides encoding the elements of the polycistronic construct disclosed herein are directly administered to the subject. In some embodiments, the particle is a viral particle. In some embodiments, the viral particle comprises anti-CD3 scFv and Cocal glycoprotein, and the polynucleotides can be delivered to cells in vivo. In some embodiments, the polynucleotides encode cytosolic FRB, synthetic cytokine receptors and chimeric antigen receptors (CAR). The polynucleotides disclosed herein can be administered to the subject, which allows various construct compositions (such as FRB, synthetic cytokine receptors and CAR) to be produced in vivo. In some embodiments, the effect of administering such polynucleotides generated in vivo is similar to the effect of directly administering ex vivo engineered cells expressing FRB, synthetic cytokine receptors and CAR. In some embodiments, administering such polynucleotides improves the in vivo transduction efficiency of particles. In some embodiments, the polynucleotides are mRNA.

[0381] The polynucleotides described herein can also be delivered to ex vivo cells. The viral particles described herein can be used ex vivo, in conventional cell manufacturing processes or in vitro or bedside processes, as described in International Patent Publication No. WO2022 / 072885A1. In one embodiment, the present disclosure provides an ex vivo method for transducing target cells, comprising contacting the target cells with particles according to the present disclosure. In some embodiments, the particles described herein can be used to transduce cells that have not been previously activated. For example, the particles described herein can be useful for transducing cells that have not previously been exposed to cell activation beads or activation reagents (such as Dynabeads or other reagents containing anti-CD3 and / or anti-CD28 antibodies or their binding fragments).

[0382] In some embodiments, the present disclosure provides a method for delivering nucleic acids to cells during an ex vivo CAR T manufacturing process. Such methods generally involve isolating PBMCs from patients via leukocyte removal. These cells are washed and optionally purified via one or more selection steps to isolate a specific T cell population of interest. In some aspects, these cells may include CD4+ and / or CD8+ T cells. The washed and / or purified cells may be optionally activated and subsequently transduced using a lentiviral vector. The activation step may include contacting the cells with an exogenous activator (such as anti-CD3 and anti-CD28 antibodies bound to a substrate) or using unbound antibodies. Illustrative activators include the presence of anti-CD3 and anti-CD28 beads and / or soluble polymers. After transduction, the cells may be optionally further washed and cultured until harvested. Methods for making engineered cell therapies including CAR T cells are known in the art (see, for example, 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 incorporated herein by reference). Illustrative methods of autologous CAR T manufacturing are disclosed in U.S. Patent Publication Nos. 2019 / 0269727, 2016 / 0122782, 2021 / 0163893, and US2017 / 0037369, each of which is incorporated herein in its entirety.

[0383] In some embodiments, the present disclosure provides a method for delivering nucleic acid to cells during an ex vivo closed-loop manufacturing process. In some embodiments, the ex vivo manufacturing process is an in vitro process. In an exemplary embodiment, the lentiviral vector disclosed herein allows for delivery of nucleic acid to target cells during a closed-loop process. Exemplary methods of closed-loop and / or in vitro circulation processes are disclosed in U.S. Patent Publication Nos. 2021 / 0244871 and WO2022072885, both of which are incorporated herein in their entirety. In some embodiments, lentiviral vectors as disclosed herein can be used for ex vivo transduction of cells. 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 a subject in a closed-loop system. In such embodiments, lentiviral particles as disclosed herein are useful even if cells have not been activated previously, and can bind to cells in a short incubation and / or contact step. In some embodiments, the incubation and / or contact step is about or less than one hour. In some embodiments, the incubation and / or contacting step is about or less than two hours, about or less than three hours, about or less than four hours, or about or less than five hours. In some embodiments, the incubation and / or contacting step is less than 12 hours or less than 24 hours. In some embodiments, the nucleic acid is delivered to the cell by transduction of 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 surface of the cell. In such embodiments, the nucleic acid can enter the cell ex vivo, or enter the cell in vivo after the cell (complexed with the lentiviral vector) is infused back into the subject.

[0384] In some embodiments, the lentiviral vectors disclosed herein eliminate the need for an ex vivo activation step. In such embodiments, the isolated cells can be directly transduced after leukapheresis, washing, or selection. It is conceivable that the surface engineering described herein enables the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein can shorten or truncate the manufacturing process by eliminating one or more unit operations (such as activation before transduction) and / or reducing the time spent in cell culture after transduction, thereby reducing the time spent in ex vivo manufacturing. B. Methods of Expressing Receptors

[0385] In some embodiments, a method for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell is provided herein. In some embodiments, the method includes contacting the target cell with a particle (such as a viral vector) comprising any polycistronic construct provided herein. In some embodiments, the contact is carried out in vitro or in vitro. In some embodiments, the contact is carried out in vivo by administering the polynucleotide construct or a particle (such as a viral vector) containing the polynucleotide construct to the subject.

[0386] In some embodiments, the target cells comprise stem cells.

[0387] In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs).

[0388] In some embodiments, the target cells comprise progenitor cells.

[0389] In some embodiments, the progenitor cells comprise peripheral blood mononuclear cells (PBMCs).

[0390] In some embodiments, the target cell comprises a T cell.

[0391] In some embodiments, the T cells comprise CD4+ or CD8+ T cells.

[0392] In some embodiments, the methods are performed ex vivo or in vitro.

[0393] In some embodiments, the method is performed in vivo.

[0394] In some embodiments, provided herein are cells produced by any of the methods disclosed herein. C. Method of Application

[0395] Also provided herein are methods for administering to a subject a cell engineered with a polycistronic construct. Also provided herein are methods for administering to a subject any of the particles provided (such as viral vectors, e.g., lentiviral vectors). In some embodiments, the subject suffers from a disease or condition, and the method of administration is used to treat the disease or condition. In any subject method, the cell or particle is 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 composition for treating a disease or condition in a subject. Such methods and uses include therapeutic methods and uses, for example, involving administering the engineered cell or particle (such as a viral particle) or a composition comprising the same to a subject suffering from a disease with a condition. In some cases, the disease or condition is a tumor or cancer. In some embodiments, the cell or its pharmaceutical composition is administered in an effective amount to effectively treat the disease or disorder. Uses include the use of the cell or its pharmaceutical composition in such methods and treatments, as well as the use of pharmaceutical drugs to perform such treatment methods. In some embodiments, the method thereby treats the disease, condition, or disorder in the subject.

[0396] In some embodiments, the engineered cells comprising any provided polycistronic constructs encoding cytoplasmic FRB, synthetic cytokine receptors, and chimeric antigen receptors (CAR) can be administered to a subject to treat a disease or condition. In some embodiments, particles, such as viral vectors (such as lentiviral vectors), can be directly administered to a subject for targeted delivery of the polycistronic construct to target cells in vivo for producing various construct compositions (such as FRB, synthetic cytokine receptors, and CAR) in vivo. Disclosed cells or particles (such as viral particles) can be administered in a variety of ways, depending on whether local or systemic treatment is desired.

[0397] In the case of adoptive cell therapy, methods of administering cells for adoptive cell therapy are known and can be used in combination with the methods and compositions provided herein.

[0398] Typically, administration can be topical, parenteral or enteral. The compositions of the present disclosure are generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically damaging the tissue of the subject and administering the pharmaceutical composition through the damaged portion in the tissue, thereby generally resulting in direct administration to the bloodstream, intramuscularly or within an internal organ. Therefore, parenteral administration includes, but is not limited to, administration of pharmaceutical compositions such as injection of the composition, application of the composition through a surgical incision, application of the composition through a non-surgical wound of a penetrating tissue, etc. In particular, parenteral administration will include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, enteral, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intrasynovial injection or infusion; and renal dialysis infusion techniques. In one embodiment, the 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 lymph nodes (such as inguinal lymph nodes). In some embodiments, the viral particles are administered by injecting 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.

[0399] The preparation of the pharmaceutical composition suitable for parenteral administration generally comprises the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such preparations can be suitable for push injection administration or for the preparation, packaging or sale of the form of continuous administration. Injectable preparations can be prepared, packaged or sold in unit dose form, such as ampoules or multi-dose containers comprising preservatives. Preparations for parenteral administration include but are not limited to suspensions, solutions, emulsions and pastes in oily or aqueous vehicles. Such preparations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of the preparation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granules) form for reorganization with a suitable carrier (such as sterile pyrogen-free water) before parenteral administration of the recombinant composition. Parenteral formulations also include aqueous solutions, which may include excipients such as salts, carbohydrates, and buffers (preferably pH 3 to 9), but for some applications, they may be more suitable for being formulated into sterile non-aqueous solutions or dried forms for use in combination with suitable vehicles (such as sterile, pyrogen-free water). Illustrative parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol solutions or glucose solutions. If necessary, such dosage forms can be appropriately buffered. Other useful parenteral formulations include those comprising active ingredients in microcrystalline form or liposome formulations. Preparations for parenteral administration can be formulated into immediate release and / or sustained release. Sustained release formulations include delayed release, sustained release, pulse release, controlled release, targeted release, and programmed release. For parenteral administration in the form of an aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should be isotonic with sufficient physiological saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injections. In some embodiments, solutions intended for subcutaneous administration include hyaluronidase.

[0400] The compositions of the present invention may additionally include other adjunct ingredients commonly found in pharmaceutical compositions. Thus, for example, the compositions may include additional, compatible, pharmaceutically active materials such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may include additional materials useful in the physical formulation of the various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, sunscreens, thickeners and stabilizers. However, when adding such materials, the biological activity of the components of the compositions of the present invention should not be excessively interfered with. The preparations may be sterilized and, if necessary, may be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavorings and / or aromatic substances, which do not interact harmfully with one or more nucleic acids in the preparations.

[0401] The present polycistronic construct can be administered in an amount for treating or preventing the disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. The therapeutic or prophylactic effect is monitored in some embodiments by regular assessment of the treated subject. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dose can be delivered by a single bolus of the composition, by multiple boluses of the composition, or by continuous boluses of the composition.

[0402] In some embodiments, in the case of infusion of differentiated cells or transgenic differentiated cells according to the present disclosure, a range of about 1 million to about 100 billion cells per kilogram of body weight of the subject is administered to the subject, such as, for example, 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 two of any of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 600 million cells, or a range defined by two of any of the foregoing values). In some embodiments, the administration of the cells or cell populations can include administering 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 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 two of any of the foregoing values), and in some cases 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 and / or number of cells therebetween. For example, in some embodiments, the administration of the cells or cell populations can include administering about 10 3 to about 10 9 cells, which includes all integer values of the number of cells within these ranges.

[0403] In some embodiments, provided herein is a method of administering any of the cells provided herein to a subject.

[0404] In some embodiments, provided herein is a method of administering any of the viral vectors provided herein to a subject.

[0405] In some embodiments, the CAR encoded by the provided polycistronic construct is targeted to an antigen associated with a disease or illness, and the method includes administering the cell (such as by adoptive cell therapy) or a viral vector (such as a lentiviral vector) to a subject suffering from or suspected of having the disease or illness. In some embodiments, the provided method includes a method for treating a patient with cancer, including administering to the patient a step of engineering a cell with any of the polycistronic constructs of the present disclosure, wherein cancer is treated in the patient. In some embodiments, the provided method includes a method for treating a patient with cancer, including administering to the patient a step of administering to the patient a viral vector (such as a lentiviral particle) comprising any polycistronic construct provided by the present disclosure, wherein cancer is treated in the patient.

[0406] 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 can include treating any cancer, including but not limited to acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, 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 sarcoma, Extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, common tumor, germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin lymphoma, Hodgkin disease, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic bile duct cancer, diffuse / invasive breast cancer, 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 carcinoma, medulloblastoma tumors, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, fungal disease, myelodysplastic syndrome, nasal cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, and penis Cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary tumor, primary central nervous system tumor, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, soft tissue sarcoma, uterine cancer, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spine cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma / thymic cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular cancer, undiagnosed cancer, ureteral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0407] In some embodiments, the CAR encoded by the provided multicistronic construct is a CAR targeting ligand that is capable of binding to an antigen on the surface of a cell associated with a disease or condition. In some embodiments, the CAR of any embodiment provided is an anti-FITC CAR for FITC, and the ligand is a bifunctional ligand consisting 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 mark the subject's cancer cells, wherein the bifunctional ligand specifically binds to a molecule expressed on a cell of the disease or condition. In some embodiments, the bifunctional ligand is FITC-folate. In some embodiments, the cancer is osteosarcoma.

[0408] In some embodiments, the method further comprises administering a bifunctional ligand to label cancer cells in the subject, wherein the bifunctional ligand specifically binds to a molecule expressed on cells of the disease or condition. In some embodiments, the bifunctional ligand is FITC-folate.

[0409] In some of any 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 is rapamycin or a rapamycin analog. 1. Non-physiological ligands

[0410] In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand. Illustrative small molecules that can be used 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.

[0411] In some embodiments, the synthetic cytokine receptor is activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0412] In some embodiments, the non-physiological ligand is a rapamycin analog.

[0413] In some embodiments, the non-physiological ligand is rapamycin.

[0414] In some embodiments, the non-physiological ligand is AP21967.

[0415] In some embodiments, the non-physiological ligand is FK506.

[0416] 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 cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is coumarins.

[0417] In some embodiments, the synthetic cytokine receptor complex is activated by folic acid, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, a rapamycin analog (analog of rapamycin), CD28 ligand, a poly (his) tag, a Strep tag, a FLAG tag, a VS tag, a Myc tag, a HA tag, a NE tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0418] In some embodiments, the non-physiological ligand can be an inorganic or organic compound less than 1000 Daltons.

[0419] In some embodiments, the ligand can be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapamycin analog includes a rapamycin variant having one or more of the following modifications relative to rapamycin: demethylation, removal or replacement of the methoxy group at C7, C42 and / or C29; removal, derivatization or replacement of the hydroxyl group at C13, C43 and / or C28; reduction, removal or derivatization of the ketone at C14, C24 and / or C30; replacement of the 6-membered piperidinol ring with a 5-membered prolyl ring; and alternative replacement of the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0420] Thus, in some embodiments, the rapamycin analog is everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-methylrapamycin, C16-(S)-3-methylindorapamycin, C16-(S)-3-methylindorapamycin (C16-iRap), AP21967 (A / C heterodimer, Takara ), mycophenolate sodium, benidipine hydrochloride, rapamycin, AP23573 (lidafenib), AP1903 (Rimiducid) or their metabolites, derivatives and / or combinations.

[0421] In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of K506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, kumarin, gibberellin, HaXS dimer (a chemical dimer of a Halo tag and a SNAP tag), TMP-H tag (trimethoprim holoenzyme protein dimer), or ABT-737 or a functional derivative thereof.

[0422] In some embodiments, the non-physiological ligand is present or provided in an amount of 0 nM to 1000 nM, such as, for example, 0.05 nM, 0.1 nM, 0.5 nM, 1 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 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 above amounts.

[0423] 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.

[0424] 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.

[0425] In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapamycin analog and is present or provided at 100 nM.

[0426] In some embodiments, the non-physiological ligand is present or provided at 1 nM.

[0427] In some embodiments, the non-physiological ligand is present or provided at 10 nM.

[0428] In some embodiments, the non-physiological ligand is present or provided at 100 nM.

[0429] In some embodiments, the non-physiological ligand is present or provided at 1000 nM. V. Definitions

[0430] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or expressions used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter relates. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of these definitions herein should not be construed as representing a substantial difference in meaning from that generally understood in the art.

[0431] As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context indicates otherwise. The conjunction "and / or" refers to all possible combinations of one or more of the listed items.

[0432] As used herein, the term "about" refers to the normal error range for each numerical value known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself.

[0433] It is to be understood that the various aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of.

[0434] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0435] The term "composition" refers to any mixture of two or more products, substances, or compounds (including cells or antibodies). It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof. The formulation is generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effectively exerted.

[0436] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0437] As used herein, a combination refers to any association between two or more items. A combination is two or more separate items, such as two compositions or two series, or a mixture thereof, such as a single mixture of two or more objects, or any variation thereof. The elements of a combination are typically functionally associated or related to each other.

[0438] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional agents and instructions for use of the composition or elements thereof, for purposes including but not limited to therapeutic use.

[0439] As used herein, "subject" refers to a recipient of a polycistronic construct or other agent. The term includes mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs, preferably humans.

[0440] As used herein, "treatment" refers to any type of action or administration that produces a benefit to a subject suffering from a disease or disorder, including improvement of the patient's condition (i.e., improvement, alleviation or relief of one or more symptoms, as well as partial or complete response to treatment).

[0441] 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 effective therapeutic amount, dosage, or dosage regimen for producing a desired therapeutic effect. As used herein, an "individual" or "subject" is a mammal. "Mammals" for therapeutic purposes include humans, livestock and farm animals, and zoo, sports, 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.

[0442] As used herein, "polynucleotide" refers to a biopolymer composed of two or more nucleotide monomers covalently bound by an ester bond between a phosphate group of a nucleotide and a hydroxyl group of the sugar portion of the next nucleotide in the chain. DNA and RNA are non-limiting examples of polynucleotides.

[0443] As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, which constitutes a portion (or all) of a protein.

[0444] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, those skilled in the art will appreciate that nucleotide substitutions can be made using conventional techniques to reflect the codon usage of any particular host organism to be expressed without affecting the polypeptide sequence encoded by the polynucleotides described herein.

[0445] Nucleic acids can comprise DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides comprising synthetic or modified nucleotides. Many different types of oligonucleotide modifications are known in the art. These include methylphosphate and thiophosphate backbones, the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it will be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made for the purpose of increasing the activity or lifetime of the polynucleotide of interest in vivo.

[0446] The term "variant" refers to a polynucleotide or polypeptide having at least one substitution, insertion, or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" refers to a variant that retains one or more functions of a reference polynucleotide or polypeptide.

[0447] As used herein, the term "sequence identity" or "identity" in relation to a polynucleotide or polypeptide sequence refers to the degree of matching of two optimally aligned polynucleotide or polypeptide sequences at each position of the alignment over the full length of the reference sequence. "Percent identity" is the number of matching positions in the optimal alignment, divided by the length of the reference sequence, plus the sum of the lengths of any intervals of the reference sequence in the alignment. Optimal alignment refers to the alignment that results in the maximum percent identity. Sequence alignment can be accomplished by many well-known methods, including, for example, by using mathematical algorithms, such as the BLAST component or the algorithm in the ClustalOmega sequence analysis program. Unless otherwise indicated, the term "sequence identity" in the claims refers to sequence identity as calculated using default parameters using BLAST version 2.12.0. Also, unless otherwise indicated, alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the full length of the reference sequence.

[0448] As used herein, "small molecule" refers to an organic compound of low molecular weight (<1000 Daltons). Small molecules can bind to specific biomacromolecules and can have a variety of biological functions or applications, including but not limited to cell signaling molecules, drugs, secondary metabolites, or other various modes of action.

[0449] The term "analog," as it relates to small molecules, refers to a compound that is similar in structure and / or function to another compound but differs in some composition. Analogs can differ in that one or more atoms, functional groups, or substructures are replaced by other atoms, functional groups, or substructures. Despite a high degree of structural and / or functional similarity, analogs can have different physical, chemical, physicochemical, biochemical, or pharmacological properties.

[0450] The term "rapamycin analogues" is a group of art-recognized analogs of rapamycin that are structurally and functionally similar to rapamycin. Some rapamycin analogues are known to share some, but not all, of the functional properties of rapamycin. For example, some rapamycin analogues are suitable for use as non-physiological ligands because they promote dimerization but have essentially no immunosuppressive activity (e.g., AP21967, AP23102, or iRAP).

[0451] An illustrative rapamycin analog of the present disclosure is AP21967

[0452] An illustrative rapamycin analog of the present disclosure is AP23102

[0453] An illustrative rapamycin analog of the present disclosure is iRAP

[0454] The term "cell population" refers to a mixture of cells suspended in solution, attached to a matrix, or stored in a container. By measuring a sample volume containing multiple cells, the overall characteristics of a cell population can be investigated. Flow cytometry can be used to reduce background fluorescence issues encountered in measuring cell populations in bulk.

[0455] As used herein, the term "engineered" refers to cells that have been stably transduced with a heterologous polynucleotide, or that have been genetically edited to introduce, delete, or modify a polynucleotide in the cell, or that have been transiently transduced with a polynucleotide in a manner that results in a stable phenotypic change in the cell. VI. Exemplary Embodiments

[0456] The implementation plans provided are: 1. A polycistronic construct comprising, in 5' to 3' order, (a) a first expression cassette comprising a nucleotide sequence encoding FRB, (b) a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine γ chain polypeptide, (c) a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine β chain polypeptide, and (d) a fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein each expression cassette is separated by a nucleotide sequence encoding a cleavage site sequence. 2. The polycistronic construct of embodiment 1, 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: 3, 13 or 50. 3. The polycistronic construct of embodiment 1 or embodiment 2, wherein the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 3, 13 or 50. 4. The polycistronic construct of any one of embodiments 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 multicistronic construct of any one of embodiments 1 to 4, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 4, 14, or 51. 6. A multicistronic construct according to any one of embodiments 1 to 5, wherein the nucleotides encoding the synthetic cytokine γ 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. 7. The multicistronic construct of any one of embodiments 1 to 6, wherein the nucleotide encoding the synthetic cytokine γ chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 15. 8. The polycistronic construct of any one of embodiments 1 to 7, wherein the synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit gamma (IL2RG). 9. The polycistronic construct of embodiment 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. The polycistronic construct of embodiment 8 or embodiment 9, wherein the IL2RG comprises the amino acid sequence of SEQ ID NO: 16. 11. The multicistronic construct of any one of embodiments 1 to 10, wherein the second expression cassette further comprises a nucleotide sequence encoding FRB. 12. The polycistronic construct of embodiment 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. The polycistronic construct of embodiment 11 or embodiment 12, wherein the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 13. 14. The polycistronic construct of any one of embodiments 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. The multicistronic construct of any one of embodiments 1 to 14, wherein the FRB comprises the amino acid sequence of SEQ ID NO: 14. 16. The multicistronic construct of any one of embodiments 1 to 15, wherein the second expression cassette is codon-optimized. 17. The polycistronic construct of any one of embodiments 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. The multicistronic construct of any one of embodiments 1 to 17, wherein the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 11. 19. The polycistronic construct of any one of embodiments 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. The multicistronic construct of any one of embodiments 1 to 19, wherein the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 12. 21. The multicistronic construct of any one of embodiments 1 to 15, wherein the second expression cassette further comprises a nucleotide sequence encoding FKBP12. 22. The polycistronic construct of embodiment 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. The polycistronic construct of embodiment 21 or embodiment 22, wherein the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21 or 55. 24. The polycistronic construct of any one of embodiments 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. The multicistronic construct of any one of embodiments 21 to 24, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 22. 26. A multicistronic construct according to any one of embodiments 1 to 25, wherein the nucleotides encoding the synthetic cytokine β 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. 27. The multicistronic construct of any one of embodiments 1 to 26, wherein the nucleotide encoding the synthetic cytokine β chain polypeptide comprises the nucleotide sequence of SEQ ID NO: 23 or 61. 28. The polycistronic construct of any one of embodiments 1 to 27, wherein the synthetic cytokine β chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB). 29. The polycistronic construct of embodiment 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. The multicistronic construct of embodiment 28 or embodiment 29, wherein the IL2RB comprises the amino acid sequence of SEQ ID NO: 24 or 62. 31. The multicistronic construct of any one of embodiments 1 to 30, wherein the third expression cassette further comprises a nucleotide sequence encoding FKBP12. 32. The polycistronic construct of embodiment 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. The polycistronic construct of embodiment 31 or embodiment 32, wherein the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 21. 34. The polycistronic construct of any one of embodiments 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: 20. 35. The multicistronic construct of any one of embodiments 31 to 34, wherein the FKBP12 comprises the amino acid sequence of SEQ ID NO: 20. 36. The multicistronic construct of any one of embodiments 1 to 35, wherein the third expression cassette is codon-optimized. 37. The polycistronic construct of any one of embodiments 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. The multicistronic construct of any one of embodiments 1 to 37, wherein the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 19. 39. The multicistronic construct of any one of embodiments 1 to 38, wherein the third expression cassette encodes an amino acid se...

Claims

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

2. The polycistronic construct of claim 1 , 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: 3, 13 or 50.

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

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 multicistronic 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. The polycistronic construct of any one of claims 1 to 5, wherein 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. 7 . The multicistronic construct according to claim 1 , wherein the nucleotide encoding the synthetic cytokine γ chain polypeptide comprises the nucleotide sequence of SEQ ID NO:

15.

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

16.

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

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

13.

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

14.

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

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

11.

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

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

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

22.

26. The polycistronic construct of any one of claims 1 to 25, wherein the nucleotides encoding the synthetic cytokine β 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.

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

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

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

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

22.

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

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

19.

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

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

13.

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

14.

46. The multicistronic construct of any one of claims 1 to 45, wherein the CAR comprises a scFv domain.

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

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

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

31.

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

35.

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

33.

61. The multicistronic construct of any one of claims 46 to 60, 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: 28 or 64.

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

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

29.

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

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

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

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

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

71. The multicistronic construct of any one of claims 68 to 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. The multicistronic 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. The multicistronic construct of any one of claims 1 to 72, wherein the CAR comprises a transmembrane domain.

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

75. The multicistronic construct of claim 74, wherein the CD8 transmembrane domain comprises a CD8 alpha transmembrane domain.

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

40.

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

41.

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

81. The polycistronic construct of claim 80, wherein the intracellular domain comprises a co-stimulatory molecule.

82. according to claim 80 or the polycistronic construct of claim 81, wherein the intracellular domain comprises 4-1BB, CD3ζ and / or CD28.

83. The polycistronic construct of claim 82, wherein the 4-1BB intracellular 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: 42 or 69.

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

85. The polycistronic construct of any one of claims 82 to 84, wherein the 4-1BB intracellular 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:

43.

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

43.

87. The multicistronic construct of any one of claims 82 to 86, wherein the CD3ζ intracellular 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: 46, 70, 100 or 118.

88. The multicistronic construct of any one of claims 82 to 87, wherein the CD3 zeta intracellular domain comprises the nucleotide sequence of SEQ ID NO: 46, 70, 100 or 118.

89. The multicistronic construct of any one of claims 82 to 88, wherein the CD3 zeta intracellular 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:

47.

90. The multicistronic construct of any one of claims 82 to 89, wherein the CD3 zeta intracellular domain comprises the amino acid sequence of SEQ ID NO:

47.

91. The multicistronic 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. The multicistronic 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. The multicistronic 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. The multicistronic 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. The polycistronic construct of claim 1, wherein each cleavage site sequence comprises a 2A cleavable linker sequence.

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

97. The polycistronic construct of claim 95 or claim 96, wherein the 2A cleavable linker is independently a T2A, P2A, E2A or F2A cleavage site.

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

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

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

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

18.

102. The method of any one of claims 101, wherein the P2A cleavable linker comprises the sequence shown in SEQ ID NO:

18.

103. The polycistronic construct of any one of claims 95 to 102, wherein at least one 2A 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.

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

9.

105. The multicistronic construct of any one of claims 97, 98, 103, and 104, wherein 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.

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

10.

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

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

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

8.

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

8.

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 (furinT2A).

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

6.

117. The multicistronic 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. The polycistronic construct of any one of claims 1 to 109, wherein the first expression cassette and the second expression cassette are separated by furinT2A, 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.

119. The polycistronic construct of any one of claims 1 to 118, wherein 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.

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

1.

121. The polycistronic construct of any one of claims 1 to 120, wherein 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.

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

2.

123. The polycistronic construct of any one of claims 1 to 118, wherein 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.

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

48.

125. The polycistronic construct of any one of claims 1 to 118, 123 or 124, wherein 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.

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

49.

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

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

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

130. The viral vector of claim 129, wherein the 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. The cell of claim 131, wherein the cell comprises a stem cell or a progenitor cell.

133. The cell of claim 132, wherein the stem cell comprises an induced pluripotent stem cell (iPSC).

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

135. The cell of claim 131, wherein the cell comprises a T cell.

136. The cell of claim 131, wherein the cell comprises a cytotoxic innate lymphocyte (CIL) cell.

137. The cell of claim 131, wherein the cell comprises a natural killer (NK) cell.

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

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

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

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

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

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

144. The method of claim 138 or claim 139, wherein the target cell comprises a T cell.

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

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

147. A method of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell.

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

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

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

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

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

153. The method of claim 147 or claim 148, wherein the target cell comprises a T cell.

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 to 154, wherein the method is performed ex vivo or in vitro.

156. The method of any one of claims 138 to 154, wherein the method is performed in vivo.

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

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

159. A method of delivering a payload to a T cell, the method comprising contacting the T cell with a viral vector comprising one or more T cell activators and a polycistronic construct according to 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 to 159, wherein the T cells comprise CD4+ or CD8+ T cells.

161. The method of any one of claims 157 to 160, wherein the method is performed ex vivo or in vitro.

162. The method of any one of claims 157 to 160, wherein the method is performed in vivo.

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

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

165. A cell produced by the method of any one of claims 138 to 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. The method of claim 166 or claim 167, wherein the method treats a disease or disorder in the subject.

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

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

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

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. The method of claim 166 or claim 167, further comprising administering a bifunctional ligand to the subject 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) portion and the chimeric antigen receptor (CAR) encoded by the polycistronic construct is an anti-FITC CAR.

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

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

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

179. The method of claim 177 or claim 178, wherein binding of the non-physiological ligand to the synthetic cytokine receptor stimulates intracellular cytokine signaling in cells transduced to express the synthetic cytokine receptor.

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

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